U.S. patent application number 15/488966 was filed with the patent office on 2017-09-28 for aav vectors targeted to oligodendrocytes.
The applicant listed for this patent is The University of North Carolina at Chapel Hill. Invention is credited to Steven Gray, Thomas McCown.
Application Number | 20170274024 15/488966 |
Document ID | / |
Family ID | 50388998 |
Filed Date | 2017-09-28 |
United States Patent
Application |
20170274024 |
Kind Code |
A1 |
McCown; Thomas ; et
al. |
September 28, 2017 |
AAV Vectors Targeted to Oligodendrocytes
Abstract
The invention relates to chimeric AAV capsids targeted to
oligodendrocytes, virus vectors comprising the same, and methods of
using the vectors to target oligodendrocytes.
Inventors: |
McCown; Thomas; (Carrboro,
NC) ; Gray; Steven; (Mebane, NC) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The University of North Carolina at Chapel Hill |
Chapel Hill |
NC |
US |
|
|
Family ID: |
50388998 |
Appl. No.: |
15/488966 |
Filed: |
April 17, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14431900 |
Mar 27, 2015 |
9636370 |
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PCT/US13/62240 |
Sep 27, 2013 |
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15488966 |
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61707108 |
Sep 28, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2750/14142
20130101; C12N 2750/14143 20130101; C07K 14/005 20130101; C12N
2750/14122 20130101; A61P 35/00 20180101; C12N 15/8645 20130101;
A61K 9/0085 20130101; C12N 7/00 20130101; C12N 15/86 20130101; A61K
39/23 20130101; C07K 14/015 20130101; A61P 25/00 20180101; C12N
2750/14145 20130101; A61P 3/00 20180101; A61P 9/10 20180101; A61P
13/00 20180101; A61K 35/76 20130101; C12N 2810/6027 20130101; A61P
31/12 20180101; A61P 25/28 20180101 |
International
Class: |
A61K 35/76 20150101
A61K035/76; A61K 9/00 20060101 A61K009/00; C07K 14/005 20060101
C07K014/005; C12N 15/86 20060101 C12N015/86; C12N 7/00 20060101
C12N007/00 |
Claims
1-27. (canceled)
28. A method of delivering a nucleic acid of interest to an
oligodendrocyte, the method comprising contacting the
oligodendrocyte with an AAV particle comprising: an AAV vector
genome comprising or encoding the nucleic acid of interest; and an
AAV capsid comprising the amino acid sequence of one of SEQ ID
NOS:2-4, wherein 25 or fewer amino acids are substituted, deleted,
and/or inserted, wherein the AAV capsid encapsidates the AAV vector
genome.
29. A method of delivering a nucleic acid of interest to an
oligodendrocyte in a mammalian subject, the method comprising:
administering an effective amount of an AAV particle comprising an
AAV vector genome comprising or encoding the nucleic acid of
interest; and an AAV capsid comprising the amino acid sequence of
one of SEQ ID NOS:2-4, wherein 25 or fewer amino acids are
substituted, deleted, and/or inserted, wherein the AAV capsid
encapsidates the AAV vector genome, or a pharmaceutical formulation
comprising the AAV particle to a mammalian subject.
30. The method of claim 29, wherein the mammalian subject is a
human subject.
31. The method of claim 29, wherein the AAV particle is delivered
to the central nervous system (CNS).
32. The method of claim 31, wherein the AAV particle is delivered
directly to the CNS by intrathecal, intracerebral,
intraventricular, intranasal, intra-aural, intra-ocular, or
peri-ocular delivery, or any combination thereof.
33. The method of claim 31, wherein the subject has a compromised
blood brain barrier and the AAV particle is delivered to the CNS by
intravenous administration.
34. A method of delivering a nucleic acid of interest to an area of
the CNS bordering a compromised blood brain barrier area in a
mammalian subject, the method comprising: intravenously
administering an effective amount of an AAV particle comprising: an
AAV vector genome comprising or encoding the nucleic acid of
interest; and an AAV capsid comprising the amino acid sequence of
one of SEQ ID NOS:2-4, wherein 25 or fewer amino acids are
substituted, deleted, and/or inserted, wherein the AAV capsid
encapsidates the AAV vector genome, or a pharmaceutical formulation
comprising the AAV particle to a mammalian subject.
35. A method of treating a disorder associated with oligodendrocyte
dysfunction in a mammalian subject in need thereof, the method
comprising administering a therapeutically effective amount of an
AAV particle comprising: an AAV vector genome; and an AAV capsid
comprising the amino acid sequence of one of SEQ ID NOS:2-4,
wherein 25 or fewer amino acids are substituted, deleted, and/or
inserted, wherein the AAV capsid encapsidates the AAV vector
genome, or a pharmaceutical formulation comprising the AAV particle
to a mammalian subject.
36. The method of claim 35, wherein the disorder associated with
oligodendrocyte dysfunction is a demyelinating disease.
37. The method of claim 35, wherein the disorder associated with
oligodendrocyte dysfunction is multiple sclerosis,
Pelizaeus-Merzbacher disease, Krabbe's disease, metachromatic
leukodystrophy, adrenoleukodystrophy, Canavan disease, Alexander
disease, orthochromatic leukodystrophy, Zellweger disease,
18q-syndrome, cerebral palsy, spinal cord injury, traumatic brain
injury, stroke, phenylketonuria, or viral infection.
38-39. (canceled)
40. The method of claim 28, wherein the AAV capsid comprises the
amino acid sequence of one of SEQ ID NOS:2-4, wherein 10 or fewer
amino acids are substituted, deleted, and/or inserted.
41. The method of claim 28, wherein the AAV capsid comprises the
amino acid sequence of one of SEQ ID NOS:2-4.
42. The method of claim 29, wherein the AAV capsid comprises the
amino acid sequence of one of SEQ ID NOS:2-4, wherein 10 or fewer
amino acids are substituted, deleted, and/or inserted.
43. The method of claim 29, wherein the AAV capsid comprises the
amino acid sequence of one of SEQ ID NOS:2-4.
44. The method of claim 34, wherein the AAV capsid comprises the
amino acid sequence of one of SEQ ID NOS:2-4, wherein 10 or fewer
amino acids are substituted, deleted, and/or inserted.
45. The method of claim 34, wherein the AAV capsid comprises the
amino acid sequence of one of SEQ ID NOS:2-4.
46. The method of claim 35, wherein the AAV capsid comprises the
amino acid sequence of one of SEQ ID NOS:2-4, wherein 10 or fewer
amino acids are substituted, deleted, and/or inserted.
47. The method of claim 35, wherein the AAV capsid comprises the
amino acid sequence of one of SEQ ID NOS:2-4.
Description
STATEMENT OF PRIORITY
[0001] This application claims the benefit of U.S. Provisional
Application Ser. No. 61/707,108, filed Sep. 28, 2012, the entire
contents of which is incorporated by reference herein.
FIELD OF THE INVENTION
[0002] The invention relates to chimeric AAV capsids targeted to
oligodendrocytes, virus vectors comprising the same, and methods of
using the vectors to target oligodendrocytes.
BACKGROUND OF THE INVENTION
[0003] Adeno-associated virus (AAV) was first reported to
efficiently transduce muscle over ten years ago (Xiao et al.,
(1996) J. Virol. 70:8098-8108). The recombinant AAV (rAAV) genome
composed of a foreign expression cassette and AAV inverted terminal
repeat (ITR) sequences exists in eukaryotic cells in an episomal
form that is responsible for persistent transgene expression
(Schnepp et al., (2003) J. Virol. 77:3495-3504). AAV vectors have a
good safety profile. No human disease has been associated with
wild-type AAV infection and low toxicity is observed in human
subjects following transduction by rAAV (Manno et al., (2003) Blood
101:2963-2972).
[0004] In the brain, the vast majority of AAV vectors exhibit a
dominant preference for neurons with a very low efficacy for other
cell types, such as oligodendrocytes. Recent advances in AAV
engineering and directed evolution have expanded the ability to
develop novel AAV serotypes, including vectors with altered tropism
(Gray et al., (2010) Mol. Ther. 18:570-578). However, in the
central nervous system, all AAV vectors and chimeras, except AAV4,
exhibit a dominant neuronal tropism. AAV vectors that efficiently
target oligodendrocytes have not been developed.
SUMMARY OF THE INVENTION
[0005] The present invention is based, in part, on the development
of a chimeric AAV capsid sequence that exhibits a dominant tropism
for oligodendrocytes. The chimeric capsid can be used to create AAV
vectors that transduce oligodendrocytes in the central nervous
system (CNS) of subjects.
[0006] Thus, one aspect of the invention relates to a nucleic acid
encoding an AAV capsid, the nucleic acid comprising an AAV capsid
coding sequence that is at least 90% identical to: (a) the
nucleotide sequence of SEQ ID NO:1; or (b) a nucleotide sequence
encoding any one of SEQ ID NOS:2-4, along with cells and viral
particles comprising the nucleic acid.
[0007] Another aspect of the invention relates to an AAV capsid
comprising an amino acid sequence at least 96% identical to any one
of SEQ ID NOS:2-4, along with AAV particles comprising an AAV
vector genome and the AAV capsid of the invention.
[0008] A further aspect of the invention relates to a method of
producing a recombinant AAV particle comprising an AAV capsid, the
method comprising: providing a cell in vitro with a nucleic acid of
the invention, an AAV rep coding sequence, an AAV vector genome
comprising a heterologous nucleic acid, and helper functions for
generating a productive AAV infection; and allowing assembly of the
recombinant AAV particle comprising the AAV capsid and
encapsidating the AAV vector genome.
[0009] An additional aspect of the invention relates to a
pharmaceutical formulation comprising the nucleic acid, virus
particle, AAV capsid, or AAV particle of the invention in a
pharmaceutically acceptable carrier.
[0010] Another aspect of the invention relates to a method of
delivering a nucleic acid of interest to an oligodendrocyte, the
method comprising contacting the oligodendrocyte with the AAV
particle of the invention.
[0011] A further aspect of the invention relates to a method of
delivering a nucleic acid of interest to an oligodendrocyte in a
mammalian subject, the method comprising administering an effective
amount of the AAV particle or pharmaceutical formulation of the
invention to a mammalian subject.
[0012] An additional aspect of the invention relates to a method of
delivering a nucleic acid of interest to an area of the CNS
bordering a compromised blood brain barrier area in a mammalian
subject, the method comprising intravenously administering an
effective amount of the AAV particle or pharmaceutical formulation
of the invention to a mammalian subject.
[0013] Another aspect of the invention relates to a method of
treating a disorder associated with oligodendrocyte dysfunction in
a mammalian subject in need thereof, the method comprising
administering a therapeutically effective amount of the AAV
particle or pharmaceutical formulation of the invention to a
mammalian subject.
[0014] A further aspect of the invention relates to a method of
preparing an AAV capsid having a tropism profile of interest, the
method comprising modifying the AAV capsid of the invention to
insert an amino acid sequence providing the tropism profile of
interest.
[0015] These and other aspects of the invention are set forth in
more detail in the description of the invention below.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1 shows the chimeric structure of the BNP61, BNP62, and
BNP63 AAV capsid clones.
[0017] FIGS. 2A-2C show the tropism of BNP61 for oligodendrocytes
in rat caudate. (A) shows GFP positive oligodendrocytes in the rat
caudate 1 week after the infusion of BNP61-CBh-GFP vectors. Note
that there are no GFP positive neurons. (B) shows a higher
magnification that reflects clear oligodendrocyte morphology, and
(C) shows that none of the GFP positive cells colocalize with the
cellular marker for astrocytes, GFAP(red).
[0018] FIGS. 3A-3B show the tropism of BNP61 for oligodendrocytes
in primary oligodendrocyte cultures.
[0019] FIG. 4 shows the biodistribution of BNP61 (MG001) and parent
capsids after intravenous injection in female wild-type mice,
determined by quantitative PCR. The Y-axis is copies of GFP per
diploid mouse genome.
[0020] FIG. 5 shows that BNP61 crosses the compromised blood-brain
barrier after peripheral administration.
[0021] FIG. 6 shows the tropism of BNP63 for oligodendrocytes in
rat piriform cortex.
DETAILED DESCRIPTION OF THE INVENTION
[0022] The present invention is based, in part, on the development
of a chimeric AAV capsid sequence that exhibits a tropism for
oligodendrocytes. The chimeric capsid can be used to create AAV
vectors that transduce oligodendrocytes in the CNS of subjects.
[0023] The present invention is explained in greater detail below.
This description is not intended to be a detailed catalog of all
the different ways in which the invention may be implemented, or
all the features that may be added to the instant invention. For
example, features illustrated with respect to one embodiment may be
incorporated into other embodiments, and features illustrated with
respect to a particular embodiment may be deleted from that
embodiment. In addition, numerous variations and additions to the
various embodiments suggested herein will be apparent to those
skilled in the art in light of the instant disclosure which do not
depart from the instant invention. Hence, the following
specification is intended to illustrate some particular embodiments
of the invention, and not to exhaustively specify all permutations,
combinations and variations thereof.
[0024] Unless the context indicates otherwise, it is specifically
intended that the various features of the invention described
herein can be used in any combination. Moreover, the present
invention also contemplates that in some embodiments of the
invention, any feature or combination of features set forth herein
can be excluded or omitted. To illustrate, if the specification
states that a complex comprises components A, B and C, it is
specifically intended that any of A, B or C, or a combination
thereof, can be omitted and disclaimed singularly or in any
combination.
[0025] Unless otherwise defined, 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. The
terminology used in the description of the invention herein is for
the purpose of describing particular embodiments only and is not
intended to be limiting of the invention.
[0026] Nucleotide sequences are presented herein by single strand
only, in the 5' to 3' direction, from left to right, unless
specifically indicated otherwise. Nucleotides and amino acids are
represented herein in the manner recommended by the IUPAC-IUB
Biochemical Nomenclature Commission, or (for amino acids) by either
the one-letter code, or the three letter code, both in accordance
with 37 C.F.R. .sctn.1.822 and established usage.
[0027] Except as otherwise indicated, standard methods known to
those skilled in the art may be used for production of recombinant
and synthetic polypeptides, antibodies or antigen-binding fragments
thereof, manipulation of nucleic acid sequences, production of
transformed cells, the construction of rAAV constructs, modified
capsid proteins, packaging vectors expressing the AAV rep and/or
cap sequences, and transiently and stably transfected packaging
cells. Such techniques are known to those skilled in the art. See,
e.g., SAMBROOK et al., MOLECULAR CLONING: A LABORATORY MANUAL 2nd
Ed. (Cold Spring Harbor, N.Y., 1989); F. M. AUSUBEL et al. CURRENT
PROTOCOLS IN MOLECULAR BIOLOGY (Green Publishing Associates, Inc.
and John Wiley & Sons, Inc., New York).
[0028] All publications, patent applications, patents, nucleotide
sequences, amino acid sequences and other references mentioned
herein are incorporated by reference in their entirety.
1. Definitions
[0029] The designation of all amino acid positions in the AAV
capsid subunits in the description of the invention and the
appended claims is with respect to VP1 capsid subunit
numbering.
[0030] As used in the description of the invention and the appended
claims, the singular forms "a," "an" and "the" are intended to
include the plural forms as well, unless the context clearly
indicates otherwise.
[0031] As used herein, "and/or" refers to and encompasses any and
all possible combinations of one or more of the associated listed
items, as well as the lack of combinations when interpreted in the
alternative ("or").
[0032] Moreover, the present invention also contemplates that in
some embodiments of the invention, any feature or combination of
features set forth herein can be excluded or omitted.
[0033] Furthermore, the term "about," as used herein when referring
to a measurable value such as an amount of a compound or agent of
this invention, dose, time, temperature, and the like, is meant to
encompass variations of .+-.20%, .+-.10%, .+-.5%, .+-.1%, .+-.0.5%,
or even .+-.0.1% of the specified amount.
[0034] The term "consisting essentially of" as used herein in
connection with a nucleic acid, protein or capsid structure means
that the nucleic acid, protein or capsid structure does not contain
any element other than the recited element(s) that significantly
alters (e.g., more than about 1%, 5% or 10%) the function of
interest of the nucleic acid, protein or capsid structure, e.g.,
tropism profile of the protein or capsid or a protein or capsid
encoded by the nucleic acid.
[0035] The term "adeno-associated virus" (AAV) in the context of
the present invention includes without limitation AAV type 1, AAV
type 2, AAV type 3 (including types 3A and 3B), AAV type 4, AAV
type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type
10, AAV type 11, avian AAV, bovine AAV, canine AAV, equine AAV, and
ovine AAV and any other AAV now known or later discovered. See,
e.g., BERNARD N. FIELDS et al., VIROLOGY, volume 2, chapter 69 (4th
ed., Lippincott-Raven Publishers). A number of additional AAV
serotypes and clades have been identified (see, e.g., Gao et al.,
(2004) J. Virol. 78:6381-6388 and Table 1), which are also
encompassed by the term "AAV."
[0036] The genomic sequences of various AAV and autonomous
parvoviruses, as well as the sequences of the ITRs, Rep proteins,
and capsid subunits are known in the art. Such sequences may be
found in the literature or in public databases such as the GenBank
database. See, e.g., GenBank.RTM. Accession Numbers NC 002077, NC
001401, NC 001729, NC 001863, NC 001829, NC 001862, NC 000883, NC
001701, NC 001510, AF063497, U89790, AF043303, AF028705, AF028704,
J02275, J01901, 102275, X01457, AF288061, AH009962, AY028226,
AY028223, NC 001358, NC 001540, AF513851, AF513852, AY530579,
AY631965, AY631966; the disclosures of which are incorporated
herein in their entirety. See also, e.g., Srivistava et al., (1983)
J. Virol 45:555; Chiorini et al., (1998) J. Virol. 71:6823;
Chiorini et al., (1999) J. Virol. 73:1309; Bantel-Schaal et al.,
(1999) J. Virol. 73:939; Xiao et al., (1999) J. Virol. 73:3994;
Muramatsu et al., (1996) Virology 221:208; Shade et at, (1986) J.
Virol 58:921; Gao et al., (2002) Proc. Nat. Acad. Sci. USA
99:11854; international patent publications WO 00/28061, WO
99/61601, WO 98/11244; U.S. Pat. No. 6,156,303; the disclosures of
which are incorporated herein in their entirety. See also Table 1.
An early description of the AAV1, AAV2 and AAV3 terminal repeat
sequences is provided by Xiao, X., (1996), "Characterization of
Adeno-associated virus (AAV) DNA replication and integration,"
Ph.D. Dissertation, University of Pittsburgh, Pittsburgh, Pa.
(incorporated herein it its entirety).
TABLE-US-00001 TABLE 1 GenBank GenBank GenBank Accession Accession
Accession Complete Genomes Number Number Number Hu T88 AY695375
Hu42 AY530605 Adeno-associated virus 1 NC_002077, AF063497 Hu T71
AY695374 Hu67 AY530627 Adeno-associated virus 2 NC_001401 Hu T70
AY695373 Hu40 AY530603 Adeno-associated virus 3 NC_001729 Hu T40
AY695372 Hu41 AY530604 Adeno-associated virus NC_001863 Hu T32
AY695371 Hu37 AY530600 3B Adeno-associated virus 4 NC_001829 Hu T17
AY695370 Rh40 AY530559 Adeno-associated virus 5 Y18065, AF085716 Hu
LG15 AY695377 Rh2 AY243007 Adeno-associated virus 6 NC_001862 Clade
C Bb1 AY243023 Avian AAV ATCC VR- AY186198, AY629583, Hu9 AY530629
Bb2 AY243022 865 NC_004828 Avian AAV strain DA-1 NC_006263,
AY629583 Hu10 AY530576 Rh10 AY243015 Bovine AAV NC_005889, AY388617
Hu11 AY530577 Hu17 AY530582 Clade A Hu53 AY530615 Hu6 AY530621 AAV1
NC_002077, AF063497 Hu55 AY530617 Rh25 AY530557 AAV6 NC_001862 Hu54
AY530616 Pi2 AY530554 Hu.48 AY530611 Hu7 AY530628 Pi1 AY530553 Hu
43 AY530606 Hu18 AY530583 Pi3 AY530555 Hu 44 AY530607 Hu15 AY530580
Rh57 AY530569 Hu 46 AY530609 Hu16 AY530581 Rh50 AY530563 Clade B
Hu25 AY530591 Rh49 AY530562 Hu. 19 AY530584 Hu60 AY530622 Hu39
AY530601 Hu. 20 AY530586 Ch5 AY243021 Rh58 AY530570 Hu 23 AY530589
Hu3 AY530595 Rh61 AY530572 Hu22 AY530588 Hu1 AY530575 Rh52 AY530565
Hu24 AY530590 Hu4 AY530802 Rh53 AY530566 Hu21 AY530587 Hu2 AY530585
Rh51 AY530564 Hu27 AY530592 Hu61 AY530623 Rh64 AY530574 Hu28
AY530593 Clade D Rh43 AY530560 Hu 29 AY530594 Rh62 AY530573 AAV8
AF513852 Hu63 AY530624 Rh48 AY530561 Rh8 AY242997 Hu64 AY530625
Rh54 AY530567 Rh1 AY530556 Hu13 AY530578 Rh55 AY530568 Clade F Hu56
AY530618 Cy2 AY243020 Hu14 (AAV9) AY530579 Hu57 AY530619 AAV7
AF513851 Hu31 AY530596 Hu49 AY530612 Rh35 AY243000 Hu32 AY530597
Hu58 AY530620 Rh37 AY242998 Clonal Isolate Hu34 AY530598 Rh36
AY242999 AAV6 Y18065, AF085716 Hu35 AY530599 Cy6 AY243016 AAV 3
NC_001729 AAV2 NC_001401 Cy4 AY243018 AAV 3B NC_001863 Hu45
AY530608 Cy3 AY243019 AAV4 NC_001829 Hu47 AY530610 Cy5 AY243017
Rh34 AY243001 Hu51 AY530613 Rh13 AY243013 Rh33 AY243002 Hu52
AY530614 Clade E Rh32 AY243003 Hu T41 AY695378 Rh38 AY530558 Hu S17
AY695376 Hu66 AY530626
[0037] A "chimeric" AAV nucleic acid capsid coding sequence or AAV
capsid protein is one that combines portions of two or more capsid
sequences. A "chimeric" AAV virion or particle comprises a chimeric
AAV capsid protein.
[0038] The term "tropism" as used herein refers to preferential
entry of the virus into certain cell or tissue type(s) and/or
preferential interaction with the cell surface that facilitates
entry into certain cell or tissue types, optionally and preferably
followed by expression (e.g., transcription and, optionally,
translation) of sequences carried by the viral genome in the cell,
e.g., for a recombinant virus, expression of the heterologous
nucleotide sequence(s). Those skilled in the art will appreciate
that transcription of a heterologous nucleic acid sequence from the
viral genome may not be initiated in the absence of trans-acting
factors, e.g., for an inducible promoter or otherwise regulated
nucleic acid sequence. In the case of a rAAV genome, gene
expression from the viral genome may be from a stably integrated
provirus and/or from a non-integrated episome, as well as any other
form which the virus nucleic acid may take within the cell.
[0039] The term "tropism profile" refers to the pattern of
transduction of one or more target cells, tissues and/or organs.
Representative examples of chimeric AAV capsids have a tropism
profile characterized by efficient transduction of oligodendrocytes
with only low transduction of neurons, astrocytes, and other CNS
cells.
[0040] The term "specific for oligodendrocytes" as used herein
refers to a viral vector that, when administered directly into the
CNS, preferentially transduces oligodendrocytes over neurons,
astrocytes, and other CNS cell types. In some embodiments, at least
about 80% of the transduced cells are oligodendrocytes, e.g., at
least about 85%, 90%, 95%, 96%, 97%, 98%, 99% or more
oligodendrocytes.
[0041] The term "disorder associated with oligodendrocyte
dysfunction" as used herein refers to a disease, disorder, or
injury in which oligodendrocytes are damaged, lost, or function
improperly. The term includes diseases, disorders, and injuries in
which oligodendmcytes are directly affected as well as diseases,
disorders, and injuries in which oligodendrocytes become
dysfunctional secondary to damage to other cells (e.g., spinal cord
injury).
[0042] The term "bordering a compromised blood-brain barrier area"
as used herein refers to CNS cells that are adjacent to a portion
of the blood-brain barrier in which the barrier function has been
compromised.
[0043] As used herein, "transduction" of a cell by a virus vector
(e.g., an AAV vector) means entry of the vector into the cell and
transfer of genetic material into the cell by the incorporation of
nucleic acid into the virus vector and subsequent transfer into the
cell via the virus vector.
[0044] Unless indicated otherwise, "efficient transduction" or
"efficient tropism," or similar terms, can be determined by
reference to a suitable positive or negative control (e.g., at
least about 50%, 60%, 70%, 80%, 85%, 90%, 95% or more of the
transduction or tropism, respectively, of a positive control or at
least about 110%, 120%, 150%, 200%, 300%, 500%, 1000% or more of
the transduction or tropism, respectively, of a negative
control).
[0045] Similarly, it can be determined if a virus "does not
efficiently transduce" or "does not have efficient tropism" for a
target tissue, or similar terms, by reference to a suitable
control. In particular embodiments, the virus vector does not
efficiently transduce (i.e., does not have efficient tropism) for
liver, kidney, gonads and/or germ cells. In particular embodiments,
undesirable transduction of tissue(s) (e.g., liver) is 20% or less,
10% or less, 5% or less, 1% or less, 0.1% or less of the level of
transduction of the desired target tissue(s) (e.g., skeletal
muscle, diaphragm muscle and/or cardiac muscle).
[0046] As used herein, the term "polypeptide" encompasses both
peptides and proteins, unless indicated otherwise.
[0047] A "nucleic acid" or "nucleotide sequence" is a sequence of
nucleotide bases, and may be RNA, DNA or DNA-RNA hybrid sequences
(including both naturally occurring and non-naturally occurring
nucleotide), but is preferably either single or double stranded DNA
sequences.
[0048] As used herein, an "isolated" nucleic acid or nucleotide
sequence (e.g., an "isolated DNA" or an "isolated RNA") means a
nucleic acid or nucleotide sequence separated or substantially free
from at least some of the other components of the naturally
occurring organism or virus, for example, the cell or viral
structural components or other polypeptides or nucleic acids
commonly found associated with the nucleic acid or nucleotide
sequence.
[0049] Likewise, an "isolated" polypeptide means a polypeptide that
is separated or substantially free from at least some of the other
components of the naturally occurring organism or virus, for
example, the cell or viral structural components or other
polypeptides or nucleic acids commonly found associated with the
polypeptide.
[0050] By the term "treat," "treating," or "treatment of" (or
grammatically equivalent terms) it is meant that the severity of
the subject's condition is reduced or at least partially improved
or ameliorated and/or that some alleviation, mitigation or decrease
in at least one clinical symptom is achieved and/or there is a
delay in the progression of the condition and/or prevention or
delay of the onset of a disease or disorder. The term "treat,"
"treats," "treating," or "treatment of" and the like also include
prophylactic treatment of the subject (e.g., to prevent the onset
of infection or cancer or a disorder). As used herein, the term
"prevent," "prevents," or "prevention" (and grammatical equivalents
thereof) are not meant to imply complete abolition of disease and
encompasses any type of prophylactic treatment that reduces the
incidence of the condition, delays the onset and/or progression of
the condition, and/or reduces the symptoms associated with the
condition. Thus, unless the context indicates otherwise, the term
"treat," "treating," or "treatment of" (or grammatically equivalent
terms) refer to both prophylactic and therapeutic regimens.
[0051] An "effective" or "therapeutically effective" amount as used
herein is an amount that is sufficient to provide some improvement
or benefit to the subject. Alternatively stated, an "effective" or
"therapeutically effective" amount is an amount that will provide
some alleviation, mitigation, or decrease in at least one clinical
symptom in the subject. Those skilled in the art will appreciate
that the therapeutic effects need not be complete or curative, as
long as some benefit is provided to the subject.
[0052] A "heterologous nucleotide sequence" or "heterologous
nucleic acid" is a sequence that is not naturally occurring in the
virus. Generally, the heterologous nucleic acid or nucleotide
sequence comprises an open reading frame that encodes a polypeptide
and/or a nontranslated RNA.
[0053] A "therapeutic polypeptide" can be a polypeptide that can
alleviate or reduce symptoms that result from an absence or defect
in a protein in a cell or subject. In addition, a "therapeutic
polypeptide" can be a polypeptide that otherwise confers a benefit
to a subject, e.g., anti-cancer effects or improvement in
transplant survivability.
[0054] As used herein, the term "vector," "virus vector," "delivery
vector" (and similar terms) generally refers to a virus particle
that functions as a nucleic acid delivery vehicle, and which
comprises the viral nucleic acid (i.e., the vector genome) packaged
within the virion. Virus vectors according to the present invention
comprise a chimeric AAV capsid according to the invention and can
package an AAV or rAAV genome or any other nucleic acid including
viral nucleic acids. Alternatively, in some contexts, the term
"vector," "virus vector," "delivery vector" (and similar terms) may
be used to refer to the vector genome (e.g., vDNA) in the absence
of the virion and/or to a viral capsid that acts as a transporter
to deliver molecules tethered to the capsid or packaged within the
capsid.
[0055] A "recombinant AAV vector genome" or "rAAV genome" is an AAV
genome (i.e., vDNA) that comprises at least one inverted terminal
repeat (e.g., one, two or three inverted terminal repeats) and one
or more heterologous nucleotide sequences. rAAV vectors generally
retain the 145 base terminal repeat(s) (TR(s)) in cis to generate
virus; however, modified AAV TRs and non-AAV TRs including
partially or completely synthetic sequences can also serve this
purpose. All other viral sequences are dispensable and may be
supplied in trans (Muzyczka, (1992) Curr. Topics Microbiol.
Immunol. 158:97). The rAAV vector optionally comprises two TRs
(e.g., AAV TRs), which generally will be at the 5' and 3' ends of
the heterologous nucleotide sequence(s), but need not be contiguous
thereto. The TRs can be the same or different from each other. The
vector genome can also contain a single ITR at its 3' or 5'
end.
[0056] The term "terminal repeat" or "TR" includes any viral
terminal repeat or synthetic sequence that forms a hairpin
structure and functions as an inverted terminal repeat (i.e.,
mediates the desired functions such as replication, virus
packaging, integration and/or provirus rescue, and the like). The
TR can be an AAV TR or a non-AAV TR. For example, a non-AAV TR
sequence such as those of other parvoviruses (e.g., canine
parvovirus (CPV), mouse parvovirus (MVM), human parvovirus B-19) or
the SV40 hairpin that serves as the origin of SV40 replication can
be used as a TR, which can further be modified by truncation,
substitution, deletion, insertion and/or addition. Further, the TR
can be partially or completely synthetic, such as the "double-D
sequence" as described in U.S. Pat. No. 5,478,745 to Samulski et
al.
[0057] An "AAV terminal repeat" or "AAV TR" may be from any AAV,
including but not limited to serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9,
10 or 11 or any other AAV now known or later discovered (see, e.g.,
Table 1). An AAV terminal repeat need not have the native terminal
repeat sequence (e.g., a native AAV TR sequence may be altered by
insertion, deletion, truncation and/or missense mutations), as long
as the terminal repeat mediates the desired functions, e.g.,
replication, virus packaging, integration, and/or provirus rescue,
and the like.
[0058] The terms "rAAV particle" and "rAAV virion" are used
interchangeably here. A "rAAV particle" or "rAAV virion" comprises
a rAAV vector genome packaged within an AAV capsid.
[0059] The AAV capsid structure is described in more detail in
BERNARD N. FIELDS et al., VIROLOGY, volume 2, chapters 69 & 70
(4th ed., Lippincott-Raven Publishers).
[0060] By "substantially retain" a property, it is meant that at
least about 75%, 85%, 90%, 95%, 97%, 98%, 99% or 100% of the
property (e.g., activity or other measurable characteristic) is
retained.
II. Chimeric AAV Capsids Targeted to Oligodendrocytes
[0061] The inventors have identified chimeric AAV capsid structures
capable of preferentially transducing oligodendrocytes over neurons
and other cells of the CNS. Thus, one aspect of the invention
relates to a nucleic acid encoding an AAV capsid, the nucleic acid
comprising, consisting essentially of, or consisting of an AAV
capsid coding sequence that is at least 90% identical to: (a) the
nucleotide sequence of SEQ ID NO:1; or (b) a nucleotide sequence
encoding SEQ ID NO:2; and viruses comprising the chimeric AAV
capsids. In some embodiments, the AAV capsid coding sequence is at
least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to
the nucleotide sequence of (a) or (b). In another embodiment, the
AAV capsid coding sequence comprises, consist essentially of, or
consist of the nucleotide sequence of (a) or (b).
TABLE-US-00002 AAV Capsid Nucleotide Sequence of BNP61 (SEQ ID NO:
1) atggctgccg atggttatct tccagattgg ctcgaggaca ctctctctga 50
aggaataaga cagtggtgga agctcaaacc tggcccacca ccaccaaagc 100
ccgcagagcg gcataaggac gacagcaggg gtcttgtgct tcctgggtac 150
aagtacctcg gacccttcaa cggactcgac aagggagagc cggtcaacga 200
ggcagacgcc gcggccctcg agcacgacaa agcctacgac cggcagctcg 250
acagcggaga caacccgtac ctcaagtaca accacgccga cgcggagttt 300
caggagcgcc ttaaagaaga tacgtctttt gggggcaacc tcgggcgagc 350
agtcttccag gccaaaaaga ggcttctpga acctcttggt ctggttgagg 400
aagcggctaa gacggctcct ggaaagaaga ggcctgtaga gcagtctcct 450
caggaaccgg actcctcctc gggcatcggc aagacaggcc agcagcccgc 500
taaaaagaga ctcaatttcg gtcagactgg cgacacagag tcagtcccag 550
accctcaacc aatcggagaa cctcccgcag ccccctcagg tgtgggatct 600
cttacaatgg cttcaggtgg tggcgcacca gtggcagaca ataacgaagg 650
tgccgatgga gtgggtagtt cctcgggaaa ttggcattgc gattcccaat 700
ggctggggga cagagtcatc accaccagca cccgaacctg ggccctgccc 750
acctacaaca atcacctcta caagcaaatc tccaacggga catcgggagg 800
agccaccaac gacaacacct acttcggcta cagcaccccc tgggggtatt 850
ttgactttaa cagattccac tgccactttt caccacgtga ctggcagcga 900
ctcatcaaca acaactgggg attccggccc aagagactca gCttcaagct 950
cttcaacatc caggtcaagg aggtcacgca gaatgaaggc accaagacca 1000
tcgccaataa ccttaccagc acggtccagg tcttcacgga ctcggagtac 1050
cagctgccgt acgttctcgg ctctgcccac cagggctgcc tgcctccgtt 1100
cccggcggac gtgttcatga ttccccagta cggctaccta acactcaaca 1150
acggtagtca ggccgtggga cgctcctcct tctactgcct ggaatacttt 1200
ccttcgcaga tgctgagaac cggcaacaac ttccagttta cttacacctt 1250
cgaggacgtg cctttccaca gcagctacgc ccacagccag agcttggacc 1300
ggctgatgaa tcctctgatt gaccagtacc tgtactactt gtctcggact 1350
caaacaacag gaggcacggc aaatacgcag actctgggct tcagccaagg 1400
tgggcctaat acaatggcca atcaggcaaa gaactggctg ccaggaccct 1450
gttaccgcca acaacgcgtc tcaacgacaa ccgggcaaaa caacaatagc 1500
aactttgcct ggactgctgg gaccaaatac catctgaatg gaagaaattc 1550
attggctaat cctggcatcg ctatggcaac acacaaagac gacaaggagc 1600
gtttttttcc cagtaacggg atcctgattt ttggcaaaca aaatgctgcc 1650
agagacaatg cggattacag cgatgtcatg ctcaccagcg aggaagaaat 1700
caaaaccact aaccctgtgg ctacagagga atacggtatc gtggcagata 1750
acttgcagca gcaaaacacg gctcctcaaa ttggaactgt caacagccag 1800
ggggccttac ccggtatggt ttggcagaac cgggacgtgt acctgcaggg 1850
tcccatgtgg gccaagattc ctcacacgga cggcaacttc cacccgtctc 1900
cgctgatggg cggctttggc ctgaaacatc ctccgcctca gatcctgatc 1950
aagaacacgc ctgtacctgc ggatcctccg accaccttca accagtcaaa 20C0
gctgaactct ttcatcacgc aatacagcac cggacaggtc agcgtggaaa 2050
ctgaatggga gctgcagaag gaaaacagca agcgctggaa ccccgagatc 2100
cagtacacct ccaactacta caaatctaca agtgtggact ttgctgttaa 2150
tacagaaggc gtgtactctg aaccccaccc cattggcacc cgttacctca 2200
cccgtcccct gtaa AAV Capsid Amino Acid Sequence of BNP61 (SEQ ID NO:
2) MAADGYLPDW LEDTLSEGIR QWWKLKPGPP PPKPAERHKD DSRGLVLPGY 50
KYLGPFNGLD KGEPVNEADA AALEHDKAYD RQLDSGDNPY LKYNHADAEF 100
QERLQGDTSF GGNLGRAVFQ AKKRVLEPLG LVEEAAKTAP GKKRPVEQSP 150
QEPDSSSGIG ETGQQPAKKR LNFGQTGDSE SVPDPQPLGE PPATPAAVGP 200
TTMASGGGAP MADNNEGADG VGSSSGNWHC DSQWLGDRVI TTSTRTWALP 250
TYNNHLYKQI SSASTGASND NHYFGYSTPW GYFDFNRFHC HFSPRDWQRL 300
INNNWGFRPK RLSFKLFNIQ VKEVTDNNGV KTIANNLTST VQVFTDSEYQ 350
LPYVLGSAHQ GCLPPFPADV FMIPQYGYLT LNNGSQAVGR SSFYCLEYFP 400
SQMLRTGNNF TFSYTFEDVP FHSSYAHSQS LDRLMNPLID QYLYYLSRTQ 450
TTGGTANTQT LGFSQGGPNT MANQAKNWLP GPCYRQQRVS TTTGQNNNSN 500
FAWTAGTKYH LNGRNSLANP GIAMATHKDD KERFFPSNGI LIFGKQNAAR 550
DNADYSDVML TSEEEIKTTN PVATEEYGIV ADNLQQQNTA PQIGTVNSQG 600
ALPGMVWQNR DVYLQGPIWA KIPHTDGNFH PSPLMGGFGL KHPPPQILIK 650
NTPVPADPPT TFNQSKLNSF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ 700
YTSNYYKSTS VDFAVNTEGV YSEPHPIGTR YLTRPL
[0062] In some embodiments, the nucleic acid encoding an AAV capsid
comprises, consists essentially of, or consists of an AAV capsid
coding sequence that is at least 90% identical to a nucleotide
sequence encoding SEQ ID NOS:3 or 4; and viruses comprising the
chimeric AAV capsids. In some embodiments, the AAV capsid coding
sequence is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%
identical to the nucleotide sequence encoding SEQ ID NOS:3 or 4. In
another embodiment, the AAV capsid coding sequence comprises,
consists essentially of, or consists of the nucleotide sequence
encoding SEQ ID NOS:3 or 4.
TABLE-US-00003 AAV Capsid Amino Acid Sequence of BNP62 (SEQ ID NO:
3) MAADGYLPDW LEDTLSEGIR QWWKLKPGPP PPKPAERHKD DSRGLVLPGY 50
KYLGPFNGLD KGEPVNEADA AALEHDKAYD RQLDSGDNPY LKYNHADAEF 100
QERLQGDTSF GGNLGRAVFQ AKKRVLEPLG LVEEAAKTAP GKKRPVEQSP 150
QEPDSSSGIG ETGQQPAKKR LNFGQTGDSE SVPDPQPLGE PPATPAAVGP 200
TTMASGGGAP MADNNEGADG VGSSSGNWHC DSQWLGDRVI TTSTRTWALP 250
TYNNHLYKQI SSASTGASND NHYFGYSTPW GYFDFNRFHC HFSPRDWQRL 300
INNNWGFRPK RLSFKLFNIQ VKEVTDNNGV KTIANNLTST VQVFTDSEYQ 350
LPYVLGSAHQ GCLPPFPADV FMIPQYGYLT LNNGSQAVGR SSFYCLEYFP 400
SQMLRTGNNF TFSYTFEDVP FHSSYAHSQS LDRLMNPLID QYLYYLSRTQ 450
TTGGTANTQT LGFSQGGPNT MANQAKNWLP GPCYRQQRVS TTTGQNNNSN 500
FAWTAGTKYH LNGRNSLANP GIAMATHKDD KERFFPSNGI LIFGKQNAAR 550
DNADYSDVML TSEEEIKTTN PVATEEYGIV ADNLQQQNTA PQIGTVNSQG 600
ALPGMVWQNR DVYLQGPIWA KIPHTDGNFH PSPLMGGFGL KHPPPQILIK 650
NTPVPADPPT TFNQSKLNSF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ 700
YTSNYYKSTS VDFAVNTEGV YSEPHPIGTR YLTRPL AAV Capsid Amino Acid
Sequence of BNP63 (SEQ ID NO: 4) MAADGYLPDW LEDTLSEGIR QWWKLKPGPP
PPKPAERHKD DSRGLVLPGY 50 KYLGPFNGLD KGEPVNEADA AALEHDKAYD
RQLDSGDNPY LKYNHADAEF 100 QERLQGDTSF GGNLGRAVFQ AKKRVLEPLG
LVEEAAKTAP GKKRPVEQSP 150 QEPDSSSGIG ETGQQPAKKR LNFGQTGDSE
SVPDPQPLGE PPATPAAVGP 200 TTMASGGGAP MADNNEGADG VGSSSGNWHC
DSQWLGDRVI TTSTRTWALP 250 TYNNHLYKQI SSASTGASND NHYFGYSTPW
GYFDFNRFHC HFSPRDWQRL 300 INNNWGFRPK RLSFKLFNIQ VKEVTDNNGV
KTIANNLTST VQVFTDSEYQ 350 LPYVLGSAHQ GCLPPFPADV FMIPQYGYLT
LNNGSQAVGR SSFYCLEYFP 400 SQMLRTGNNF TFSYTFEDVP FHSSYAHSQS
LDRLMNPLID QYLYYLSRTQ 450 TTGGTANTQT LGFSQGGPNT MANQAKNWLP
GPCYRQQRVS TTTGQNNNSN 500 FAWTAGTKYH LNGRNSLANP GIAMATHKDD
KERFFPSNGI LIFGKQNAAR 550 DNADYSDVML TSEEEIKTTN PVATEEYGIV
ADNLQQQNTA PQIGTVNSQG 600 ALPGMVWQNR DVYLQGPIWA KIPHTDGNFH
PSPLMGGFGL KHPPPQILIK 650 NTPVPADPPT TFNQSKLNSF ITQYSTGQVS
VEIEWELQKE NSKRWNPEIQ 700 YTSNYYKSTS VDFAVNTEGV YSEPHPIGTR
YLTRPL
[0063] SEQ ID NOS:2-4 show examples of the VP1 capsid protein
sequences of the invention. The designation of all amino acid
positions in the description of the invention and the appended
claims is with respect to VP1 numbering. Those skilled in the art
will understand that the AAV capsid generally contains the smaller
VP2 and VP3 capsid proteins as well. Due to the overlap of the
coding sequences for the AAV capsid proteins, the nucleic acid
coding sequences and amino acid sequences of the VP2 and VP3 capsid
proteins will be apparent from the VP1 sequences shown in SEQ ID
NOS:1-4. In particular, VP2 starts at nucleotide 412 (acg) of SEQ
ID NO:1 and threonine 148 of SEQ ID NO:2. VP3 starts at nucleotide
607 (atg) of SEQ ID NO:1 and methionine 203 of SEQ ID NO:2. In
certain embodiments, isolated VP2 and VP3 capsid proteins
comprising the sequence from SEQ ID NOS:2-4 and isolated nucleic
acids encoding the VP2 or VP3 proteins, or both, are
contemplated.
[0064] The invention also provides chimeric AAV capsid proteins and
chimeric capsids, wherein the capsid protein comprises, consists
essentially of, or consists of an amino acid sequence as shown in
one of SEQ ID NOS:2-4, wherein 1, 2 or fewer, 3 or fewer, 4 or
fewer, 5 or fewer, 6 or fewer, 7 or fewer, 8 or fewer, 9 or fewer,
10 or fewer, 12 or fewer, 15 or fewer, 20 or fewer, 25 or fewer, 30
or fewer, 40 or fewer, or 50 or fewer of the amino acids within the
capsid protein coding sequence of one of SEQ ID NOS:2-4 is
substituted by another amino acid (naturally occurring, modified
and/or synthetic), optionally a conservative amino acid
substitution, and/or are deleted and/or there are insertions
(including N-terminal and C-terminal extensions) of 1, 2 or fewer,
3 or fewer, 4 or fewer, 5 or fewer, 6 or fewer, 7 or fewer, 8 or
fewer, 9 or fewer, 10 or fewer, 12 or fewer, 15 or fewer, 20 or
fewer, 25 or fewer, 30 or fewer, 40 or fewer, or 50 or fewer amino
acids or any combination of substitutions, deletions and/or
insertions, wherein the substitutions, deletions and/or insertions
do not unduly impair the structure and/or function of a virion
(e.g., an AAV virion) comprising the variant capsid protein or
capsid. For example, in representative embodiments of the
invention, an AAV virion comprising the chimeric capsid protein
substantially retains at least one property of a chimeric virion
comprising a chimeric capsid protein as shown in one of SEQ ID
NOS:2-4. For example, the virion comprising the chimeric capsid
protein can substantially retain the oligodendrocyte tropism
profile of a virion comprising the chimeric AAV capsid protein as
shown in one of SEQ ID NOS:2-4. Methods of evaluating biological
properties such as virus transduction are well-known in the art
(see, e.g., the Examples).
[0065] Conservative amino acid substitutions are known in the art.
In particular embodiments, a conservative amino acid substitution
includes substitutions within one or more of the following groups:
glycine, alanine; valine, isoleucine, leucine; aspartic acid,
glutamic acid; asparagine, glutamine; serine, threonine; lysine,
arginine; and/or phenylalanine, tyrosine.
[0066] It will be apparent to those skilled in the art that the
amino acid sequences of the chimeric AAV capsid protein of SEQ ID
NOS:2-4 can further be modified to incorporate other modifications
as known in the art to impart desired properties. As nonlimiting
possibilities, the capsid protein can be modified to incorporate
targeting sequences (e.g., ROD) or sequences that facilitate
purification and/or detection. For example, the capsid protein can
be fused to all or a portion of glutathione-S-transferase,
maltose-binding protein, a heparin/heparan sulfate binding domain,
poly-His, a ligand, and/or a reporter protein (e.g., Green
Fluorescent Protein, .beta.-glucuronidase, .beta.-galactosidase,
luciferase, etc), an immunoglobulin Fc fragment, a single-chain
antibody, hemagglutinin, c-myc, FLAG epitope, and the like to form
a fusion protein. Methods of inserting targeting peptides into the
AAV capsid are known in the art (see, e.g., international patent
publication WO 00/28004; Nicklin et at, (2001) Mol. Ther. 474-181;
White et al., (2004) Circulation 109:513-319; Muller et al., (2003)
Nature Biotech. 21:1040-1046.
[0067] The viruses of the invention can further comprise a duplexed
viral genome as described in international patent publication WO
01/92551 and U.S. Pat. No. 7,465,583.
[0068] The invention also provides AAV capsids comprising the
chimeric AAV capsid proteins of the invention and virus particles
(i.e., virions) comprising the same, wherein the virus particle
packages (i.e., encapsidates) a vector genome, optionally an AAV
vector genome. In particular embodiments, the invention provides an
AAV particle comprising an AAV capsid comprising an AAV capsid
protein of the invention, wherein the AAV capsid packages an AAV
vector genome. The invention also provides an AAV particle
comprising an AAV capsid or AAV capsid protein encoded by the
chimeric nucleic acid capsid coding sequences of the invention.
[0069] In particular embodiments, the virion is a recombinant
vector comprising a heterologous nucleic acid of interest, e.g.,
for delivery to a cell. Thus, the present invention is useful for
the delivery of nucleic acids to cells in vitro, ex vivo, and in
vivo. In representative embodiments, the recombinant vector of the
invention can be advantageously employed to deliver or transfer
nucleic acids to animal (e.g., mammalian) cells.
[0070] Any heterologous nucleotide sequence(s) may be delivered by
a virus vector of the present invention. Nucleic acids of interest
include nucleic acids encoding polypeptides, optionally therapeutic
(e.g., for medical or veterinary uses) and/or immunogenic (e.g.,
for vaccines) polypeptides.
[0071] In some embodiments, the polypeptide is one that stimulates
growth and/or differentiation of oligodendrocytes. Examples
include, without limitation, insulin-like growth factor-1,
glial-derived neurotrophic factor, neurotrophin-3, artemin,
transforming growth factor alpha, platelet-derived growth factor,
leukemia inhibitory factor, prolactin, monocarboxylate transporter
1, or nuclear factor 1A.
[0072] Therapeutic polypeptides include, but are not limited to,
cystic fibrosis transmembrane regulator protein (CFTR), dystrophin
(including the protein product of dystrophin mini-genes or
micro-genes, see, e.g., Vincent et al., (1993) Nature Genetics
5:130; U.S. Patent Publication No. 2003017131; Wang et al., (2000)
Proc. Natl. Acad. Sci. USA 97:13714-9 [mini-dystrophin]; Harper et
al., (2002) Nature Med. 8:253-61 [micro-dystrophin]); mini-agrin, a
laminin-.alpha.2, a sarcoglycan (.alpha., .beta., .gamma. or
.delta.), Fukutin-related protein, myostatin pro-peptide,
follistatin, dominant negative myostatin, an angiogenic factor
(e.g., VEGF, angiopoietin-1 or 2), an anti-apoptotic factor (e.g.,
heme-oxygenase-1, TGF-.beta., inhibitors of pro-apoptotic signals
such as caspases, proteases, kinases, death receptors [e.g.,
CD-095], modulators of cytochrome C release, inhibitors of
mitochondrial pore opening and swelling); activin type II soluble
receptor, anti-inflammatory polypeptides such as the Ikappa B
dominant mutant, sarcospan, utrophin, mini-utrophin, antibodies or
antibody fragments against myostatin or myostatin propeptide, cell
cycle modulators, Rho kinase modulators such as Cethrin, which is a
modified bacterial C3 exoenzyme [available from BioAxone
Therapeutics, Inc., Saint-Lauren, Quebec, Canada], BCL-xL, BCL2,
XIAP, FLICEc-s, dominant-negative caspase-8, dominant negative
caspase-9, SPI-6 (see, e.g., U.S. Patent Application No.
20070026076), transcriptional factor PGC-.alpha.1, Pinch gene, ILK
gene and thymosin 34 gene), clotting factors (e.g., Factor VIII,
Factor IX, Factor X, etc.), erythropoietin, angiostatin,
endostatin, catalase, tyrosine hydroxylase, an intracellular and/or
extracellular superoxide dismutase, leptin, the LDL receptor,
neprilysin, lipoprotein lipase, ornithine transcarbamylase,
.beta.-globin, .alpha.-globin, spectrin, .alpha..sub.1-antitrypsin,
adenosine deaminase, hypoxanthine guanine phosphoribosyl
transferase, .beta.-glucocerebrosidase, sphingomyelinase, lysosomal
hexosaminidase A, branched-chain keto acid dehydrogenase, RP65
protein, a cytokine (e.g., .alpha.-interferon, .beta.-interferon,
interferon-.gamma., interleukins-1 through-14,
granulocyte-macrophage colony stimulating factor, lymphotoxin, and
the like), peptide growth factors, neurotrophic factors and
hormones (e.g., somatotropin, insulin, insulin-like growth factors
including IGF-1 and IGF-2, GLP-1, platelet derived growth factor,
epidermal growth factor, fibroblast growth factor, nerve growth
factor, neurotrophic factor-3 and -4, brain-derived neurotrophic
factor, glial derived growth factor, transforming growth
factor-.alpha. and -.beta., and the like), bone morphogenic
proteins (including RANKL and VEGF), a lysosomal protein, a
glutamate receptor, a lymphokine, soluble CD4, an Fe receptor, a T
cell receptor, ApoE, ApoC, inhibitor 1 of protein phosphatase
inhibitor 1 (I-1), phospholamban, serca2a, lysosomal acid
.alpha.-glucosidase, .alpha.-galactosidase A, Barket,
.beta.2-adrenergic receptor, .beta.2-adrenergic receptor kinase
(BARK), phosphoinositide-3 kinase (PI3 kinase), calsarcin, a
receptor (e.g., the tumor necrosis growth factor-.alpha. soluble
receptor), an anti-inflammatory factor such as IRAP, Pim-1,
PGC-1.alpha., SOD-1, SOD-2, ECF-SOD, kallikrein, thymosin-.beta.4,
hypoxia-inducible transcription factor [HIF], an angiogenic factor,
S100A1, parvalbumin, adenylyl cyclase type 6, a molecule that
effects G-protein coupled receptor kinase type 2 knockdown such as
a truncated constitutively active bARKct; phospholamban inhibitory
or dominant-negative molecules such as phospholamban S16E, a
monoclonal antibody (including single chain monoclonal antibodies)
or a suicide gene product (e.g., thymidine kinase, cytosine
deaminase, diphtheria toxin, and tumor necrosis factors such as
TNF-.alpha.), and any other polypeptide that has a therapeutic
effect in a subject in need thereof.
[0073] Heterologous nucleotide sequences encoding polypeptides
include those encoding reporter polypeptides (e.g., an enzyme).
Reporter polypeptides are known in the art and include, but are not
limited to, Green Fluorescent Protein, .beta.-galactosidase,
alkaline phosphatase, luciferase, and chloramphenicol
acetyltransferase.
[0074] Alternatively, the heterologous nucleic acid may encode an
antisense oligonucleotide, a ribozyme (e.g., as described in U.S.
Pat. No. 5,877,022), RNAs that effect spliceosome-mediated
trans-splicing (see, Puttaerju et al., (1999) Nature Biotech.
17:246; U.S. Pat. No. 6,013,487; U.S. Pat. No. 6,083,702),
interfering RNAs (RNAi) including small interfering RNAs (siRNA)
that mediate gene silencing (see, Sharp et al., (2000) Science
287:2431), microRNA, or other non-translated "functional" RNAs,
such as "guide" RNAs (Gorman et al., (1998) Proc. Nat. Acad. Sci.
USA 95:4929; U.S. Pat. No. 5,869,248 to Yuan et al.), and the like.
Exemplary untranslated RNAs include RNAi or antisense RNA against
the multiple drug resistance (MDR) gene product (e.g., to treat
tumors and/or for administration to the heart to prevent damage by
chemotherapy), RNAi or antisense RNA against myostatin (Duchenne or
Becker muscular dystrophy), RNAi or antisense RNA against VEGF or a
tumor immunogen including but not limited to those tumor immunogens
specifically described herein (to treat tumors), RNAi or antisense
oligonucleotides targeting mutated dystrophins (Duchenne or Becker
muscular dystrophy), RNAi or antisense RNA against the hepatitis B
surface antigen gene (to prevent and/or treat hepatitis B
infection), RNAi or antisense RNA against the HIV tat and/or rev
genes (to prevent and/or treat HIV) and/or RNAi or antisense RNA
against any other immunogen from a pathogen (to protect a subject
from the pathogen) or a defective gene product (to prevent or treat
disease). RNAi or antisense RNA against the targets described above
or any other target can also be employed as a research reagent.
[0075] As is known in the art, anti-sense nucleic acids (e.g., DNA
or RNA) and inhibitory RNA (e.g., microRNA and RNAi such as siRNA
or shRNA) sequences can be used to induce "exon skipping" in
patients with muscular dystrophy arising from defects in the
dystrophin gene. Thus, the heterologous nucleic acid can encode an
antisense nucleic acid or inhibitory RNA that induces appropriate
exon skipping. Those skilled in the art will appreciate that the
particular approach to exon skipping depends upon the nature of the
underlying defect in the dystrophin gene, and numerous such
strategies are known in the art. Exemplary antisense nucleic acids
and inhibitory RNA sequences target the upstream branch point
and/or downstream donor splice site and/or internal splicing
enhancer sequence of one or more of the dystrophin exons (e.g.,
exons 19 or 23). For example, in particular embodiments, the
heterologous nucleic acid encodes an antisense nucleic acid or
inhibitory RNA directed against the upstream branch point and
downstream splice donor site of exon 19 or 23 of the dystrophin
gene. Such sequences can be incorporated into an AAV vector
delivering a modified U7 snRNA and the antisense nucleic acid or
inhibitory RNA (see, e.g., Goyenvalle et al., (2004) Science
306:1796-1799). As another strategy, a modified U1 snRNA can be
incorporated into an AAV vector along with siRNA, microRNA or
antisense RNA complementary to the upstream and downstream splice
sites of a dystrophin exon (e.g., exon 19 or 23) (see, e.g., Denti
et al., (2006) Proc. Nat. Acad. Sci. USA 103:3758-3763). Further,
antisense nucleic acids and inhibitory RNA can target the splicing
enhancer sequences within exons 19, 43, 45 or 53 (see, e.g., U.S.
Pat. No. 6,653,467; U.S. Pat. No. 6,727,355; and U.S. Pat. No.
6,653,466).
[0076] Ribozymes are RNA-protein complexes that cleave nucleic
acids in a site-specific fashion. Ribozymes have specific catalytic
domains that possess endonuclease activity (Kim et al., (1987)
Proc. Nat. Acad. Sci. USA 84; 8788; Gerlach et al., (1987) Nature
328:802; Forster and Symons, (1987) Cell 49:211). For example, a
large number of ribozymes accelerate phosphoester transfer
reactions with a high degree of specificity, often cleaving only
one of several phosphoesters in an oligonucleotide substrate
(Michel and Westhof, (1990), J. Mol. Biol. 216:585; Reinhold-Hurek
and Shub, (1992) Nature 357:173). This specificity has been
attributed to the requirement that the substrate bind via specific
base-pairing interactions to the internal guide sequence ("IGS") of
the ribozyme prior to chemical reaction.
[0077] Ribozyme catalysis has primarily been observed as part of
sequence-specific cleavage/ligation reactions involving nucleic
acids (Joyce, (1989) Nature 338:217). For example, U.S. Pat. No.
5,354,855 reports that certain ribozymes can act as endonucleases
with a sequence specificity greater than that of known
ribonucleases and approaching that of the DNA restriction enzymes.
Thus, sequence-specific ribozyme-mediated inhibition of nucleic
acid expression may be particularly suited to therapeutic
applications (Scanlon et al., (1991) Proc. Natl. Acad. Sci. USA
88:10591; Sarver et al., (1990) Science 247:1222; Sioud et al.,
(1992) J. Mol. Biol. 223:831).
[0078] MicroRNAs (mir) are natural cellular RNA molecules that can
regulate the expression of multiple genes by controlling the
stability of the mRNA. Over-expression or diminution of a
particular microRNA can be used to treat a dysfunction and has been
shown to be effective in a number of disease states and animal
models of disease (see, e.g., Couzin, (2008) Science 319:1782-4).
The chimeric AAV can be used to deliver microRNA into cells,
tissues and subjects for the treatment of genetic and acquired
diseases, or to enhance functionality and promote growth of certain
tissues. For example, mir-1, mir-133, mir-206 and/or mir-208 can be
used to treat cardiac and skeletal muscle disease (see, e.g., Chen
et al., (2006) Genet. 38:228-33; van Rooij et al., (2008) Trends
Genet. 24; 159-66). MicroRNA can also be used to modulate the
immune system after gene delivery (Brown et at, (2007) Blood
110:4144-52).
[0079] The term "antisense oligonucleotide" (including "antisense
RNA") as used herein, refers to a nucleic acid that is
complementary to and specifically hybridizes to a specified DNA or
RNA sequence. Antisense oligonucleotides and nucleic acids that
encode the same can be made in accordance with conventional
techniques. See, e.g., U.S. Pat. No. 5,023,243 to Tullis; U.S. Pat.
No. 5,149,797 to Pederson et a.
[0080] Those skilled in the art will appreciate that it is not
necessary that the antisense oligonucleotide be fully complementary
to the target sequence as long as the degree of sequence similarity
is sufficient for the antisense nucleotide sequence to specifically
hybridize to its target (as defined above) and reduce production of
the protein product (e.g., by at least about 30%, 40%, 50%, 60%,
70%, 80%, 90%, 95% or more).
[0081] To determine the specificity of hybridization, hybridization
of such oligonucleotides to target sequences can be carried out
under conditions of reduced stringency, medium stringency or even
stringent conditions. Suitable conditions for achieving reduced,
medium and stringent hybridization conditions are as described
herein.
[0082] Alternatively stated, in particular embodiments, antisense
oligonucleotides of the invention have at least about 60%, 70%,
80%, 90%, 95%, 97%, 98% or higher sequence identity with the
complement of the target sequence and reduce production of the
protein product (as defined above). In some embodiments, the
antisense sequence contains 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10
mismatches as compared with the target sequence.
[0083] Methods of determining percent identity of nucleic acid
sequences are described in more detail elsewhere herein.
[0084] The length of the antisense oligonucleotide is not critical
as long as it specifically hybridizes to the intended target and
reduces production of the protein product (as defined above) and
can be determined in accordance with routine procedures. In
general, the antisense oligonucleotide is at least about eight, ten
or twelve or fifteen nucleotides in length and/or less than about
20, 30, 40, 50, 60, 70, 80, 100 or 150 nucleotides in length.
[0085] RNA interference (RNAi) is another useful approach for
reducing production of a protein product (e.g., shRNA or siRNA).
RNAi is a mechanism of post-transcriptional gene silencing in which
double-stranded RNA (dsRNA) corresponding to a target sequence of
interest is introduced into a cell or an organism, resulting in
degradation of the corresponding mRNA. The mechanism by which RNAi
achieves gene silencing has been reviewed in Sharp et al., (2001)
Genes Dev 15: 485-490; and Hammond et al., (2001) Nature Rev. Gen.
2:110-119). The RNAi effect persists for multiple cell divisions
before gene expression is regained. RNAi is therefore a powerful
method for making targeted knockouts or "knockdowns" at the RNA
level. RNAi has proven successful in human cells, including human
embryonic kidney and HeLa cells (see, e.g., Elbashir et al., Nature
(2001) 411:494-8).
[0086] Initial attempts to use RNAi in mammalian cells resulted in
antiviral defense mechanisms involving PKR in response to the dsRNA
molecules (see, e.g., Gil et al., (2000) Apoptosis 5:107). It has
since been demonstrated that short synthetic dsRNA of about 21
nucleotides, known as "short interfering RNAs" (siRNA) can mediate
silencing in mammalian cells without triggering the antiviral
response (see, e.g., Elbashir et al., Nature (2001) 411:494-8;
Caplen et al., (2001) Proc. Nat. Acad. Sci. USA 98; 9742).
[0087] The RNAi molecule (including an siRNA molecule) can be a
short hairpin RNA (shRNA; see Paddison et al., (2002), Proc. Nat.
Acad. Sci. USA 99:1443-1448), which is believed to be processed in
the cell by the action of the RNase III like enzyme Dicer into
20-25mer siRNA molecules. The shRNAs generally have a stem-loop
structure in which two inverted repeat sequences are separated by a
short spacer sequence that loops out. There have been reports of
shRNAs with loops ranging from 3 to 23 nucleotides in length. The
loop sequence is generally not critical. Exemplary loop sequences
include the following motifs: AUG, CCC, UUCG, CCACC, CTCGAG,
AAGCUU, CCACACC and UUCAAGAGA.
[0088] The RNAi can further comprise a circular molecule comprising
sense and antisense regions with two loop regions on either side to
form a "dumbbell" shaped structure upon dsRNA formation between the
sense and antisense regions. This molecule can be processed in
vitro or in vivo to release the dsRNA portion, e.g., a siRNA.
[0089] International patent publication WO 01/77350 describes a
vector for bi-directional transcription to generate both sense and
antisense transcripts of a heterologous sequence in a eukaryotic
cell. This technique can be employed to produce RNAi for use
according to the invention.
[0090] Shinagawa et al., (2003) Genes Dev. 17:1340 reported a
method of expressing long dsRNAs from a CMV promoter (a pol II
promoter), which method is also applicable to tissue specific pol
II promoters. Likewise, the approach of Xia at al., (2002) Nature
Biotech. 20:1006, avoids poly(A) tailing and can be used in
connection with tissue-specific promoters.
[0091] Methods of generating RNAi include chemical synthesis, in
vitro transcription, digestion of long dsRNA by Dicer (in vitro or
in vivo), expression in vivo from a delivery vector, and expression
in vivo from a PCR-derived RNAi expression cassette (see, e.g.,
TechNotes 10(3) "Five Ways to Produce siRNAs," from Ambion. Inc.,
Austin Tex.; available at www.ambion.com).
[0092] Guidelines for designing siRNA molecules are available (see
e.g., literature from Ambion, Inc., Austin Tex.; available at
www.ambion.com). In particular embodiments, the siRNA sequence has
about 30-50% 0/C content. Further, long stretches of greater than
four T or A residues are generally avoided if RNA polymerase III is
used to transcribe the RNA. Online siRNA target finders are
available, e.g., from Ambion, Inc. (www.ambion.com), through the
Whitehead Institute of Biomedical Research (www.jura.wi.mit.edu) or
from Dharmacon Research, Inc. (www.dharmacon.com).
[0093] The antisense region of the RNAi molecule can be completely
complementary to the target sequence, but need not be as long as it
specifically hybridizes to the target sequence (as defined above)
and reduces production of the protein product (e.g., by at least
about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more). In some
embodiments, hybridization of such oligonucleotides to target
sequences can be carried out under conditions of reduced
stringency, medium stringency or even stringent conditions, as
defined above.
[0094] In other embodiments, the antisense region of the RNAi has
at least about 60%, 70%, 80%, 90%, 95%, 97%, 98% or higher sequence
identity with the complement of the target sequence and reduces
production of the protein product (e.g., by at least about 30%,
40%, 50%, 60%, 70%, 80%, 90%, 95% or more). In some embodiments,
the antisense region contains 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10
mismatches as compared with the target sequence. Mismatches are
generally tolerated better at the ends of the dsRNA than in the
center portion.
[0095] In particular embodiments, the RNAi is formed by
intermolecular complexing between two separate sense and antisense
molecules. The RNAi comprises a double stranded region formed by
the intermolecular basepairing between the two separate strands. In
other embodiments, the RNAi comprises a ds region formed by
intramolecular basepairing within a single nucleic acid molecule
comprising both sense and antisense regions, typically as an
inverted repeat (e.g., a shRNA or other stem loop structure, or a
circular RNAi molecule). The RNAi can further comprise a spacer
region between the sense and antisense regions.
[0096] Generally, RNAi molecules are highly selective. If desired,
those skilled in the art can readily eliminate candidate RNAi that
are likely to interfere with expression of nucleic acids other than
the target by searching relevant databases to identify RNAi
sequences that do not have substantial sequence homology with other
known sequences, for example, using BLAST (available at
www.ncbi.nlm.nih.gov/BLAST).
[0097] Kits for the production of RNAi are commercially available,
e.g., from New England Biolabs, Inc. and Ambion, Inc.
[0098] The recombinant virus vector may also comprise a
heterologous nucleotide sequence that shares homology with and
recombines with a locus on the host chromosome. This approach may
be utilized to correct a genetic defect in the host cell.
[0099] The present invention also provides recombinant virus
vectors that express an immunogenic polypeptide, e.g., for
vaccination. The heterologous nucleic acid may encode any immunogen
of interest known in the art including, but are not limited to,
immunogens from human immunodeficiency virus, influenza virus, gag
proteins, tumor antigens, cancer antigens, bacterial antigens,
viral antigens, and the like. Alternatively, the immunogen can be
presented in the virus capsid (e.g., incorporated therein) or
tethered to the virus capsid (e.g., by covalent modification).
[0100] The use of parvoviruses as vaccines is known in the art
(see, e.g., Miyamura et at, (1994) Proc. Nat. Acad. Sci. USA
91:8507; U.S. Pat. No. 5,916,563 to Young er al., U.S. Pat. No.
5,905,040 to Mazzara et al., U.S. Pat. No. 5,882,652, U.S. Pat. No.
5,863,541 to Samulski et al; the disclosures of which are
incorporated herein in their entireties by reference). The antigen
may be presented in the virus capsid. Alternatively, the antigen
may be expressed from a heterologous nucleic acid introduced into a
recombinant vector genome.
[0101] An immunogenic polypeptide, or immunogen, may be any
polypeptide suitable for protecting the subject against a disease,
including but not limited to microbial, bacterial, protozoal,
parasitic, fungal and viral diseases. For example, the immunogen
may be an orthomyxovirus immunogen (e.g., an influenza virus
immunogen, such as the influenza virus hemagglutinin (HA) surface
protein or the influenza virus nucleoprotein gene, or an equine
influenza virus immunogen), or a lentivirus immunogen (e.g., an
equine infectious anemia virus immunogen, a Simian Immunodeficiency
Virus (SIV) immunogen, or a Human Immunodeficiency Virus (HIV)
immunogen, such as the HIV or SIV envelope GP160 protein, the HIV
or SIV matrix/capsid proteins, and the HIV or SIV gag, pol and env
genes products). The immunogen may also be an arenavirus immunogen
(e.g., Lassa fever virus immunogen, such as the Lassa fever virus
nucleocapsid protein gene and the Lassa fever envelope glycoprotein
gene), a poxvirus immunogen (e.g., vaccinia, such as the vaccinia
L1 or L8 genes), a flavivirus immunogen (e.g., a yellow fever virus
immunogen or a Japanese encephalitis virus immunogen), a filovirus
immunogen (e.g., an Ebola virus immunogen, or a Marburg virus
immunogen, such as NP and GP genes), a bunyavirus immunogen (e.g.,
RVFV, CCHF, and SFS viruses), or a coronavirus immunogen (e.g., an
infectious human coronavirus immunogen, such as the human
coronavirus envelope glycoprotein gene, or a porcine transmissible
gastroenteritis virus immunogen, or an avian infectious bronchitis
virus immunogen, or a severe acute respiratory syndrome (SARS)
immunogen such as a S [S1 or S2], M, E, or N protein or an
immunogenic fragment thereof). The immunogen may further be a polio
immunogen, herpes immunogen (e.g., CMV, EBV, HSV immunogens) mumps
immunogen, measles immunogen, rubella immunogen, diphtheria toxin
or other diphtheria immunogen, pertussis antigen, hepatitis (e.g.,
hepatitis A, hepatitis B or hepatitis C) immunogen, or any other
vaccine immunogen known in the art.
[0102] Alternatively, the immunogen may be any tumor or cancer cell
antigen. Optionally, the tumor or cancer antigen is expressed on
the surface of the cancer cell. Exemplary cancer and tumor cell
antigens are described in S. A. Rosenberg, (1999) Immunity 10:281).
Illustrative cancer and tumor antigens include, but are not limited
to: BRCA1 gene product, BRCA2 gene product, gp100, tyrosinase,
GAGE-1/2, BAGE, RAGE, NY-ESO-1, CDK-4, .beta.-catenin, MUM-1,
Caspase-8, KIAA0205, HPVE, SART-1, PRAME, p15, melanoma tumor
antigens (Kawakami et al., (1994) Proc. Natl. Acad. Sci. USA
91:3515; Kawakami et al., (1994) J. Exp. Med., 180:347; Kawakami et
al., (1994) Cancer Res. 54:3124) including MART-1 (Coulie et al.,
(1991) J. Exp. Med. 180:35), gp100 (Wick et al., (1988) J. Cutan.
Pathol. 4:201) and MAGE antigen (MAGE-1, MAGE-2 and MAGE-3) (Van
der Bruggen et al., (1991) Science, 254:1643), CEA, TRP-1; TRP-2;
P-15 and tyrosinase (Brichard et al., (1993) J. Exp. Med. 178:489);
HER-2/neu gene product (U.S. Pat. No. 4,968,603); CA 125; HE4;
LK26; FB5 (endosialin); TAG 72; AFP; CA19-9; NSE; DU-PAN-2; CA50;
Span-1; CA72-4; HCG; STN (sialyl Tn antigen); c-erbB-2 proteins;
PSA; L-CanAg; estrogen receptor; milk fat globulin; p53 tumor
suppressor protein (Levine, (1993) Ann. Rev. Biochem. 62:623);
mucin antigens (international patent publication WO 90/05142);
telomerases; nuclear matrix proteins; prostatic acid phosphatase;
papilloma virus antigens; and antigens associated with the
following cancers: melanomas, adenocarcinoma, thymoma, sarcoma,
lung cancer, liver cancer, colorectal cancer, non-Hodgkin's
lymphoma, Hodgkin's lymphoma, leukemias, uterine cancer, breast
cancer, prostate cancer, ovarian cancer, cervical cancer, bladder
cancer, kidney cancer, pancreatic cancer, brain cancer, kidney
cancer, stomach cancer, esophageal cancer, head and neck cancer and
others (see, e.g., Rosenberg, (1996) Annu. Rev. Med.
47:481-91).
[0103] Alternatively, the heterologous nucleotide sequence may
encode any polypeptide that is desirably produced in a cell in
vitro, ex vivo, or in vivo. For example, the virus vectors may be
introduced into cultured cells and the expressed protein product
isolated therefrom.
[0104] It will be understood by those skilled in the art that the
heterologous nucleic acid(s) of interest may be operably associated
with appropriate control sequences. For example, the heterologous
nucleic acid may be operably associated with expression control
elements, such as transcription/translation control signals,
origins of replication, polyadenylation signals, internal ribosome
entry sites (IRES), promoters, enhancers, and the like.
[0105] Those skilled in the art will further appreciate that a
variety of promoter/enhancer elements may be used depending on the
level and tissue-specific expression desired. The promoter/enhancer
may be constitutive or inducible, depending on the pattern of
expression desired. The promoter/enhancer may be native or foreign
and can be a natural or a synthetic sequence. By foreign, it is
intended that the transcriptional initiation region is not found in
the wild-type host into which the transcriptional initiation region
is introduced.
[0106] Promoter/enhancer elements can be native to the target cell
or subject to be treated and/or native to the heterologous nucleic
acid sequence. The promoter/enhancer element is generally chosen so
that it will function in the target cell(s) of interest. In
representative embodiments, the promoter/enhancer element is a
mammalian promoter/enhancer element. The promoter/enhance element
may be constitutive or inducible.
[0107] Inducible expression control elements are generally used in
those applications in which it is desirable to provide regulation
over expression of the heterologous nucleic acid sequence(s).
Inducible promoters/enhancer elements for gene delivery can be
tissue-specific or tissue-preferred promoter/enhancer elements, and
include muscle specific or preferred (including cardiac, skeletal
and/or smooth muscle), neural tissue specific or preferred
(including brain-specific), eye (including retina-specific and
cornea-specific), liver specific or preferred, bone marrow specific
or preferred, pancreatic specific or preferred, spleen specific or
preferred, and lung specific or preferred promoter/enhancer
elements. In one embodiment, an oligodendrocyte-specified or
oligodendrocyte-preferred promoter is used. Examples include,
without limitation, myelin basic protein, cyclic nucleotide
phosphodiesterase, proteolipid protein, Gtx, and Sox10. Use of an
oligodendrocyte-specific or preferred promoter can increase the
specificity achieved by the chimeric AAV vector by further limiting
expression of the heterologous nucleic acid to oligodendrocytes.
Other inducible promoter/enhancer elements include
hormone-inducible and metal-inducible elements. Exemplary inducible
promoters/enhancer elements include, but are not limited to, a Tet
on/off element, a RU486-inducible promoter, an ecdysone-inducible
promoter, a rapamycin-inducible promoter, and a metallothionein
promoter.
[0108] In embodiments wherein the heterologous nucleic acid
sequence(s) is transcribed and then translated in the target cells,
specific initiation signals are generally employed for efficient
translation of inserted protein coding sequences. These exogenous
translational control sequences, which may include the ATG
initiation codon and adjacent sequences, can be of a variety of
origins, both natural and synthetic.
[0109] The invention also provides chimeric AAV particles
comprising an AAV capsid and an AAV genome, wherein the AAV genome
"corresponds to" (i.e., encodes) the AAV capsid. Also provided are
collections or libraries of such chimeric AAV particles, wherein
the collection or library comprises 2 or more, 10 or more, 50 or
more, 100 or more, 1000 or more, 10.sup.4 or more, 10.sup.5 or
more, or 10.sup.6 or more distinct sequences.
[0110] The present invention further encompasses "empty" capsid
particles (I.e., in the absence of a vector genome) comprising,
consisting of, or consisting essentially of the chimeric AAV capsid
proteins of the invention. The chimeric AAV capsids of the
invention can be used as "capsid vehicles," as has been described
in U.S. Pat. No. 5,863,541. Molecules that can be covalently
linked, bound to or packaged by the virus capsids and transferred
into a cell include DNA, RNA, a lipid, a carbohydrate, a
polypeptide, a small organic molecule, or combinations of the same.
Further, molecules can be associated with (e.g., "tethered to") the
outside of the virus capsid for transfer of the molecules into host
target cells. In one embodiment of the invention the molecule is
covalently linked (i.e., conjugated or chemically coupled) to the
capsid proteins. Methods of covalently linking molecules are known
by those skilled in the art.
[0111] The virus capsids of the invention also find use in raising
antibodies against the novel capsid structures. As a further
alternative, an exogenous amino acid sequence may be inserted into
the virus capsid for antigen presentation to a cell, e.g., for
administration to a subject to produce an immune response to the
exogenous amino acid sequence.
[0112] The invention also provides nucleic acids (e.g., isolated
nucleic acids) encoding the chimeric virus capsids and chimeric
capsid proteins of the invention. Further provided are vectors
comprising the nucleic acids, and cells (in vivo or in culture)
comprising the nucleic acids and/or vectors of the invention. Such
nucleic acids, vectors and cells can be used, for example, as
reagents (e.g., helper constructs or packaging cells) for the
production of virus vectors as described herein.
[0113] In exemplary embodiments, the invention provides nucleic
acid sequences encoding the AAV capsid of SEQ ID NOS:2-4 or at
least 90% identical to the nucleotide sequence of SEQ ID NO:1. The
invention also provides nucleic acids encoding the AAV capsid
variants, capsid protein variants and fusion proteins as described
above. In particular embodiments, the nucleic acid hybridizes to
the complement of the nucleic acid sequences specifically disclosed
herein under standard conditions as known by those skilled in the
art and encodes a variant capsid and/or capsid protein. Optionally,
the variant capsid or capsid protein substantially retains at least
one property of the capsid and/or capsid or capsid protein encoded
by the nucleic acid sequence of SEQ ID NO:1. For example, a virus
particle comprising the variant capsid or variant capsid protein
can substantially retain the oligodendrocyte tropism profile of a
virus particle comprising a capsid or capsid protein encoded by a
nucleic acid coding sequence of SEQ ID NO:1.
[0114] For example, hybridization of such sequences may be carried
out under conditions of reduced stringency, medium stringency or
even stringent conditions. Exemplary conditions for reduced, medium
and stringent hybridization are as follows: (e.g., conditions
represented by a wash stringency of 35-40% Formamide with
5.times.Denhardt's solution, 0.5% SDS and 1.times.SSPE at
37.degree. C.; conditions represented by a wash stringency of
40-45% Formamide with 5.times.Denhardt's solution, 0.5% SDS, and
1.times.SSPE at 42.degree. C.; and conditions represented by a wash
stringency of 50% Formamide with 5.times.Denhardt's solution, 0.5%
SDS and 1.times.SSPE at 42.degree. C., respectively). See, e.g.,
Sambrook et al., Molecular Cloning A Laboratory Manual (2d Ed.
1989) (Cold Spring Harbor Laboratory).
[0115] In other embodiments, nucleic acid sequences encoding a
variant capsid or capsid protein of the invention have at least
about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher
sequence identity with the nucleic acid sequence of SEQ ID NO:1 and
optionally encode a variant capsid or capsid protein that
substantially retains at least one property of the capsid or capsid
protein encoded by a nucleic acid of SEQ ID NO:1.
[0116] As is known in the art, a number of different programs can
be used to identify whether a nucleic acid or polypeptide has
sequence identity to a known sequence. Percent identity as used
herein means that a nucleic acid or fragment thereof shares a
specified percent identity to another nucleic acid, when optimally
aligned (with appropriate nucleotide insertions or deletions) with
the other nucleic acid (or its complementary strand), using BLASTN.
To determine percent identity between two different nucleic acids,
the percent identity is to be determined using the BLASTN program
"BLAST 2 sequences". This program is available for public use from
the National Center for Biotechnology information (NCBI) over the
Internet (Altschul et al., (1997) Nucleic Acids Res.
25(17):3389-3402). The parameters to be used are whatever
combination of the following yields the highest calculated percent
identity (as calculated below) with the default parameters shown in
parentheses: Program--blastn Matrix--0 BLOSUM62 Reward for a
match--0 or 1 (1) Penalty for a mismatch--0, -1, -2 or -3 (-2) Open
gap penalty--0, 1, 2, 3, 4 or 5 (5) Extension gap penalty--0 or 1
(1) Gap x_dropoff--0 or 50 (50) Expect--10.
[0117] Percent identity or similarity when referring to
polypeptides, indicates that the polypeptide in question exhibits a
specified percent identity or similarity when compared with another
protein or a portion thereof over the common lengths as determined
using BLASTP. This program is also available for public use from
the National Center for Biotechnology Information (NCBI) over the
Internet (Altschul et al., (1997) Nucleic Acids Res.
25(17):3389-3402). Percent identity or similarity for polypeptides
is typically measured using sequence analysis software. See, e.g.,
the Sequence Analysis Software Package of the Genetics Computer
Group. University of Wisconsin Biotechnology Center, 910 University
Avenue, Madison, Wis. 53705. Protein analysis software matches
similar sequences using measures of homology assigned to various
substitutions, deletions and other modifications. Conservative
substitutions typically include substitutions within the following
groups: glycine, alanine; valine, isoleucine, leucine; aspartic
acid, glutamic acid; asparagine, glutamine; serine, threonine;
lysine, arginine; and phenylalanine, tyrosine.
[0118] In particular embodiments, the nucleic acid can comprise,
consist essentially of, or consist of a vector including but not
limited to a plasmid, phage, viral vector (e.g., AAV vector, an
adenovirus vector, a herpesvirus vector, or a baculovirus vector),
bacterial artificial chromosome (BAC), or yeast artificial
chromosome (YAC). For example, the nucleic acid can comprise,
consist of, or consist essentially of an AAV vector comprising a 5'
and/or 3 terminal repeat (e.g., 5' and/or 3' AAV terminal
repeat).
[0119] In some embodiments, the nucleic acid encoding the chimeric
AAV capsid protein further comprises an AAV rep coding sequence.
For example, the nucleic acid can be a helper construct for
producing viral stocks.
[0120] The invention also provides packaging cells stably
comprising a nucleic acid of the invention. For example, the
nucleic acid can be stably incorporated into the genome of the cell
or can be stably maintained in an episomal form (e.g., an "EBV
based nuclear episome").
[0121] The nucleic acid can be incorporated into a delivery vector,
such as a viral delivery vector. To illustrate, the nucleic acid of
the invention can be packaged in an AAV particle, an adenovirus
particle, a herpesvirus particle, a baculovirus particle, or any
other suitable virus particle.
[0122] Moreover, the nucleic acid can be operably associated with a
promoter element. Promoter elements are described in more detail
herein.
[0123] The present invention further provides methods of producing
the virus vectors of the invention. In a representative embodiment,
the present invention provides a method of producing a recombinant
virus vector, the method comprising providing to a cell in vitro,
(a) a template comprising (i) a heterologous nucleic acid, and (ii)
packaging signal sequences sufficient for the encapsidation of the
AAV template into virus particles (e.g., one or more (e.g., two)
terminal repeats, such as AAV terminal repeats), and (b) AAV
sequences sufficient for replication and encapsidation of the
template into viral particles (e.g., the AAV rep and AAV cap
sequences encoding an AAV capsid of the invention). The template
and AAV replication and capsid sequences are provided under
conditions such that recombinant virus particles comprising the
template packaged within the capsid are produced in the cell. The
method can further comprise the step of collecting the virus
particles from the cell. Virus particles may be collected from the
medium and/or by lysing the cells.
[0124] In one illustrative embodiment, the invention provides a
method of producing a rAAV particle comprising an AAV capsid, the
method comprising: providing a cell in vitro with a nucleic acid
encoding a chimeric AAV capsid of the invention, an AAV rep coding
sequence, an AAV vector genome comprising a heterologous nucleic
acid, and helper functions for generating a productive AAV
infection; and allowing assembly of the AAV particles comprising
the AAV capsid and encapsidating the AAV vector genome.
[0125] The cell is typically a cell that is permissive for AAV
viral replication. Any suitable cell known in the art may be
employed, such as mammalian cells. Also suitable are
trans-complementing packaging cell lines that provide functions
deleted from a replication-defective helper virus, e.g., 293 cells
or other E1a trans-complementing cells.
[0126] The AAV replication and capsid sequences may be provided by
any method known in the art. Current protocols typically express
the AAV rep/cap genes on a single plasmid. The AAV replication and
packaging sequences need not be provided together, although it may
be convenient to do so. The AAV rep and/or cap sequences may be
provided by any viral or non-viral vector. For example, the rep/cap
sequences may be provided by a hybrid adenovirus or herpesvirus
vector (e.g., inserted into the E1a or E3 regions of a deleted
adenovirus vector). EBV vectors may also be employed to express the
AAV cap and rep genes. One advantage of this method is that EBV
vectors are episomal, yet will maintain a high copy number
throughout successive cell divisions (i.e., are stably integrated
into the cell as extra-chromosomal elements, designated as an EBV
based nuclear episome.
[0127] As a further alternative, the rep/cap sequences may be
stably carried (episomal or integrated) within a cell.
[0128] Typically, the AAV rep/cap sequences will not be flanked by
the AAV packaging sequences (e.g., AAV ITRs), to prevent rescue
and/or packaging of these sequences.
[0129] The template (e.g., an rAAV vector genome) can be provided
to the cell using any method known in the art. For example, the
template may be supplied by a non-viral (e.g., plasmid) or viral
vector. In particular embodiments, the template is supplied by a
herpesvirus or adenovirus vector (e.g., inserted into the E1a or E3
regions of a deleted adenovirus). As another illustration, Palombo
et al., (1998) J. Virol. 72:5025, describe a baculovirus vector
carrying a reporter gene flanked by the AAV ITRs. EBV vectors may
also be employed to deliver the template, as described above with
respect to the rep/cap genes.
[0130] In another representative embodiment, the template is
provided by a replicating rAAV virus. In still other embodiments,
an AAV provirus is stably integrated into the chromosome of the
cell.
[0131] To obtain maximal virus titers, helper virus functions
(e.g., adenovirus or herpesvirus) essential for a productive AAV
infection are generally provided to the cell. Helper virus
sequences necessary for AAV replication are known in the art.
Typically, these sequences are provided by a helper adenovirus or
herpesvirus vector. Alternatively, the adenovirus or herpesvirus
sequences can be provided by another non-viral or viral vector,
e.g., as a non-infectious adenovirus miniplasmid that carries all
of the helper genes required for efficient AAV production as
described by Ferrari et al., (1997) Nature Med. 3:1295, and U.S.
Pat. Nos. 6,040,183 and 6,093,570.
[0132] Further, the helper virus functions may be provided by a
packaging cell with the helper genes integrated in the chromosome
or maintained as a stable extrachromosomal element. In
representative embodiments, the helper virus sequences cannot be
packaged into AAV virions, e.g., are not flanked by AAV ITRs.
[0133] Those skilled in the art will appreciate that it may be
advantageous to provide the AAV replication and capsid sequences
and the helper virus sequences (e.g., adenovirus sequences) on a
single helper construct. This helper construct may be a non-viral
or viral construct, but is optionally a hybrid adenovirus or hybrid
herpesvirus comprising the AAV rep/cap genes.
[0134] In one particular embodiment, the AAV rep/cap sequences and
the adenovirus helper sequences are supplied by a single adenovirus
helper vector. This vector further contains the rAAV template. The
AAV rep/cap sequences and/or the rAAV template may be inserted into
a deleted region (e.g., the E1a or E3 regions) of the
adenovirus.
[0135] In a further embodiment, the AAV rep/cap sequences and the
adenovirus helper sequences are supplied by a single adenovirus
helper vector. The rAAV template is provided as a plasmid
template.
[0136] In another illustrative embodiment, the AAV rep/cap
sequences and adenovirus helper sequences are provided by a single
adenovirus helper vector, and the rAAV template is integrated into
the cell as a provirus. Alternatively, the rAAV template is
provided by an EBV vector that is maintained within the cell as an
extrachromosomal element (e.g., as a "EBV based nuclear episome,"
see Margolski, (1992) Curr. Top. Microbiol. Immun. 158:67).
[0137] In a further exemplary embodiment, the AAV rep/cap sequences
and adenovirus helper sequences are provided by a single adenovirus
helper. The rAAV template is provided as a separate replicating
viral vector. For example, the rAAV template may be provided by a
rAAV particle or a second recombinant adenovirus particle.
[0138] According to the foregoing methods, the hybrid adenovirus
vector typically comprises the adenovirus 5' and 3' cis sequences
sufficient for adenovirus replication and packaging (i.e., the
adenovirus terminal repeats and PAC sequence). The AAV rep/cap
sequences and, if present, the rAAV template are embedded in the
adenovirus backbone and are flanked by the 5' and 3' cis sequences,
so that these sequences may be packaged into adenovirus capsids. As
described above, in representative embodiments, the adenovirus
helper sequences and the AAV rep/cap sequences are not flanked by
the AAV packaging sequences (e.g., the AAV ITRs), so that these
sequences are not packaged into the AAV virions.
[0139] Herpesvirus may also be used as a helper virus in AAV
packaging methods. Hybrid herpesviruses encoding the AAV rep
protein(s) may advantageously facilitate for more scalable AAV
vector production schemes. A hybrid herpes simplex virus type I
(HSV-1) vector expressing the AAV-2 rep and cap genes has been
described (Conway et al., (1999) Gene Therapy 6:986 and WO
00/17377, the disclosures of which are incorporated herein in their
entireties).
[0140] As a further alternative, the virus vectors of the invention
can be produced in insect cells using baculovirus vectors to
deliver the rep/cap genes and rAAV template as described by Urabe
et al., (2002) Human Gene Therapy 13:1935-43.
[0141] Other methods of producing AAV use stably transformed
packaging cells (see, e.g., U.S. Pat. No. 5,658,785).
[0142] AAV vector stocks free of contaminating helper virus may be
obtained by any method known in the art. For example, AAV and
helper virus may be readily differentiated based on size. AAV may
also be separated away from helper virus based on affinity for a
heparin substrate (Zolotukhin et al., (1999) Gene Therapy 6:973).
In representative embodiments, deleted replication-defective helper
viruses are used so that any contaminating helper virus is not
replication competent. As a further alternative, an adenovirus
helper lacking late gene expression may be employed, as only
adenovirus early gene expression is required to mediate packaging
of AAV virus. Adenovirus mutants defective for late gene expression
are known in the art (e.g., ts100K and ts149 adenovirus
mutants).
[0143] The inventive packaging methods may be employed to produce
high titer stocks of virus particles, in particular embodiments,
the virus stock has a titer of at least about 10.sup.5 transducing
units (tu)/ml, at least about 10.sup.6 tu/ml, at least about
10.sup.7 tu/ml, at least about 10.sup.8 tu/ml, at least about
10.sup.9 tu/ml, or at least about 10.sup.10 tu/ml.
[0144] The novel capsid protein and capsid structures find use in
raising antibodies, for example, for diagnostic or therapeutic uses
or as a research reagent. Thus, the invention also provides
antibodies against the novel capsid proteins and capsids of the
invention.
[0145] The term "antibody" or "antibodies" as used herein refers to
all types of immunoglobulins, including IgG, IgM, IgA, IgD, and
IgE. The antibody can be monoclonal or polyclonal and can be of any
species of origin, including (for example) mouse, rat, rabbit,
horse, goat, sheep or human, or can be a chimeric antibody. See,
e.g., Walker et al., Mol. Immunol. 26, 403-11 (1989), The
antibodies can be recombinant monoclonal antibodies, for example,
produced according to the methods disclosed in U.S. Pat. No.
4,474,893 or U.S. Pat. No. 4,816,567. The antibodies can also be
chemically constructed, for example, according to the method
disclosed in U.S. Pat. No. 4,676,980.
[0146] Antibody fragments included within the scope of the present
invention include, for example, Fab, F(ab')2, and Fe fragments, and
the corresponding fragments obtained from antibodies other than
IgG. Such fragments can be produced by known techniques. For
example, F(ab')2 fragments can be produced by pepsin digestion of
the antibody molecule, and Fab fragments can be generated by
reducing the disulfide bridges of the F(ab')2 fragments.
Alternatively, Fab expression libraries can be constructed to allow
rapid and easy identification of monoclonal Fab fragments with the
desired specificity (Huse et al., (1989) Science 254,
1275-1281).
[0147] Polyclonal antibodies can be produced by immunizing a
suitable animal (e.g., rabbit, goat, etc.) with an antigen to which
a monoclonal antibody to the target binds, collecting immune serum
from the animal, and separating the polyclonal antibodies from the
immune serum, in accordance with known procedures.
[0148] Monoclonal antibodies can be produced in a hybridoma cell
line according to the technique of Kohler and Milstein, (1975)
Nature 265, 495-97. For example, a solution containing the
appropriate antigen can be injected into a mouse and, after a
sufficient time, the mouse sacrificed and spleen cells obtained.
The spleen cells are then immortalized by fusing them with myeloma
cells or with lymphoma cells, typically in the presence of
polyethylene glycol, to produce hybridoma cells. The hybridoma
cells are then grown in a suitable medium and the supernatant
screened for monoclonal antibodies having the desired specificity.
Monoclonal Fab fragments can be produced in E. coli by recombinant
techniques known to those skilled in the art. See, e.g., W. Huse,
(1989) Science 246, 1275-81.
[0149] Antibodies specific to a target polypeptide can also be
obtained by phage display techniques known in the art.
[0150] Various immunoassays can be used for screening to identify
antibodies having the desired specificity, Numerous protocols for
competitive binding or immunoradiometric assays using either
polyclonal or monoclonal antibodies with established specificity
are well known in the art. Such immunoassays typically involve the
measurement of complex formation between an antigen and its
specific antibody (e.g., antigen/antibody complex formation). A
two-site, monoclonal-based immunoassay utilizing monoclonal
antibodies reactive to two non-interfering epitopes can be used as
well as a competitive binding assay.
[0151] Antibodies can be conjugated to a solid support (e.g.,
beads, plates, slides or wells formed from materials such as latex
or polystyrene) in accordance with known techniques. Antibodies can
likewise be directly or indirectly conjugated to detectable groups
such as radiolabels (e.g., .sup.35S, .sup.125I, .sup.131I), enzyme
labels (e.g., horseradish peroxidase, alkaline phosphatase), and
fluorescence labels (e.g., fluorescein) in accordance with known
techniques. Determination of the formation of an antibody/antigen
complex in the methods of this invention can be by detection of,
for example, precipitation, agglutination, flocculation,
radioactivity, color development or change, fluorescence,
luminescence, etc., as is well known in the art.
III. Methods of Using Chimeric AAV Capsids
[0152] The present invention also relates to methods for delivering
heterologous nucleotide sequences into oligodendrocytes. The virus
vectors of the invention may be employed, e.g., to deliver a
nucleotide sequence of interest to an oligodendrocyte in vitro,
e.g., to produce a polypeptide or nucleic acid in vitro or for ex
vivo gene therapy. The vectors are additionally useful in a method
of delivering a nucleotide sequence to a subject in need thereof,
e.g., to express a therapeutic or immunogenic polypeptide or
nucleic acid. In this manner, the polypeptide or nucleic acid may
thus be produced in vivo in the subject. The subject may be in need
of the polypeptide or nucleic acid because the subject has a
deficiency of the polypeptide, or because the production of the
polypeptide or nucleic acid in the subject may impart some
therapeutic effect, as a method of treatment or otherwise, and as
explained further below.
[0153] In particular embodiments, the vectors are useful to express
a polypeptide or nucleic acid that provides a beneficial effect to
oligodendrocytes, e.g., to promote growth and/or differentiation of
oligodendrocytes. The ability to target vectors to oligodendrocytes
may be particularly useful to treat diseases or disorders involving
oligodendrocyte dysfunction and/or demyelination of neurons. In
other embodiments, the vectors are useful to express a polypeptide
or nucleic acid that provides a beneficial effect to cells near the
oligodendrocytes (e.g., neurons).
[0154] Thus, one aspect of the invention relates to a method of
delivering a nucleic acid of interest to an oligodendrocyte, the
method comprising contacting the oligodendrocyte with the AAV
particle of the invention.
[0155] In another aspect, the invention relates to a method of
delivering a nucleic acid of interest to an oligodendrocyte in a
mammalian subject, the method comprising administering an effective
amount of the AAV particle or pharmaceutical formulation of the
invention to a mammalian subject.
[0156] A further aspect of the invention relates to a method of
treating a disorder associated with oligodendrocyte dysfunction in
a subject in need thereof, the method comprising administering a
therapeutically effective amount of the AAV particle of the
invention to the subject. In one embodiment, the disorder
associated with oligodendrocyte dysfunction is a demyelinating
disease. In one embodiment, the disorder associated with
oligodendrocyte dysfunction is multiple sclerosis,
Pelizaeus-Merzbacher disease, Krabbe's disease, metachromatic
leukodystrophy, adrenoleukodystrophy, Canavan disease, Alexander
disease, orthochromatic leukodystrophy, Zellweger disease,
18q-syndrome, cerebral palsy, spinal cord injury, traumatic brain
injury, stroke, phenylketonuria, or viral infection, or any other
disorder known or later found to be associated with oligodendrocyte
dysfunction. In another embodiment, the methods of the invention
are used to treat a disorder that is not directly associated with
oligodendrocyte dysfunction but would benefit by expression of a
heterologous polypeptide or nucleic acid in oligodendrocytes in
addition to or instead of expression in neurons, astrocytes, or
other CNS cell types. Examples include, without limitation,
neurodegenerative disorders such as Alzheimer's disease,
Parkinson's disease, and Huntington's disease, CNS tumors, and
other CNS disorders.
[0157] CNS disorders include but are not limited to disorders of
thinking and cognition such as schizophrenia and delirium; amnestic
disorders; disorders of mood, such as affective disorders and
anxiety disorders (including post-traumatic stress disorder,
separation anxiety disorder, selective mutism, reactive attachment
disorder, stereotypic movement disorder, panic disorders,
agoraphobia, specific phobias, social phobia, obsessive-compulsive
disorder, acute stress disorder, generalized anxiety disorder,
substance-induced anxiety disorder and/or anxiety disorder not
otherwise specified); disorders of social behavior; disorders of
learning and memory, such as learning disorders (e.g., dyslexia);
motor skills disorders; communication disorders (e.g., stuttering);
pervasive developmental disorders (e.g., autistic disorder, Rett's
disorder, childhood disintegrative disorder, Asperger's disorder,
and/or pervasive developmental disorder not otherwise specified)
and dementia. Accordingly, the term "central nervous system
disorder" encompasses the disorders listed above as well as
depressive disorders (including major depressive disorder,
dysthmyic disorder, depressive disorder not otherwise specified,
postpartum depression); seasonal affective disorder, mania; bipolar
disorders (including bipolar I disorder, bipolar II disorder,
cyclothymic disorder, bipolar disorder not otherwise specified);
attention-deficit and disruptive behavior disorders (including
attention deficit disorder with hyperactivity disorder, conduct
disorder, oppositional defiant disorder and/or disruptive behavior
disorder not otherwise specified); drug addiction/substance abuse
(including abuse of opiates, amphetamines, alcohol, hallucinogens,
cannabis, inhalants, phencyclidine, sedatives, hypnotics,
anxyolytics and/or cocaine); alcohol-induced disorders;
amphetamine-induced disorders; caffeine-induced disorders;
cannabis-induced disorders; cocaine-induced disorders;
hallucinogen-induced disorders; inhalant-induced disorders;
nicotine-induced disorders; opioid-induced disorders;
phencyclidine-induced disorders; sedative, hypnotic or
anxyolytic-induced disorders; agitation; apathy; psychoses;
irritability; disinhibition; schizophreniform disorder;
schizoaffective disorder; delusional disorder; brief psychotic
disorder, shared psychotic disorder, substance-induced psychotic
disorder; psychotic disorder not otherwise specified; unipolar
disorders, mood disorders (e.g., mood disorder with psychotic
features); somatoform disorders; factitious disorders;
disassociative disorders; mental retardation; feeding and eating
disorders of infancy or early childhood; eating disorders such as
anorexia nervosa, bulimnia nervosa and/or eating disorder not
otherwise specified; sleeping disorders (e.g., dyssomnias such as
primary insomnia, primary hypersomnia, narcolepsy,
breathing-related sleep disorder and circadian rhythm sleep
disorder and/or parasomnias); impulse control disorders (e.g.,
kleptomania, pyromania, trichotillomania, pathological gambling
and/or intermittent explosive disorder); adjustment disorders;
personality disorders (e.g., paranoid personality disorder,
schizoid personality disorder, schizotypal personality disorder,
antisocial personality disorder, borderline personality disorder,
histrionic personality disorder, narcissistic personality disorder,
avoidant personality disorder, dependent personality disorder
and/or obsessive-compulsive personality disorder); Tic disorders
(e.g., Tourette's disorder, chronic motor or vocal tic disorder,
transient tic disorder and/or tic disorder not otherwise
specified); elimination disorders; and any combination of the
foregoing as well as any other disorder or group of disorders
described in the Diagnostic and Statistical Manual of Mental
Disorders--Fourth Edition (DSM-IV; the American Psychiatric
Association, Washington D.C., 1994). "Central Nervous System
disorders" also include other conditions that implicate the CNS
including but not limited to neurodegenerative disorders such as
Alzheimer's disease, involuntary movement disorders such as
Parkinson's disease, Huntington's disease, amyotrophic lateral
sclerosis (ALS), and the like. Other CNS disorders include without
limitation epilepsy, multiple sclerosis, neurogenic pain,
psychogenic pain, and migraines. In other embodiments, the CNS
disorder encompasses any subset of the foregoing diseases or
excludes any one or more of the foregoing conditions. In particular
embodiments, the term "central nervous system disorder" does not
encompass benign and/or malignant tumors of the CNS.
[0158] In another aspect of the invention, the chimeric AAV capsid
and vectors of the invention are fully- or nearly fully-detargeted
vectors that can be further modified to a desirable tropic profile
for targeting of one or more peripheral organs or tissues as
discussed below. In this aspect, the present invention also relates
to methods for delivering heterologous nucleotide sequences into a
broad range of cells, including dividing and non-dividing cells.
The virus vectors of the invention may be employed to deliver a
nucleotide sequence of interest to a cell in vitro, e.g., to
produce a polypeptide in vitro or for ex vivo gene therapy. The
vectors are additionally useful in a method of delivering a
nucleotide sequence to a subject in need thereof, e.g., to express
a therapeutic or immunogenic polypeptide or nucleic acid. In this
manner, the polypeptide or nucleic acid may thus be produced in
vivo in the subject. The subject may be in need of the polypeptide
or nucleic acid because the subject has a deficiency of the
polypeptide, or because the production of the polypeptide or
nucleic acid in the subject may impart some therapeutic effect, as
a method of treatment or otherwise, and as explained further
below.
[0159] In general, the virus vectors of the invention may be
employed to deliver any foreign nucleic acid with a biological
effect to treat or ameliorate the symptoms associated with any
disorder related to gene expression. Further, the invention can be
used to treat any disease state for which it is beneficial to
deliver a therapeutic polypeptide. Illustrative disease states
include, but are not limited to; cystic fibrosis (cystic fibrosis
transmembrane regulator protein) and other diseases of the lung,
hemophilia A (Factor VIII), hemophilia B (Factor IX), thalassemia
(.beta.-globin), anemia (erythropoietin) and other blood disorders,
Alzheimer's disease (GDF; neprilysin), multiple sclerosis
(.beta.-interferon), Parkinson's disease (glial-cell line derived
neurotrophic factor [GDNF]), Huntington's disease (inhibitory RNA
including without limitation RNAi such as siRNA or shRNA, antisense
RNA or microRNA to remove repeats), amyotrophic lateral sclerosis,
epilepsy (galanin, neurotrophic factors), and other neurological
disorders, cancer (endostatin, angiostatin, TRAIL, FAS-ligand,
cytokines including interferons; inhibitory RNA including without
limitation RNAi (such as siRNA or shRNA), antisense RNA and
microRNA including inhibitory RNA against VEGF, the multiple drug
resistance gene product or a cancer immunogen), diabetes mellitus
(insulin, PGC-.alpha.1, GLP-1, myostatin pro-peptide, glucose
transporter 4), muscular dystrophies including Duchenne and Becker
(e.g., dystrophin, mini-dystrophin, micro-dystrophin, insulin-like
growth factor I, a sarcoglycan [e.g., .alpha., .beta., .gamma.],
Inhibitory RNA [e.g., RNAi, antisense RNA or microRNA] against
myostatin or myostatin propeptide, laminin-alpha2, Fukutin-related
protein, dominant negative myostatin, follistatin, activin type II
soluble receptor, anti-inflammatory polypeptides such as the Ikappa
B dominant mutant, sarcospan, utrophin, mini-utrophin, inhibitory
RNA [e.g., RNAi, antisense RNA or microRNA] against splice
junctions in the dystrophin gene to induce exon skipping [see,
e.g., WO/2003/095647], inhibitory RNA (e.g., RNAi, antisense RNA or
micro RNA] against U7 shRNAs to induce exon skipping [see, e.g.,
WO/2006/021724], and antibodies or antibody fragments against
myostatin or myostatin propeptide), Gaucher disease
(glucocerebrosidase), Hurler's disease (.alpha.-L-iduronidase),
adenosine deaminase deficiency (adenosine deaminase), glycogen
storage diseases (e.g., Fabry disease [.alpha.-galactosidase] and
Pompe disease [lysosomal acid .alpha.-glucosidase]) and other
metabolic defects including other lysosomal storage disorders and
glycogen storage disorders, congenital emphysema (al-antitrypsin),
Lesch-Nyhan Syndrome (hypoxanthine guanine phosphoribosyl
transferase), Niemann-Pick disease (sphingomyelinase), Maple Syrup
Urine Disease (branched-chain keto acid dehydrogenase), retinal
degenerative diseases (and other diseases of the eye and retina;
e.g., PDGF, endostatin and/or angiostatin for macular
degeneration), diseases of solid organs such as brain (including
Parkinson's Disease [GDNF], astrocytomas [endostatin, angiostatin
and/or RNAi against VEGF], glioblastomas [endostatin, angiostatin
and/or RNAi against VEGF]), liver (RNAi such as siRNA or shRNA,
microRNA or antisense RNA for hepatitis B and/or hepatitis C
genes), kidney, heart including congestive heart failure or
peripheral artery disease (PAD) (e.g., by delivering protein
phosphatase inhibitor I [I-1], phospholamban, sarcoplasmic
endoreticulum Ca.sup.2+-ATPase [serca2a], zinc finger proteins that
regulate the phospholamban gene, Pim-1, PGC-1.alpha., SOD-1, SOD-2,
ECF-SOD, kallikrein, thymosin-.beta.4, hypoxia-inducible
transcription factor [HIF], .beta.arket, .beta.2-adrenergic
receptor, .beta.2-adrenergic receptor kinase [.beta.ARK],
phosphoinositide-3 kinase [PI3 kinase], calsarcin, an angiogenic
factor, S100A1, parvalbumin, adenylyl cyclase type 6, a molecule
that effects G-protein coupled receptor kinase type 2 knockdown
such as a truncated constitutively active bARKct, an inhibitory RNA
[e.g., RNAi, antisense RNA or microRNA] against phospholamban;
phospholamban inhibitory or dominant-negative molecules such as
phospholamban S16E, etc.), arthritis (insulin-like growth factors),
joint disorders (insulin-like growth factors), intimal hyperplasia
(e.g., by delivering enos, inos), improve survival of heart
transplants (superoxide dismutase), AIDS (soluble CD4), muscle
wasting (insulin-like growth factor 1, myostatin pro-peptide, an
anti-apoptotic factor, follistatin), limb ischemia (VEGF, FGF,
PGC-1.alpha., EC-SOD, HIF), kidney deficiency (erythropoietin),
anemia (erythropoietin), arthritis (anti-inflammatory factors such
as IRAP and TNF.alpha. soluble receptor), hepatitis
(.alpha.-interferon), LDL receptor deficiency (LDL receptor),
hyperammonemia (ornithine transcarbamylase), spinal cerebral
ataxias including SCA1, SCA2 and SCA3, phenylketonuria
(phenylalanine hydroxylase), autoimmune diseases, and the like. The
invention can further be used following organ transplantation to
increase the success of the transplant and/or to reduce the
negative side effects of organ transplantation or adjunct therapies
(e.g., by administering immunosuppressant agents or inhibitory
nucleic acids to block cytokine production). As another example,
bone morphogenic proteins (including RANKL and/or VEGF) can be
administered with a bone allograft, for example, following a break
or surgical removal in a cancer patient.
[0160] Exemplary lysosomal storage diseases that can be treated
according to the present invention include without limitation:
Hurler's Syndrome (MPS IH), Scheie's Syndrome (MPS IS), and
Hurler-Scheie Syndrome (MPS 1H/S) (.alpha.-L-iduronidase); Hunter's
Syndrome (MPS II) (iduronate sulfate sulfatase); Sanfilippo A
Syndrome (MPS IIIA) (Heparan-S-sulfate sulfaminidase), Sanfilippo B
Syndrome (MPS IIIB) (N-acetyl-D-glucosaminidase). Sanfilippo C
Syndrome (MPS IIIC) (Acetyl-CoA-glucosaminide N-acetyltransferase),
Sanfilippo D Syndrome (MPS HID) (N-acetyl-glucosaminine-6-sulfate
sulfatase); Morquio A disease (MPS IVA) (Galactosamine-6-sulfate
sulfatase), Morquio B disease (MPS IV B) (.beta.-Galactosidase);
Maroteaux-lmay disease (MPS VI) (arylsulfatase B); Sly Syndrome
(MPS VII) (s-glucuronidase); hyaluronidase deficiency (MPS IX)
(hyaluronidase); sialidosis (mucolipidosis I), mucolipidosis 1
(I-Cell disease) (N-actylglucos-aminyl-1-phosphotransfcrase
catalytic subunit), mucolipidosis III (pseudo-Hurler polydystrophy)
(N-acetylglucos-aminyl-1-phosphotransferase; type IIIA [catalytic
subunit] and type IIIC [substrate recognition subunit]); GM1
gangliosidosis (ganglioside .beta.-galactosidase), GM2
gangliosidosis Type I (Tay-Sachs disease) (.beta.-hexaminidase A),
GM2 gangliosidosis type II (Sandhoff's disease)
(.beta.-hexosaminidase B); Niemann-Pick disease (Types A and B)
(sphingomyelinase); Gaucher's disease (glucocerebrosidase);
Farber's disease (ceraminidase); Fabry's disease
(.alpha.-galactosidase A); Krabbe's disease (galactosylceramide
.beta.-galactosidase); metachromatic leukodystrophy (arylsulfatase
A); lysosomal acid lipase deficiency including Wolman's disease
(lysosomal acid lipase); Batten disease (juvenile neuronal ceroid
lipofuscinosis) (lysosomal trans-membrane CLN3 protein) sialidosis
(neuranainidase 1); galactosialidosis (Goldberg's syndrome)
(protective protein/cathepsin A); .alpha.-mannosidosis
(.alpha.-D-mannosidase); .beta.-mannosidosis
(.beta.-D-mannosidosis); fucosidosis (.alpha.-D-fucosidase);
aspartylglucosaminuria (N-Aspartylglucosaminidase); and sialuria
(Na phosphate cotransporter).
[0161] Exemplary glycogen storage diseases that can be treated
according to the present invention include, but are not limited to,
Type Ia GSD (von Gierke disease) (glucose-6-phosphatase), Type Ib
GSD (glucose-6-phosphate translocase), Type Ic GSD (microsomal
phosphate or pyrophosphate transporter), Type Id GSD (microsomal
glucose transporter), Type II GSD including Pompe disease or
infantile Type IIa GSD (lysosomal acid .alpha.-glucosidase) and
Type IIb (Danon) (lysosomal membrane protein-2), Type IIIa and IIIb
GSD (Debrancher enzyme; amyloglucosidase and
oligoglucanotransferase), Type IV GSD (Andersen's disease)
(branching enzyme), Type V GSD (McArdle disease) (muscle
phosphorylase), Type VI GSD (Hers' disease) (liver phosphorylase),
Type VII GSD (Tarui's disease) (phosphofructokinase), GSD Type
VIII/IXa (X-linked phosphorylase kinase), GSD Type IXb (Liver and
muscle phosphorylase kinase), GSD Type IXc (liver phosphorylase
kinase), GSD Type IXd (muscle phosphorylase kinase), GSD O
(glycogen synthase), Fanconi-Bickel syndrome (glucose
transporter-2), phosphoglucoisomerase deficiency, muscle
phosphoglycerate kinase deficiency, phosphoglycerate mutase
deficiency, fructose 1,6-diphosphatase deficiency,
phosphoenolpyruvate carboxykinase deficiency, and lactate
debydrogenase deficiency.
[0162] Nucleic acids and polypeptides that can be delivered to
cardiac muscle include those that are beneficial in the treatment
of damaged, degenerated or atrophied cardiac muscle and/or
congenital cardiac defects. For example, angiogenic factors useful
for facilitating vascularization in the treatment of heart disease
include but are not limited to vascular endothelial growth factor
(VEGF), VEGF II, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF.sub.121,
VEGF.sub.138, VEGF.sub.145, VEGF.sub.165, VEGF.sub.189,
VEGF.sub.206, hypoxia inducible factor 1.alpha. (HIP 1.alpha.),
endothelial NO synthase (eNOS), iNOS, VEFGR-1 (Flt1), VEGFR-2
(KDR/Flk1), VEGFR-3 (Flt4), angiogenin, epidermal growth factor
(EGF), angiopoietin, platelet-derived growth factor, angiogenic
factor, transforming growth factor-.alpha. (TGF-.alpha.),
transforming growth factor-.beta. (TGF-.beta.), vascular
permeability factor (VPF), tumor necrosis factor alpha
(TNF-.alpha.), interleukin-3 (IL-3), interleukin-8 (IL-8),
platelet-derived endothelial growth factor (PD-EGF), granulocyte
colony stimulating factor (G-CSF), hepatocyte growth factor (HGF),
scatter factor (SF), pleitrophin, proliferin, follistatin,
placental growth factor (PIGF), midkine, platelet-derived growth
factor-BB-(PDGF), fractalkine, ICAM-1, angiopoietin-1 and -2 (Ang1
and Ang2), Tie-2, neumpilin-1, ICAM-1, chemokines and cytokines
that stimulate smooth muscle cell, monocyte, or leukocyte
migration, anti-apoptotic peptides and proteins, fibroblast growth
factors (FGF), FGF-1, FGF-1b, FGF-1c, FGF-2, FGF-2b, FGF-2c, FGF-3,
FGF-3b, FGF-3c, FGF-4, FGF-5, FGF-7, FGF-9, acidic FGF, basic FGF,
monocyte chemotactic protein-1, granulocyte macrophage-colony
stimulating factor, insulin-like growth factor-1 (IGF-1), IGF-2,
early growth response factor-1 (EGR-1), ETS-1, human tissue
kallikrein (HK), matrix metalloproteinase, chymase, urokinase-type
plasminogen activator and heparinase. (see, e.g., U.S. Patent
Application No. 20060287259 and U.S. Patent Application No.
20070059288).
[0163] The most common congenital heart disease found in adults is
bicuspid aortic valve, whereas atrial septal defect is responsible
for 30-40% of congenital heart disease seen in adults. The most
common congenital cardiac defect observed in the pediatric
population is ventricular septal defect. Other congenital heart
diseases include Eisenmenger's syndrome, patent ductus arteriosus,
pulmonary stenosis, coarctation of the aorta, transposition of the
great arteries, tricuspid atresia, univentricular heart, Ebstein's
anomaly, and double-outlet right ventricle. A number of studies
have identified putative genetic loci associated with one or more
of these congenital heart diseases. For example, the putative
gene(s) for congenital heart disease associated with Down syndrome
is 21q22.2-q22.3, between ETS2 and MX1. Similarly, most cases of
DiGeorge syndrome result from a deletion of chromosome 22q11.2 (the
DiGeorge syndrome chromosome region, or DGCR). Several genes are
lost in this deletion including the putative transcription factor
TUPLE1. This deletion is associated with a variety of phenotypes,
e.g., Shprintzen syndrome; conotruncal anomaly face (or Takao
syndrome); and isolated outflow tract defects of the heart
including Tetralogy of Fallot, truncus arteriosus, and interrupted
aortic arch. All of the foregoing disorders can be treated
according to the present invention.
[0164] Other significant diseases of the heart and vascular system
are also believed to have a genetic, typically polygenic,
etiological component. These diseases include, for example,
hypoplastic left heart syndrome, cardiac valvular dysplasia,
Pfeiffer cardiocranial syndrome, oculofaciocardiodental syndrome,
Kapur-Toriello syndrome, Sonoda syndrome, Ohdo Blepharophimosis
syndrome, heart-hand syndrome, Pierre-Robin syndrome, Hirschsprung
disease, Kousseff syndrome, Grange occlusive arterial syndrome,
Kearns-Sayre syndrome, Kartagener syndrome, Alagille syndrome,
Ritscher-Schinzel syndrome, Ivemark syndrome, Young-Simpson
syndrome, hemochromatosis, Holzgreve syndrome, Barth syndrome,
Smith-Lemli-Opitz syndrome, glycogen storage disease, Gaucher-like
disease, Fabry disease, Lowry-Maclean syndrome, Rett syndrome,
Opitz syndrome, Marfan syndrome, Miller-Dieker lissencephaly
syndrome, mucopolysaccharidosis, Bruada syndrome, humerospinal
dysostosis, Phaver syndrome, McDonough syndrome, Marfanoid
hypermobility syndrome, atransferrinemia, Cornelia de Lange
syndrome, Leopard syndrome, Diamond-Blackfan anemia, Steinfeld
syndrome, progeria, and Williams-Beuren syndrome. All of these
disorders can be treated according to the present invention.
[0165] Anti-apoptotic factors can be delivered to skeletal muscle,
diaphragm muscle and/or cardiac muscle to treat muscle wasting
diseases, limb ischemia, cardiac infarction, heart failure,
coronary artery disease and/or type I or type II diabetes.
[0166] Nucleic acids that can be delivered to skeletal muscle
include those that are beneficial in the treatment of damaged,
degenerated and/or atrophied skeletal muscle. The genetic defects
that cause muscular dystrophy are known for many forms of the
disease. These defective genes either fail to produce a protein
product, produce a protein product that fails to function properly,
or produce a dysfunctional protein product that interferes with the
proper function of the cell. The heterologous nucleic acid may
encode a therapeutically functional protein or a polynucleotide
that inhibits production or activity of a dysfunctional protein.
Polypeptides that may be expressed from delivered nucleic acids, or
inhibited by delivered nucleic acids (e.g., by delivering RNAi,
microRNA or antisense RNA), include without limitation dystrophin,
a mini-dystrophin or a micro-dystrophin (Duchene's and Becker MD);
dystrophin-associated glycoproteins i-sarcoglycan (limb-girdle MD
2E), .delta.-sarcoglycan (limb-girdle MD 22F), .alpha.-sarcoglycan
(limb girdle MI) 2D) and 1-sarcoglycan (limb-girdle MD 2C),
utrophin, calpain (autosomal recessive limb-girdle MD type 2A),
caveolin-3 (autosomal-dominant limb-girdle MD), laminin-alpha2
(merosin-deficient congenital MD), miniagrin (laminin-alpha2
deficient congenital MD), fukutin (Fukuyama type congenital MD),
emerin (Emery-Dreifuss MD), myotilin, lamin A/C, calpain-3,
dysferlin, and/or telethonin. Further, the heterologous nucleic
acid can encode mir-1, mir-133, mir-206, mir-208 or an antisense
RNA, RNAi (e.g., siRNA or shRNA) or microRNA to induce exon
skipping in a defective dystrophin gene.
[0167] In particular embodiments, the nucleic acid is delivered to
tongue muscle (e.g., to treat dystrophic tongue). Methods of
delivering to the tongue can be by any method known in the art
including direct injection, oral administration, topical
administration to the tongue, intravenous administration,
intra-articular administration and the like.
[0168] The foregoing proteins can also be administered to diaphragm
muscle to treat muscular dystrophy.
[0169] Alternatively, a gene transfer vector may be administered
that encodes any other therapeutic polypeptide.
[0170] In particular embodiments, a virus vector according to the
present invention is used to deliver a nucleic acid of interest as
described herein to skeletal muscle, diaphragm muscle and/or
cardiac muscle, for example, to treat a disorder associated with
one or more of these tissues such as muscular dystrophy, heart
disease (including PAD and congestive heart failure), and the
like.
[0171] Gene transfer has substantial potential use in understanding
and providing therapy for disease states. There are a number of
inherited diseases in which defective genes are known and have been
cloned. In general, the above disease states fall into two classes:
deficiency states, usually of enzymes, which are generally
inherited in a recessive manner, and unbalanced states, which may
involve regulatory or structural proteins, and which are typically
inherited in a dominant manner. For deficiency state diseases, gene
transfer can be used to bring a normal gene into affected tissues
for replacement therapy, as well as to create animal models for the
disease using inhibitory RNA such as RNAi (e.g., siRNA or shRNA),
microRNA or antisense RNA. For unbalanced disease states, gene
transfer can be used to create a disease state in a model system,
which can then be used in efforts to counteract the disease state.
Thus, the virus vectors according to the present invention permit
the treatment of genetic diseases. As used herein, a disease state
is treated by partially or wholly remedying the deficiency or
imbalance that causes the disease or makes it more severe. The use
of site-specific recombination of nucleic sequences to cause
mutations or to correct defects is also possible.
[0172] The virus vectors according to the present invention may
also be employed to provide an antisense nucleic acid or inhibitory
RNA (e.g., microRNA or RNAi such as a siRNA or shRNA) to a cell in
vitro or in vivo. Expression of the inhibitory RNA in the target
cell diminishes expression of a particular protein(s) by the cell.
Accordingly, inhibitory RNA may be administered to decrease
expression of a particular protein in a subject in need thereof.
Inhibitory RNA may also be administered to cells in vitro to
regulate cell physiology, e.g., to optimize cell or tissue culture
systems.
[0173] As a further aspect, the virus vectors of the present
invention may be used to produce an immune response in a subject.
According to this embodiment, a virus vector comprising a nucleic
acid encoding an immunogen may be administered to a subject, and an
active immune response (optionally, a protective immune response)
is mounted by the subject against the immunogen. Immunogens are as
described hereinabove.
[0174] Alternatively, the virus vector may be administered to a
cell ex vivo and the altered cell is administered to the subject.
The heterologous nucleic acid is introduced into the cell, and the
cell is administered to the subject, where the heterologous nucleic
acid encoding the immunogen is optionally expressed and induces an
immune response in the subject against the immunogen. In particular
embodiments, the cell is an antigen-presenting cell (e.g., a
dendritic cell).
[0175] An "active immune response" or "active immunity" is
characterized by "participation of host tissues and cells after an
encounter with the immunogen. It involves differentiation and
proliferation of immunocompetent cells in lymphoreticular tissues,
which lead to synthesis of antibody or the development of
cell-mediated reactivity, or both." Herbert B. Herscowitz,
Immunophysiology: Cell Function and Cellular Interactions in
Antibody Formation, in IMMUNOLOGY: BASIC PROCESSES 117 (Joseph A.
Bellanti ed., 1985). Alternatively stated, an active immune
response is mounted by the host after exposure to immunogens by
infection or by vaccination. Active immunity can be contrasted with
passive immunity, which is acquired through the "transfer of
preformed substances (antibody, transfer factor, thymic graft,
interleukin-2) from an actively immunized host to a non-immune
host;" Id
[0176] A "protective" immune response or "protective" immunity as
used herein indicates that the immune response confers some benefit
to the subject in that it prevents or reduces the incidence of
disease. Alternatively, a protective immune response or protective
immunity may be useful in the treatment of disease, in particular
cancer or tumors (e.g., by causing regression of a cancer or tumor
and/or by preventing metastasis and/or by preventing growth of
metastatic nodules), The protective effects may be complete or
partial, as long as the benefits of the treatment outweigh any
disadvantages thereof.
[0177] The virus vectors of the present invention may also be
administered for cancer immunotherapy by administration of a viral
vector expressing a cancer cell antigen (or an immunologically
similar molecule) or any other immunogen that produces an immune
response against a cancer cell. To illustrate, an immune response
may be produced against a cancer cell antigen in a subject by
administering a viral vector comprising a heterologous nucleotide
sequence encoding the cancer cell antigen, for example to treat a
patient with cancer. The virus vector may be administered to a
subject in vivo or by using ex vivo methods, as described
herein.
[0178] As used herein, the term "cancer" encompasses tumor-forming
cancers. Likewise, the term "cancerous tissue" encompasses tumors.
A "cancer cell antigen" encompasses tumor antigens.
[0179] The term "cancer" has its understood meaning in the art, for
example, an uncontrolled growth of tissue that has the potential to
spread to distant sites of the body (i.e., metastasize). Exemplary
cancers include, but are not limited to, leukemia, lymphoma (e.g.,
Hodgkin and non-Hodgkin lymphomas), colorectal cancer, renal
cancer, liver cancer, breast cancer, lung cancer, prostate cancer,
testicular cancer, ovarian cancer, uterine cancer, cervical cancer,
brain cancer (e.g., gliomas and glioblastoma), bone cancer,
sarcoma, melanoma, head and neck cancer, esophageal cancer, thyroid
cancer, and the like. In embodiments of the invention, the
invention is practiced to treat and/or prevent tumor-forming
cancers.
[0180] The term "tumor" is also understood in the art, for example,
as an abnormal mass of undifferentiated cells within a
multicellular organism. Tumors can be malignant or benign. In
representative embodiments, the methods disclosed herein are used
to prevent and treat malignant tumors.
[0181] Cancer cell antigens have been described hereinabove. By the
terms "treating cancer" or "treatment of cancer," it is intended
that the severity of the cancer is reduced or the cancer is
prevented or at least partially eliminated. For example, in
particular contexts, these terms indicate that metastasis of the
cancer is prevented or reduced or at least partially eliminated. In
further representative embodiments, these terms indicate that
growth of metastatic nodules (e.g., after surgical removal of a
primary tumor) is prevented or reduced or at least partially
eliminated. By the terms "prevention of cancer" or "preventing
cancer" it is intended that the methods at least partially
eliminate or reduce the incidence or onset of cancer. Alternatively
stated, the onset or progression of cancer in the subject may be
slowed, controlled, decreased in likelihood or probability, or
delayed.
[0182] In particular embodiments, cells may be removed from a
subject with cancer and contacted with a virus vector according to
the present invention. The modified cell is then administered to
the subject, whereby an immune response against the cancer cell
antigen is elicited. This method is particularly advantageously
employed with immunocompromised subjects that cannot mount a
sufficient immune response in vivo (i.e., cannot produce enhancing
antibodies in sufficient quantities).
[0183] It is known in the art that immune responses may be enhanced
by immunomodulatory cytokines (e.g., .alpha.-interferon,
.beta.-interferon, .gamma.-interferon, .omega.-interferon,
.tau.-interferon, interleukin-1.alpha., interleukin-1.beta.,
interleukin-2, interleukin-3, interleukin-4, interleukin 5,
interleukin-6, interleukin-7, interleukin-8, interleukin-9,
interleukin-10, interleukin-11, interleukin 12, interleukin-13,
interleukin-14, interleukin-18, B cell Growth factor, CD40 Ligand,
tumor necrosis factor-.alpha., tumor necrosis factor-.beta.,
monocyte chemoattractant protein-1, granulocyte-macrophage colony
stimulating factor, and lymphotoxin). Accordingly, immunomodulatory
cytokines (e.g., CTL inductive cytokines) may be administered to a
subject in conjunction with the virus vectors.
[0184] Cytokines may be administered by any method known in the
art. Exogenous cytokines may be administered to the subject, or
alternatively, a nucleotide sequence encoding a cytokine may be
delivered to the subject using a suitable vector, and the cytokine
produced in vivo.
[0185] The viral vectors are further useful for targeting
oligodendrocytes for research purposes, e.g., for study of CNS
function in vitro or in animals or for use in creating and/or
studying animal models of disease. For example, the vectors can be
used to deliver heterologous nucleic acids to oligodendrocytes in
animal models of demyelinating diseases. Demyelination can be
induced in animals by a variety of means, including without
limitation administration of viruses (e.g., Semliki virus, murine
hepatitis virus, or Theiler's murine encephalomyelitis virus) and
administration of chemicals (e.g., cuprizone, ethidium bromide, or
lysolecithin). In some embodiments, the vector can also be used in
animal models of experimental autoimmune encephalomyelitis. This
condition can be induced by, for example, administration of
kainite, SIN-1, anti-galactocerebroside, or irradiation. In other
embodiments, the vital vector can be used to specifically deliver
to oligodendrocytes a toxic agent or an enzyme that produces a
toxic agent (e.g., thymidine kinase) in order to kill some or all
of the cells.
[0186] Further, the virus vectors according to the present
invention find further use in diagnostic and screening methods,
whereby a gene of interest is transiently or stably expressed in a
cell culture system, or alternatively, a transgenic animal model.
The invention can also be practiced to deliver a nucleic acid for
the purposes of protein production, e.g., for laboratory,
industrial or commercial purposes.
[0187] Recombinant virus vectors according to the present invention
find use in both veterinary and medical applications. Suitable
subjects include both avians and mammals. The term "avian" as used
herein includes, but is not limited to, chickens, ducks, geese,
quail, turkeys, pheasant, parrots, parakeets. The term "mammal" as
used herein includes, but is not limited to, humans, primates
non-human primates (e.g., monkeys and baboons), cattle, sheep,
goats, pigs, horses, cats, dogs, rabbits, rodents (e.g., rats,
mice, hamsters, and the like), etc. Human subjects include
neonates, infants, juveniles, and adults. Optionally, the subject
is "in need of" the methods of the present invention, e.g., because
the subject has or is believed at risk for a disorder including
those described herein or that would benefit from the delivery of a
nucleic acid including those described herein. For example, in
particular embodiments, the subject has (or has had) or is at risk
for a demyelinating disorder or a spinal cord or brain injury. As a
further option, the subject can be a laboratory animal and/or an
animal model of disease.
[0188] In particular embodiments, the present invention provides a
pharmaceutical composition comprising a virus vector of the
invention in a pharmaceutically acceptable carrier and, optionally,
other medicinal agents, pharmaceutical agents, stabilizing agents,
buffers, carriers, adjuvants, diluents, etc. For injection, the
carrier will typically be a liquid. For other methods of
administration, the carrier may be either solid or liquid. For
inhalation administration, the carrier will be respirable, and will
preferably be in solid or liquid particulate form.
[0189] By "pharmaceutically acceptable" it is meant a material that
is not toxic or otherwise undesirable, i.e., the material may be
administered to a subject without causing any undesirable
biological effects.
[0190] One aspect of the present invention is a method of
transferring a nucleotide sequence to a cell in vitro. The virus
vector may be introduced to the cells at the appropriate
multiplicity of infection according to standard transduction
methods appropriate for the particular target cells. Titers of the
virus vector or capsid to administer can vary, depending upon the
target cell type and number, and the particular virus vector or
capsid, and can be determined by those of skill in the art without
undue experimentation. In particular embodiments, at least about
10.sup.3 infectious units, more preferably at least about 10.sup.5
infectious units are introduced to the cell.
[0191] The cell(s) into which the virus vector can be introduced
may be of any type, including but not limited to neural cells
(including cells of the peripheral and central nervous systems, in
particular, brain cells such as neurons, oligodendrocytes, glial
cells, astrocytes), lung cells, cells of the eye (including retinal
cells, retinal pigment epithelium, and corneal cells), epithelial
cells (e.g., gut and respiratory epithelial cells), skeletal muscle
cells (including myoblasts, myotubes and myofibers), diaphragm
muscle cells, dendritic cells, pancreatic cells (including islet
cells), hepatic cells, a cell of the gastrointestinal tract
(including smooth muscle cells, epithelial cells), heart cells
(including cardiomyocytes), bone cells (e.g., bone marrow stem
cells), hematopoietic stem cells, spleen cells, keratinocytes,
fibroblasts, endothelial cells, prostate cells, joint cells
(including, e.g., cartilage, meniscus, synovium and bone marrow),
germ cells, and the like. Alternatively, the cell may be any
progenitor cell. As a further alternative, the cell can be a stem
cell (e.g., neural stem cell, liver stem cell). As still a further
alternative, the cell may be a cancer or tumor cell (cancers and
tumors are described above). Moreover, the cells can be from any
species of origin, as indicated above.
[0192] The virus vectors may be introduced to cells in vitro for
the purpose of administering the modified cell to a subject. In
particular embodiments, the cells have been removed from a subject,
the virus vector is introduced therein, and the cells are then
replaced back into the subject. Methods of removing cells from
subject for treatment ex vivo, followed by introduction back into
the subject are known in the art (see, e.g., U.S. Pat. No.
5,399,346). Alternatively, the recombinant virus vector is
introduced into cells from another subject, into cultured cells, or
into cells from any other suitable source, and the cells are
administered to a subject in need thereof.
[0193] Suitable cells for ex vivo gene therapy are as described
above. Dosages of the cells to administer to a subject will vary
upon the age, condition and species of the subject, the type of
cell, the nucleic acid being expressed by the cell, the mode of
administration, and the like. Typically, at least about 10.sup.2 to
about 10.sup.8 or about 10.sup.3 to about 10.sup.6 cells will be
administered per dose in a pharmaceutically acceptable carrier. In
particular embodiments, the cells transduced with the virus vector
are administered to the subject in an effective amount in
combination with a pharmaceutical carrier.
[0194] In some embodiments, cells that have been transduced with
the virus vector may be administered to elicit an immunogenic
response against the delivered polypeptide (e.g., expressed as a
transgene or in the capsid). Typically, a quantity of cells
expressing an effective amount of the polypeptide in combination
with a pharmaceutically acceptable carrier is administered.
Optionally, the dosage is sufficient to produce a protective immune
response (as defined above). The degree of protection conferred
need not be complete or permanent, as long as the benefits of
administering the immunogenic polypeptide outweigh any
disadvantages thereof.
[0195] A further aspect of the invention is a method of
administering the virus vectors or capsids of the invention to
subjects. In particular embodiments, the method comprises a method
of delivering a nucleic acid of interest to an animal subject, the
method comprising: administering an effective amount of a virus
vector according to the invention to an animal subject.
Administration of the virus vectors of the present invention to a
human subject or an animal in need thereof can be by any means
known in the art. Optionally, the virus vector is delivered in an
effective dose in a pharmaceutically acceptable carrier.
[0196] The virus vectors of the invention can further be
administered to a subject to elicit an immunogenic response (e.g.,
as a vaccine). Typically, vaccines of the present invention
comprise an effective amount of virus in combination with a
pharmaceutically acceptable carrier. Optionally, the dosage is
sufficient to produce a protective immune response (as defined
above). The degree of protection conferred need not be complete or
permanent, as long as the benefits of administering the immunogenic
polypeptide outweigh any disadvantages thereof. Subjects and
immunogens are as described above.
[0197] Dosages of the virus vectors to be administered to a subject
will depend upon the mode of administration, the disease or
condition to be treated, the individual subject's condition, the
particular virus vector, and the nucleic acid to be delivered, and
can be determined in a routine manner. Exemplary doses for
achieving therapeutic effects are virus titers of at least about
10.sup.5, 10.sup.6, 10.sup.7, 10.sup.8, 10.sup.9, 10.sup.10,
10.sup.11, 10.sup.12, 10.sup.3, 10.sup.14, 10.sup.15 transducing
units or more, preferably about 10.sup.7 or 10.sup.8, 10.sup.9,
10.sup.10, 10.sup.11, 10.sup.12, 10.sup.13 or 10.sup.14 transducing
units, yet more preferably about 10.sup.12 transducing units.
[0198] In particular embodiments, more than one administration
(e.g., two, three, four or more administrations) may be employed to
achieve the desired level of gene expression over a period of
various intervals, e.g., daily, weekly, monthly, yearly, etc.
[0199] Exemplary modes of administration include oral, rectal,
transmucosal, topical, intranasal, inhalation (e.g., via an
aerosol), buccal (e.g., sublingual), vaginal, intrathecal,
intraocular, transdermal, in utero (or in ovo), parenteral (e.g.,
intravenous, subcutaneous, intradermal, intramuscular [including
administration to skeletal, diaphragm and/or cardiac muscle],
intradermal, intrapleural, intracerebral, and intraarticular),
topical (e.g., to both skin and mucosal surfaces, including airway
surfaces, and transdermal administration), intro-lymphatic, and the
like, as well as direct tissue or organ injection (e.g., to liver,
skeletal muscle, cardiac muscle, diaphragm muscle or brain).
Administration can also be to a tumor (e.g., in or a near a tumor
or a lymph node). The most suitable route in any given case will
depend on the nature and severity of the condition being treated
and on the nature of the particular vector that is being used.
[0200] In some embodiments, the viral vector is administered
directly to the CNS, e.g., the brain or the spinal cord. Direct
administration can result in high specificity of transduction of
oligodendrocytes, e.g., wherein at least 80%, 85%, 90%, 95% or more
of the transduced cells are oligodendrocytes. Any method known in
the art to administer vectors directly to the CNS can be used. The
vector may be introduced into the spinal cord, brainstem (medulla
oblongata, pons), midbrain (hypothalamus, thalamus, epithalamus,
pituitary gland, substantia nigra, pineal gland), cerebellum,
telencephalon (corpus striatum, cerebrum including the occipital,
temporal, parietal and frontal lobes, cortex, basal ganglia,
hippocampus and amygdala), limbic system, neocortex, corpus
striatum, cerebrum, and inferior colliculus. The vector may also be
administered to different regions of the eye such as the retina,
cornea or optic nerve. The vector may be delivered into the
cerebrospinal fluid (e.g., by lumbar puncture) for more disperse
administration of the vector.
[0201] The delivery vector may be administered to the desired
region(s) of the CNS by any route known in the art, including but
not limited to, intrathecal, intracerebral, intraventricular,
intranasal, intra-aural, intra-ocular (e.g., intra-vitreous,
sub-retinal, anterior chamber) and peri-ocular (e.g., sub-Tenon's
region) delivery or any combination thereof.
[0202] Typically, the viral vector will be administered in a liquid
formulation by direct injection (e.g., stereotactic injection) to
the desired region or compartment in the CNS. In some embodiments,
the vector can be delivered via a reservoir and/or pump. In other
embodiments, the vector may be provided by topical application to
the desired region or by intra-nasal administration of an aerosol
formulation. Administration to the eye or into the ear, may be by
topical application of liquid droplets. As a further alternative,
the vector may be administered as a solid, slow-release
formulation. Controlled release of parvovirus and AAV vectors is
described by international patent publication WO 01/91803.
[0203] In some embodiments where the subject has a compromised
blood-brain barrier (BBB), the viral vector can be delivered
systemically (e.g., intravenously) to the subject, wherein the
vector transduces oligodendrocytes in the area of (e.g., bordering)
the BBB compromise. In certain embodiments, the vector transduces
cells in the compromised area but not cells in uncompromised areas.
Thus, one aspect of the invention relates to a method of delivering
a nucleic acid of interest to an area of the CNS bordering a
compromised blood brain barrier area in a mammalian subject, the
method comprising intravenously administering an effective amount
of the AAV particle of the invention.
[0204] In some embodiments, the compromise in the BBB is due to a
disease or disorder. Examples include, without limitation,
neurodegenerative diseases such as Alzheimer's, Parkinson's
disease, disease, amyotrophic lateral sclerosis, and multiple
sclerosis, epilepsy, CNS tumors, or cerebral infarcts. In other
embodiments, the BBB compromise can be an induced disruption, e.g.,
to promote delivery of agents to the CNS. Temporary BBB compromises
can be induced by, for example, toxic chemicals (such as metrazol,
VP-16, cisplatin, hydroxyurea, fluorouracil, and etoposide),
osmotic agents (such as mannitol and arabinose), biological agents
(such as retinoic acid, phorbol myristate acetate, leukotriene C4,
bradykinin, histamine, RMP-7, and alkylglycerols), or irradiation
(such as ultrasound or electromagnetic radiation).
[0205] Administration to skeletal muscle according to the present
invention includes but is not limited to administration to skeletal
muscles in the limbs (e.g., upper arm, lower arm, upper leg, and/or
lower leg), back, neck, head (e.g., tongue), thorax, abdomen,
pelvis/perineum, and/or digits. Suitable skeletal muscle tissues
include but are not limited to abductor digiti minimi (in the
hand), abductor digiti minimi (in the foot), abductor hallucis,
abductor ossis metatarsi quinti, abductor pollicis brevis, abductor
pollicis longus, adductor brevis, adductor hallucis, adductor
longus, adductor magnus, adductor pollicis, anconeus, anterior
scalene, articularis genus, biceps brachii, biceps femoris,
brachialis, brachioradialis, buccinator, coracobrachialis,
corrugator supercilii, deltoid, depressor anguli oris, depressor
labii inferioris, digastric, dorsal interossei (in the hand),
dorsal interossei (in the foot), extensor carpi radialis brevis,
extensor carpi radialis longus, extensor carpi ulnaris, extensor
digiti minimi, extensor digitorum, extensor digitorum brevis,
extensor digitorum longus, extensor hallucis brevis, extensor
hallucis longus, extensor indicis, extensor pollicis brevis,
extensor pollicis longus, flexor carpi radialis, flexor carpi
ulnaris, flexor digiti minimi brevis (in the hand), flexor digiti
minimi brevis (in the foot), flexor digitorum brevis, flexor
digitorum longus, flexor digitorum profundus, flexor digitorum
superficialis, flexor hallucis brevis, flexor hallucis longus,
flexor pollicis brevis, flexor pollicis longus, frontalis,
gastrocnemius, geniohyoid, gluteus maximus, gluteus medius, gluteus
minimus, gracilis, iliocostalis cervicis, iliocostalis lumborum,
iliocostalis thoracis, illiacus, inferior gemellus, inferior
oblique, inferior rectus, infraspinatus, interspinalis,
intertransversi, lateral pterygoid, lateral rectus, latissimus
dorsi, levator anguli oris, levator labii superioris, levator labii
superioris alaeque nasi, levator palpebrae superioris, levator
scapulae, long rotators, longissimus capitis, longissimus cervicis,
longissimus thoracis, longus capitis, longus colli, lumbricals (in
the hand), lumbricals (in the foot), masseter, medial pterygoid,
medial rectus, middle scalene, muitifidus, mylohyoid, obliquus
capitis inferior, obliquus capitis superior, obturator externus,
obturator internus, occipitalis, omohyoid, opponens digiti minimi,
opponens pollicis, orbicularis oculi, orbicularis oris, palmar
interossei, palmaris brevis, palmaris longus, pectineus, pectoralis
major, pectoralis minor, peroneus brevis, peroneus longus, peroneus
tertius, piriformis, plantar interossei, plantaris, platysma,
popliteus, posterior scalene, pronator quadratus, pronator teres,
psoas major, quadratus femoris, quadratus plantae, rectus capitis
anterior, rectus capitis lateralis, rectus capitis posterior major,
rectus capitis posterior minor, rectus femoris, rhomboid major,
rhomboid minor, risorius, sartorius, scalenus minimus,
semimembranosus, semispinalis capitis, semispinalis cervicis,
semispinalis thoracis, semitendinosus, serratus anterior, short
rotators, soleus, spinalis capitis, spinalis cervicis, spinalis
thoracis, splenius capitis, splenius cervicis, sternocleidomastoid,
sternohyoid, sternothyroid, stylohyoid, subclavius, subscapularis,
superior gemellus, superior oblique, superior rectus, supinator,
supraspinatus, temporalis, tensor fascia lata, teres major, teres
minor, thoracis, thyrohyoid, tibialis anterior, tibialis posterior,
trapezius, triceps brachii, vastus intennedius, vastus lateralis,
vastus medialis, zygomaticus major, and zygomaticus minor and any
other suitable skeletal muscle as known in the art.
[0206] The virus vector can be delivered to skeletal muscle by any
suitable method including without limitation intravenous
administration, intra-arterial administration, intraperitoneal
administration, isolated limb perfusion (of leg and/or arm; see,
e.g. Arruda et al., (2005) Blood 105:3458-3464), and/or direct
intramuscular injection.
[0207] Administration to cardiac muscle includes without limitation
administration to the left atrium, right atrium, left ventricle,
right ventricle and/or septum. The virus vector can be delivered to
cardiac muscle by any method known in the art including, e.g.,
intravenous administration, intra-arterial administration such as
intra-aortic administration, direct cardiac injection (e.g., into
left atrium, right atrium, left ventricle, right ventricle), and/or
coronary artery perfusion.
[0208] Administration to diaphragm muscle can be by any suitable
method including intravenous administration, intra-arterial
administration, and/or intra-peritoneal administration.
[0209] Delivery to any of these tissues can also be achieved by
delivering a depot comprising the virus vector, which can be
implanted into the skeletal, cardiac and/or diaphragm muscle tissue
or the tissue can be contacted with a film or other matrix
comprising the virus vector. Examples of such implantable matrices
or substrates are described in U.S. Pat. No. 7,201,898.
[0210] In particular embodiments, a virus vector according to the
present invention is administered to skeletal muscle, diaphragm
muscle and/or cardiac muscle (e.g., to treat muscular dystrophy or
heart disease [for example, PAD or congestive heart failure]).
[0211] The invention can be used to treat disorders of skeletal,
cardiac and/or diaphragm muscle. Alternatively, the invention can
be practiced to deliver a nucleic acid to skeletal, cardiac and/or
diaphragm muscle, which is used as a platform for production of a
protein product (e.g., an enzyme) or non-translated RNA (e.g.,
RNAi, microRNA, antisense RNA) that normally circulates in the
blood or for systemic delivery to other tissues to treat a disorder
(e.g., a metabolic disorder, such as diabetes (e.g., insulin),
hemophilia (e.g., Factor IX or Factor VIII), or a lysosomal storage
disorder (such as Gaucher's disease [glucocerebrosidase], Pompe
disease [lysosomal acid .alpha.-glucosidase] or Fabry disease
[.alpha.-galactosidase A]) or a glycogen storage disorder (such as
Pompe disease [lysosomal acid .alpha. glucosidase]). Other suitable
proteins for treating metabolic disorders are described above.
[0212] In a representative embodiment, the invention provides a
method of treating muscular dystrophy in a subject in need thereof,
the method comprising: administering an effective amount of a virus
vector of the invention to a mammalian subject, wherein the virus
vector comprises a heterologous nucleic acid effective to treat
muscular dystrophy. In an exemplary embodiment, the method
comprises: administering an effective amount of a virus vector of
the invention to a mammalian subject, wherein the virus vector
comprises a heterologous nucleic acid encoding dystrophin, a
mini-dystrophin, a micro-dystrophin, utrophin, mini-utrophin,
laminin-.alpha.2, mini-agrin, Fukutin-related protein, follistatin,
dominant negative myostatin, .alpha.-sarcoglycan,
.beta.-sarcoglycan, .gamma.-sarcoglycan, .delta.-sarcoglycan,
IGF-1, myostatin pro-peptide, activin type II soluble receptor,
anti-inflammatory polypeptides such as the Ikappa B dominant
mutant, sarcospan, antibodies or antibody fragments against
myostatin or myostatin propeptide, or an inhibitory RNA (e.g.,
antisense RNA, microRNA or RNAi) against myostatin, mir-1, mir-133,
mir-206, mir-208 or an inhibitory RNA (e.g., microRNA, RNAi or
antisense RNA) to induce exon skipping in a defective dystrophin
gene. In particular embodiments, the virus vector can be
administered to skeletal, diaphragm and/or cardiac muscle as
described elsewhere herein.
[0213] The invention further encompasses a method of treating a
metabolic disorder in a subject in need thereof. In representative
embodiments, the method comprises: administering an effective
amount of a virus vector of the invention to skeletal muscle of a
subject, wherein the virus vector comprises, a heterologous nucleic
acid encoding a polypeptide, wherein the metabolic disorder is a
result of a deficiency and/or defect in the polypeptide.
Illustrative metabolic disorders and heterologous nucleic acids
encoding polypeptides are described herein. As a further option,
the heterologous nucleic acid can encode a secreted protein.
The invention can also be practiced to produce inhibitory RNA
(e.g., antisense RNA, microRNA or RNAi) for systemic delivery.
[0214] The invention also provides a method of treating congenital
heart failure in a subject in need thereof, the method comprising
administering an effective amount of a virus vector of the
invention to a mammalian subject, wherein the virus vector
comprises a heterologous nucleic acid effective to treat congenital
heart failure. In representative embodiments, the method comprises
administering an effective amount of a virus vector of the
invention to a mammalian subject, wherein the virus vector
comprises a heterologous nucleic acid encoding a sarcoplasmic
endoreticulum Ca.sup.2+-ATPase (SERCA2a), an angiogenic factor,
phospholamban, PI3 kinase, calsarcan, a .beta.-adrenergic receptor
kinase (.beta.ARK), .beta.ARKct, inhibitor 1 of protein phosphatase
1, Pim-1, PGC-1.alpha., SOD-1, SOD-2, EC-SOD, Kallikrein, HIF,
thymosin-.beta.4, S100A1, parvalbumin, adenylyl cyclase type 6, a
molecule that effects G-protein coupled receptor kinase type 2
knockdown such as a truncated constitutively active bARKet;
phospholamban inhibitory or dominant-negative molecules such as
phospholamban S16E, mir-1, mir-133, mir-206, mir-208.
[0215] Injectables can be prepared in conventional forms, either as
liquid solutions or suspensions, solid forms suitable for solution
or suspension in liquid prior to injection, or as emulsions.
Alternatively, one may administer the virus vector in a local
rather than systemic manner, for example, in a depot or
sustained-release formulation. Further, the virus vector can be
delivered dried to a surgically implantable matrix such as a bone
graft substitute, a suture, a stent, and the like (e.g., as
described in U.S. Pat. No. 7,201,898).
[0216] Pharmaceutical compositions suitable for oral administration
can be presented in discrete units, such as capsules, cachets,
lozenges, or tablets, each containing a predetermined amount of the
composition of this invention; as a powder or granules; as a
solution or a suspension in an aqueous or non-aqueous liquid; or as
an oil-in-water or water-in-oil emulsion. Oral delivery can be
performed by complexing a virus vector of the present invention to
a carrier capable of withstanding degradation by digestive enzymes
in the gut of an animal. Examples of such carriers include plastic
capsules or tablets, as known in the art. Such formulations are
prepared by any suitable method of pharmacy, which includes the
step of bringing into association the composition and a suitable
carrier (which may contain one or more accessory ingredients as
noted above). In general, the pharmaceutical composition according
to embodiments of the present invention are prepared by uniformly
and intimately admixing the composition with a liquid or finely
divided solid carrier, or both, and then, if necessary, shaping the
resulting mixture. For example, a tablet can be prepared by
compressing or molding a powder or granules containing the
composition, optionally with one or more accessory ingredients.
Compressed tablets are prepared by compressing, in a suitable
machine, the composition in a free-flowing form, such as a powder
or granules optionally mixed with a binder, lubricant, inert
diluent, and/or surface active/dispersing agent(s). Molded tablets
are made by molding, in a suitable machine, the powdered compound
moistened with an inert liquid binder.
[0217] Pharmaceutical compositions suitable for buccal
(sub-lingual) administration include lozenges comprising the
composition of this invention in a flavored base, usually sucrose
and acacia or tragacanth; and pastilles comprising the composition
in an inert base such as gelatin and glycerin or sucrose and
acacia.
[0218] Pharmaceutical compositions suitable for parenteral
administration can comprise sterile aqueous and non-aqueous
injection solutions of the composition of this invention, which
preparations are optionally isotonic with the blood of the intended
recipient. These preparations can contain anti-oxidants, buffers,
bacteriostats and solutes, which render the composition isotonic
with the blood of the intended recipient. Aqueous and non-aqueous
sterile suspensions, solutions and emulsions can include suspending
agents and thickening agents. Examples of non-aqueous solvents are
propylene glycol, polyethylene glycol, vegetable oils such as olive
oil, and injectable organic esters such as ethyl oleate. Aqueous
carriers include water, alcoholic/aqueous solutions, emulsions or
suspensions, including saline and buffered media. Parenteral
vehicles include sodium chloride solution, Ringer's dextrose,
dextrose and sodium chloride, lactated Ringer's, or fixed oils.
Intravenous vehicles include fluid and nutrient replenishers,
electrolyte replenishers (such as those based on Ringer's
dextrose), and the like. Preservatives and other additives may also
be present such as, for example, antimicrobials, anti-oxidants,
chelating agents, and inert gases and the like.
[0219] The compositions can be presented in unit/dose or multi-dose
containers, for example, in sealed ampoules and vials, and can be
stored in a freeze-dried (lyophilized) condition requiring only the
addition of the sterile liquid carrier, for example, saline or
water-for-injection immediately prior to use.
[0220] Extemporaneous injection solutions and suspensions can be
prepared from sterile powders, granules and tablets of the kind
previously described. For example, an injectable, stable, sterile
composition of this invention in a unit dosage form in a sealed
container can be provided. The composition can be provided in the
form of a lyophilizate, which can be reconstituted with a suitable
pharmaceutically acceptable carrier to form a liquid composition
suitable for injection into a subject. The unit dosage form can be
from about 1 .mu.g to about 10 grams of the composition of this
invention. When the composition is substantially water-insoluble, a
sufficient amount of emulsifying agent, which is physiologically
acceptable, can be included in sufficient quantity to emulsify the
composition in an aqueous carrier. One such useful emulsifying
agent is phosphatidyl choline.
[0221] Pharmaceutical compositions suitable for rectal
administration can be presented as unit dose suppositories. These
can be prepared by admixing the composition with one or more
conventional solid carriers, such as for example, cocoa butter and
then shaping the resulting mixture.
[0222] Pharmaceutical compositions of this invention suitable for
topical application to the skin can take the form of an ointment,
cream, lotion, paste, gel, spray, aerosol, and/or oil. Carriers
that can be used include, but are not limited to, petroleum jelly,
lanoline, polyethylene glycols, alcohols, transdermal enhancers,
and combinations of two or more thereof. In some embodiments, for
example, topical delivery can be performed by mixing a
pharmaceutical composition of the present invention with a
lipophilic reagent (e.g., DMSO) that is capable of passing into the
skin.
[0223] Pharmaceutical compositions suitable for transdermal
administration can be in the form of discrete patches adapted to
remain in intimate contact with the epidermis of the subject for a
prolonged period of time. Compositions suitable for transdermal
administration can also be delivered by iontophoresis (see, for
example, Pharm. Res. 3:318 (1986)) and typically take the form of
an optionally buffered aqueous solution of the composition of this
invention. Suitable formulations can comprise citrate or bis\tris
buffer (pH 6) or ethanol/water and can contain from 0.1 to 0.2M
active ingredient.
[0224] The virus vectors disclosed herein may be administered to
the lungs of a subject by any suitable means, for example, by
administering an aerosol suspension of respirable particles
comprised of the virus vectors, which the subject inhales. The
respirable particles may be liquid or solid. Aerosols of liquid
particles comprising the virus vectors may be produced by any
suitable means, such as with a pressure-driven aerosol nebulizer or
an ultrasonic nebulizer, as is known to those of skill in the art.
See, e.g., U.S. Pat. No. 4,501,729. Aerosols of solid particles
comprising the virus vectors may likewise be produced with any
solid particulate medicament aerosol generator, by techniques known
in the pharmaceutical art.
IV. Use of the AAV Capsid to Target Peripheral Tissues
[0225] The AAV capsids and vectors of the present invention have
been demonstrated to be fully or nearly fully detargeted for
peripheral organs and tissues (see FIG. 4). This detargeting makes
the vectors ideal as a "blank" vector that can be altered to
produce the desired tropic profile, e.g., to target specific organs
and tissues and/or detarget other organs and tissues. Thus, one
aspect of the invention relates to a method of preparing an AAV
capsid having a tropism profile of interest, the method comprising
modifying the AAV capsid of the present invention to insert an
amino acid sequence providing the tropism profile of interest. In
some embodiments, the tropism profile of interest is enhanced
selectivity for a tissue selected from skeletal muscle, cardiac
muscle, diaphragm, kidney, liver, pancreas, spleen,
gastrointestinal tract, lung, joint tissue, tongue, ovary, testis,
a germ cell, a cancer cell, or a combination thereof and/or reduced
selectivity for a tissue selected from liver, ovary, testis, a germ
cell, or a combination thereof.
[0226] Examples of specific targeting and detargeting sequences are
known in the art. One example is the molecular basis for
preferential liver tropism, which has been mapped, in the case of
AAV2 and AAV6, to a continuous basic footprint that appears to be
involved in the interaction of either serotype with heparin.
Specifically, it has previously been demonstrated that a single
lysine residue on AAV6 (K531) dictates heparin binding ability and
consequently, liver tropism. In corollary, substitutional
mutagenesis of the corresponding glutamate/aspartate residue on
other serotypes with a lysine residue confers heparin binding,
possibly by forming a minimum continuous basic footprint on the
capsid surface. Another example is the capsid mutants comprising
alterations in the three-fold axis loop 4 as disclosed in
International Publication No. WO 2012/093784, incorporated herein
by reference in its entirety. These mutants exhibit one or more
properties including (i) reduced transduction of liver, (ii)
enhanced movement across endothelial cells, (iii) systemic
transduction; (iv) enhanced transduction of muscle tissue (e.g.,
skeletal muscle, cardiac muscle and/or diaphragm muscle), and/or
(v) reduced transduction of brain tissues (e.g., neurons). Other
tropic sequences are described in Li et al., (2012) J. Virol.
86:7752-7759; Pulicherla et al., (2011) Mol. Ther, 19:1070-1078;
Bowles et al., (2012) Mol. Ther. 20:443-455; Asokan et al., (2012)
Mol. Ther. 20:699-708; and Asokan et al., (2010) Nature Biotechnol.
28:79-82; each incorporated by reference in its entirety.
[0227] In some embodiments, the AAV capsid of the present invention
can be modified through DNA scrambling and/or directed evolution to
identify modified capsids having the desired tropism profile.
Techniques for DNA scrambling and directed evolution of AAV capsids
are described in International Publication No. WO 2009/137006,
incorporated herein by reference in its entirety.
[0228] Having described the present invention, the same will be
explained in greater detail in the following examples, which are
included herein for illustration purposes only, and which are not
intended to be limiting to the invention.
Example 1
Discovery and Characterization of the BNP61 AAV Clone
[0229] A mutant DNA shuffled AAV capsid library was injected
intravenously into a rat model of Parkinson's disease and 3 days
later cells were dissociated from the caudate nucleus. Using PCR
rescue, a single clone emerged (BNP61). Additional shuffling and
selection yielded the same clone. As seen in FIG. 1, this clone is
a chimera of several AAV serotypes.
[0230] Direct infusion of this BNP61 clone into the rat brain
produced a surprising cellular transduction pattern. To date,
almost all diverse AAV serotypes and chimeras exhibit a >95%
tropism for neurons, when gene expression is driven by a
constitutive promoter. In marked contrast, BNP61 exhibited a
>95% tropism for oligodendrocytes with no evidence of astrocyte
or microglial transduction and minimal neuronal transduction (FIGS.
2A-2C). FIG. 2A shows GFP positive oligodendrocytes in the rat
caudate 1 week after the infusion of BNP61-CBh-GFP vectors. Note
that there are no GP positive neurons. FIG. 2B shows a higher
magnification that reflects clear oligodendrocyte morphology, and
FIG. 2C shows that none of the GFP positive cells colocalize with
the cellular marker for astrocytes, GFAP (red).
[0231] Also, in primary oligodendrocyte cultures, BNP61 transduces
the oligodendrocytes but not the underlying bed of astrocytes
(FIGS. 3A-3B). Mixed glial cultures at day 10 were transduced with
BNP61-GFP at a MOI of 100 viral particles and images were taken 72
hr later. FIG. 3A is a light image of the glial culture. FIG. 3B is
an image of fluorescent AAV GFP-positive cells taken from the same
frame as FIG. 3A. The underlayer bed of astrocytes was not
transduced and nearly all GFP-expressing cells appear
morphologically as oligodendrocytes. Arrows indicate near-focused
oligodendrocytes showing processes consistent with oligodendrocyte
progenitor culture morphology. Thus, the BNP61 AAV vector exhibits
properties that are distinctly different from other AAV vectors
characterized to date.
[0232] In order to assess peripheral biodistribution, mice received
intravenous administration of BNP61-CBh-GFP vectors and
subsequently the peripheral organs were harvested. All mice were
injected as adults with 5.times.10.sup.10 vg except as indicated.
Neonatal injections were with 2.5.times.10.sup.10 vg. Adults were
sacrificed 10 days post-injection, and neonates were sacrificed at
4 weeks post-injection. * indicates samples not tested. In the
legend, the number of animals for each vector is shown in
parentheses. Error bars are S.E.M. As seen in FIG. 4, BNP61 did not
accumulate in any of the peripheral organs.
Example 2
BNP61 Crosses the Compromised Blood-Brain Barrier
[0233] After the first selection round, the BNP61 clone was
packaged with GFP and recombinant virus was produced. Then, 2 weeks
post-6-OHDA treatment the recombinant virus was administered
intravenously at a dose of 8.times.10.sup.11 vector genomes/kg. One
month later, the rats were sacrificed and the brains sectioned. In
these 2 week post treatment rats, substantial gene expression was
found in oligodendrocytes and some neurons within the 6-OHDA
treated striatum (FIG. 5), while no gene expression was found in
the contralateral striatum, the injector tract in the cortex or
distal brain structures. Note the lack of NeuN co-localization with
the many oligodendrocytes that surround the lone NeuN positive
neuron (FIG. 5). Thus, it appears that after intravenous
administration this novel AAV clone exhibits the ability to cross
the 6-OHDA compromised blood-brain barrier, but not the intact
blood-brain barrier.
Example 3
Generation of Additional Oligodendrocyte Targeted Clones
[0234] Using the same AAV DNA capsid shuffling and in vivo directed
evolution process described in Example 1, two additional
oligodendrocyte-targeted clones were identified. BNP62 and BNP63.
As shown in FIG. 1 and similar to BNP61, these clones are chimeras
of several AAV serotypes. The amino acid sequence identity between
the three clones is shown in Table 2. All three clones are greater
than 95% identical to each other at the amino acid level.
TABLE-US-00004 TABLE 2 Sequence identity between clones BNP61 BNP62
BNP63 BNP61 100% 96% 97% BNP62 96% 100% 96% BNP63 97% 96% 100%
[0235] FIG. 6 shows that the BNP63 clone transduces
oligodendrocytes in the rat piriform cortex. The BNP63 transduced
cells (green) do not co-localize with a cellular marker for
astrocytes (GFAP, red). Also, the transduced cells exhibit a clear
oligodendrocyte morphology.
[0236] The foregoing is illustrative of the present invention, and
is not to be construed as limiting thereof. The invention is
defined by the following claims, with equivalents of the claims to
be included therein.
Sequence CWU 1
1
412214DNAArtificialChimeric AAV capsid nucleotide sequence
1atggctgccg atggttatct tccagattgg ctcgaggaca ctctctctga aggaataaga
60cagtggtgga agctcaaacc tggcccacca ccaccaaagc ccgcagagcg gcataaggac
120gacagcaggg gtcttgtgct tcctgggtac aagtacctcg gacccttcaa
cggactcgac 180aagggagagc cggtcaacga ggcagacgcc gcggccctcg
agcacgacaa agcctacgac 240cggcagctcg acagcggaga caacccgtac
ctcaagtaca accacgccga cgcggagttt 300caggagcgcc ttaaagaaga
tacgtctttt gggggcaacc tcgggcgagc agtcttccag 360gccaaaaaga
ggcttcttga acctcttggt ctggttgagg aagcggctaa gacggctcct
420ggaaagaaga ggcctgtaga gcagtctcct caggaaccgg actcctcctc
gggcatcggc 480aagacaggcc agcagcccgc taaaaagaga ctcaatttcg
gtcagactgg cgacacagag 540tcagtcccag accctcaacc aatcggagaa
cctcccgcag ccccctcagg tgtgggatct 600cttacaatgg cttcaggtgg
tggcgcacca gtggcagaca ataacgaagg tgccgatgga 660gtgggtagtt
cctcgggaaa ttggcattgc gattcccaat ggctggggga cagagtcatc
720accaccagca cccgaacctg ggccctgccc acctacaaca atcacctcta
caagcaaatc 780tccaacggga catcgggagg agccaccaac gacaacacct
acttcggcta cagcaccccc 840tgggggtatt ttgactttaa cagattccac
tgccactttt caccacgtga ctggcagcga 900ctcatcaaca acaactgggg
attccggccc aagagactca gcttcaagct cttcaacatc 960caggtcaagg
aggtcacgca gaatgaaggc accaagacca tcgccaataa ccttaccagc
1020acggtccagg tcttcacgga ctcggagtac cagctgccgt acgttctcgg
ctctgcccac 1080cagggctgcc tgcctccgtt cccggcggac gtgttcatga
ttccccagta cggctaccta 1140acactcaaca acggtagtca ggccgtggga
cgctcctcct tctactgcct ggaatacttt 1200ccttcgcaga tgctgagaac
cggcaacaac ttccagttta cttacacctt cgaggacgtg 1260cctttccaca
gcagctacgc ccacagccag agcttggacc ggctgatgaa tcctctgatt
1320gaccagtacc tgtactactt gtctcggact caaacaacag gaggcacggc
aaatacgcag 1380actctgggct tcagccaagg tgggcctaat acaatggcca
atcaggcaaa gaactggctg 1440ccaggaccct gttaccgcca acaacgcgtc
tcaacgacaa ccgggcaaaa caacaatagc 1500aactttgcct ggactgctgg
gaccaaatac catctgaatg gaagaaattc attggctaat 1560cctggcatcg
ctatggcaac acacaaagac gacaaggagc gtttttttcc cagtaacggg
1620atcctgattt ttggcaaaca aaatgctgcc agagacaatg cggattacag
cgatgtcatg 1680ctcaccagcg aggaagaaat caaaaccact aaccctgtgg
ctacagagga atacggtatc 1740gtggcagata acttgcagca gcaaaacacg
gctcctcaaa ttggaactgt caacagccag 1800ggggccttac ccggtatggt
ttggcagaac cgggacgtgt acctgcaggg tcccatctgg 1860gccaagattc
ctcacacgga cggcaacttc cacccgtctc cgctgatggg cggctttggc
1920ctgaaacatc ctccgcctca gatcctgatc aagaacacgc ctgtacctgc
ggatcctccg 1980accaccttca accagtcaaa gctgaactct ttcatcacgc
aatacagcac cggacaggtc 2040agcgtggaaa ttgaatggga gctgcagaag
gaaaacagca agcgctggaa ccccgagatc 2100cagtacacct ccaactacta
caaatctaca agtgtggact ttgctgttaa tacagaaggc 2160gtgtactctg
aaccccaccc cattggcacc cgttacctca cccgtcccct gtaa
22142737PRTArtificialChimeric AAV capsid amino acid sequence 2Met
Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Thr Leu Ser 1 5 10
15 Glu Gly Ile Arg Gln Trp Trp Lys Leu Lys Pro Gly Pro Pro Pro Pro
20 25 30 Lys Pro Ala Glu Arg His Lys Asp Asp Ser Arg Gly Leu Val
Leu Pro 35 40 45 Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp
Lys Gly Glu Pro 50 55 60 Val Asn Glu Ala Asp Ala Ala Ala Leu Glu
His Asp Lys Ala Tyr Asp 65 70 75 80 Arg Gln Leu Asp Ser Gly Asp Asn
Pro Tyr Leu Lys Tyr Asn His Ala 85 90 95 Asp Ala Glu Phe Gln Glu
Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly 100 105 110 Asn Leu Gly Arg
Ala Val Phe Gln Ala Lys Lys Arg Leu Leu Glu Pro 115 120 125 Leu Gly
Leu Val Glu Glu Ala Ala Lys Thr Ala Pro Gly Lys Lys Arg 130 135 140
Pro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser Ser Gly Ile Gly 145
150 155 160 Lys Thr Gly Gln Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly
Gln Thr 165 170 175 Gly Asp Thr Glu Ser Val Pro Asp Pro Gln Pro Ile
Gly Glu Pro Pro 180 185 190 Ala Ala Pro Ser Gly Val Gly Ser Leu Thr
Met Ala Ser Gly Gly Gly 195 200 205 Ala Pro Val Ala Asp Asn Asn Glu
Gly Ala Asp Gly Val Gly Ser Ser 210 215 220 Ser Gly Asn Trp His Cys
Asp Ser Gln Trp Leu Gly Asp Arg Val Ile 225 230 235 240 Thr Thr Ser
Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu 245 250 255 Tyr
Lys Gln Ile Ser Asn Gly Thr Ser Gly Gly Ala Thr Asn Asp Asn 260 265
270 Thr Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg
275 280 285 Phe His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile
Asn Asn 290 295 300 Asn Trp Gly Phe Arg Pro Lys Arg Leu Ser Phe Lys
Leu Phe Asn Ile 305 310 315 320 Gln Val Lys Glu Val Thr Gln Asn Glu
Gly Thr Lys Thr Ile Ala Asn 325 330 335 Asn Leu Thr Ser Thr Val Gln
Val Phe Thr Asp Ser Glu Tyr Gln Leu 340 345 350 Pro Tyr Val Leu Gly
Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro 355 360 365 Ala Asp Val
Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn 370 375 380 Gly
Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe 385 390
395 400 Pro Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Gln Phe Thr Tyr
Thr 405 410 415 Phe Glu Asp Val Pro Phe His Ser Ser Tyr Ala His Ser
Gln Ser Leu 420 425 430 Asp Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr
Leu Tyr Tyr Leu Ser 435 440 445 Arg Thr Gln Thr Thr Gly Gly Thr Ala
Asn Thr Gln Thr Leu Gly Phe 450 455 460 Ser Gln Gly Gly Pro Asn Thr
Met Ala Asn Gln Ala Lys Asn Trp Leu 465 470 475 480 Pro Gly Pro Cys
Tyr Arg Gln Gln Arg Val Ser Thr Thr Thr Gly Gln 485 490 495 Asn Asn
Asn Ser Asn Phe Ala Trp Thr Ala Gly Thr Lys Tyr His Leu 500 505 510
Asn Gly Arg Asn Ser Leu Ala Asn Pro Gly Ile Ala Met Ala Thr His 515
520 525 Lys Asp Asp Lys Glu Arg Phe Phe Pro Ser Asn Gly Ile Leu Ile
Phe 530 535 540 Gly Lys Gln Asn Ala Ala Arg Asp Asn Ala Asp Tyr Ser
Asp Val Met 545 550 555 560 Leu Thr Ser Glu Glu Glu Ile Lys Thr Thr
Asn Pro Val Ala Thr Glu 565 570 575 Glu Tyr Gly Ile Val Ala Asp Asn
Leu Gln Gln Gln Asn Thr Ala Pro 580 585 590 Gln Ile Gly Thr Val Asn
Ser Gln Gly Ala Leu Pro Gly Met Val Trp 595 600 605 Gln Asn Arg Asp
Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro 610 615 620 His Thr
Asp Gly Asn Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly 625 630 635
640 Leu Lys His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro
645 650 655 Ala Asp Pro Pro Thr Thr Phe Asn Gln Ser Lys Leu Asn Ser
Phe Ile 660 665 670 Thr Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile
Glu Trp Glu Leu 675 680 685 Gln Lys Glu Asn Ser Lys Arg Trp Asn Pro
Glu Ile Gln Tyr Thr Ser 690 695 700 Asn Tyr Tyr Lys Ser Thr Ser Val
Asp Phe Ala Val Asn Thr Glu Gly 705 710 715 720 Val Tyr Ser Glu Pro
His Pro Ile Gly Thr Arg Tyr Leu Thr Arg Pro 725 730 735 Leu
3736PRTArtificialChimeric AAV capsid amino acid sequence 3Met Ala
Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser 1 5 10 15
Glu Gly Ile Arg Glu Trp Trp Asp Leu Lys Pro Gly Ala Pro Lys Pro 20
25 30 Lys Ala Asn Gln Gln Lys Gln Asp Asp Gly Arg Gly Leu Val Leu
Pro 35 40 45 Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys
Gly Glu Pro 50 55 60 Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His
Asp Lys Ala Tyr Asp 65 70 75 80 Gln Gln Leu Lys Ala Gly Asp Asn Pro
Tyr Leu Arg Tyr Asn His Ala 85 90 95 Asp Ala Glu Phe Gln Glu Arg
Leu Gln Glu Asp Thr Ser Phe Gly Gly 100 105 110 Asn Leu Gly Arg Ala
Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro 115 120 125 Leu Gly Leu
Val Glu Glu Gly Ala Lys Thr Ala Pro Gly Lys Lys Arg 130 135 140 Pro
Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser Ser Gly Ile Gly 145 150
155 160 Lys Thr Gly Gln Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln
Thr 165 170 175 Gly Asp Thr Glu Ser Val Pro Asp Pro Gln Pro Ile Gly
Glu Pro Pro 180 185 190 Ala Ala Pro Ser Gly Val Gly Ser Leu Thr Met
Ala Ser Gly Gly Gly 195 200 205 Ala Pro Val Ala Asp Asn Asn Glu Gly
Ala Asp Gly Val Gly Ser Ser 210 215 220 Ser Gly Asn Trp His Cys Asp
Ser Gln Trp Leu Gly Asp Arg Val Ile 225 230 235 240 Thr Thr Ser Thr
Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu 245 250 255 Tyr Lys
Gln Ile Ser Ser Ala Ser Thr Gly Ala Ser Asn Asp Asn His 260 265 270
Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe 275
280 285 His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn
Asn 290 295 300 Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe
Asn Ile Gln 305 310 315 320 Val Lys Glu Val Thr Asp Asn Asn Gly Val
Lys Thr Ile Ala Asn Asn 325 330 335 Leu Thr Ser Thr Val Gln Val Phe
Thr Asp Ser Asp Tyr Gln Leu Pro 340 345 350 Tyr Val Leu Gly Ser Ala
His Gln Gly Cys Leu Pro Pro Phe Pro Ala 355 360 365 Asp Val Phe Met
Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly 370 375 380 Ser Gln
Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro 385 390 395
400 Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Gln Phe Thr Tyr Thr Phe
405 410 415 Glu Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser
Leu Asp 420 425 430 Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr
Tyr Leu Ser Arg 435 440 445 Thr Gln Thr Thr Gly Gly Thr Ala Asn Thr
Gln Thr Leu Gly Phe Ser 450 455 460 Gln Gly Gly Pro Asn Thr Met Ala
Asn Gln Ala Lys Asn Trp Leu Pro 465 470 475 480 Gly Pro Cys Tyr Arg
Gln Gln Arg Val Ser Thr Thr Thr Gly Gln Asn 485 490 495 Asn Asn Ser
Asn Phe Ala Trp Thr Ala Gly Thr Lys Tyr His Leu Asn 500 505 510 Gly
Arg Asn Ser Leu Ala Asn Pro Gly Ile Ala Met Ala Thr His Lys 515 520
525 Asp Asp Lys Glu Arg Phe Phe Pro Ser Asn Gly Ile Leu Ile Phe Gly
530 535 540 Lys Gln Asn Ala Ala Arg Asp Asn Ala Asp Tyr Ser Asp Val
Met Leu 545 550 555 560 Thr Ser Glu Glu Glu Ile Lys Thr Thr Asn Pro
Val Ala Thr Glu Glu 565 570 575 Tyr Gly Ile Val Ala Asp Asn Leu Gln
Gln Gln Asn Thr Ala Pro Gln 580 585 590 Ile Gly Thr Val Asn Ser Gln
Gly Ala Leu Pro Gly Met Val Trp Gln 595 600 605 Asn Arg Asp Val Tyr
Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His 610 615 620 Thr Asp Gly
Asn Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Leu 625 630 635 640
Lys His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala 645
650 655 Asp Pro Pro Thr Thr Phe Asn Gln Ser Lys Leu Asn Ser Phe Ile
Thr 660 665 670 Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp
Glu Leu Gln 675 680 685 Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile
Gln Tyr Thr Ser Asn 690 695 700 Tyr Tyr Lys Ser Thr Ser Val Asp Phe
Ala Val Asn Thr Glu Gly Val 705 710 715 720 Tyr Ser Glu Pro His Pro
Ile Gly Thr Arg Tyr Leu Thr Arg Pro Leu 725 730 735
4736PRTArtificialChimeric AAV capsid amino acid sequence 4Met Ala
Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Thr Leu Ser 1 5 10 15
Glu Gly Ile Arg Gln Trp Trp Lys Leu Lys Pro Gly Pro Pro Pro Pro 20
25 30 Lys Pro Ala Glu Arg His Lys Asp Asp Ser Arg Gly Leu Val Leu
Pro 35 40 45 Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys
Gly Glu Pro 50 55 60 Val Asn Glu Ala Asp Ala Ala Ala Leu Glu His
Asp Lys Ala Tyr Asp 65 70 75 80 Arg Gln Leu Asp Ser Gly Asp Asn Pro
Tyr Leu Lys Tyr Asn His Ala 85 90 95 Asp Ala Glu Phe Gln Glu Arg
Leu Gln Gly Asp Thr Ser Phe Gly Gly 100 105 110 Asn Leu Gly Arg Ala
Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro 115 120 125 Leu Gly Leu
Val Glu Glu Gly Ala Lys Thr Ala Pro Gly Lys Lys Arg 130 135 140 Pro
Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser Ser Gly Ile Gly 145 150
155 160 Glu Thr Gly Gln Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln
Thr 165 170 175 Gly Asp Ser Glu Ser Val Pro Asp Pro Gln Pro Leu Gly
Glu Pro Pro 180 185 190 Ala Thr Pro Ala Ala Val Gly Pro Thr Thr Met
Ala Ser Gly Gly Gly 195 200 205 Ala Pro Met Ala Asp Asn Asn Glu Gly
Ala Asp Gly Val Gly Ser Ser 210 215 220 Ser Gly Asn Trp His Cys Asp
Ser Gln Trp Leu Gly Asp Arg Val Ile 225 230 235 240 Thr Thr Ser Thr
Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu 245 250 255 Tyr Lys
Gln Ile Ser Ser Ala Ser Thr Gly Ala Ser Asn Asp Asn His 260 265 270
Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe 275
280 285 His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn
Asn 290 295 300 Trp Gly Phe Arg Pro Lys Arg Leu Ser Phe Lys Leu Phe
Asn Ile Gln 305 310 315 320 Val Lys Glu Val Thr Asp Asn Asn Gly Val
Lys Thr Ile Ala Asn Asn 325 330 335 Leu Thr Ser Thr Val Gln Val Phe
Thr Asp Ser Glu Tyr Gln Leu Pro 340 345 350 Tyr Val Leu Gly Ser Ala
His Gln Gly Cys Leu Pro Pro Phe Pro Ala 355 360 365 Asp Val Phe Met
Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly 370 375 380 Ser Gln
Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro 385 390 395
400 Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr Phe
405 410 415 Glu Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser
Leu Asp 420 425 430 Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr
Tyr Leu Ser Arg 435 440 445 Thr Gln Thr Thr Gly Gly Thr Ala Asn Thr
Gln Thr Leu Gly Phe Ser 450 455
460 Gln Gly Gly Pro Asn Thr Met Ala Asn Gln Ala Lys Asn Trp Leu Pro
465 470 475 480 Gly Pro Cys Tyr Arg Gln Gln Arg Val Ser Thr Thr Thr
Gly Gln Asn 485 490 495 Asn Asn Ser Asn Phe Ala Trp Thr Ala Gly Thr
Lys Tyr His Leu Asn 500 505 510 Gly Arg Asn Ser Leu Ala Asn Pro Gly
Ile Ala Met Ala Thr His Lys 515 520 525 Asp Asp Lys Glu Arg Phe Phe
Pro Ser Asn Gly Ile Leu Ile Phe Gly 530 535 540 Lys Gln Asn Ala Ala
Arg Asp Asn Ala Asp Tyr Ser Asp Val Met Leu 545 550 555 560 Thr Ser
Glu Glu Glu Ile Lys Thr Thr Asn Pro Val Ala Thr Glu Glu 565 570 575
Tyr Gly Ile Val Ala Asp Asn Leu Gln Gln Gln Asn Thr Ala Pro Gln 580
585 590 Ile Gly Thr Val Asn Ser Gln Gly Ala Leu Pro Gly Met Val Trp
Gln 595 600 605 Asn Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys
Ile Pro His 610 615 620 Thr Asp Gly Asn Phe His Pro Ser Pro Leu Met
Gly Gly Phe Gly Leu 625 630 635 640 Lys His Pro Pro Pro Gln Ile Leu
Ile Lys Asn Thr Pro Val Pro Ala 645 650 655 Asp Pro Pro Thr Thr Phe
Asn Gln Ser Lys Leu Asn Ser Phe Ile Thr 660 665 670 Gln Tyr Ser Thr
Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln 675 680 685 Lys Glu
Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Ser Asn 690 695 700
Tyr Tyr Lys Ser Thr Ser Val Asp Phe Ala Val Asn Thr Glu Gly Val 705
710 715 720 Tyr Ser Glu Pro His Pro Ile Gly Thr Arg Tyr Leu Thr Arg
Pro Leu 725 730 735
* * * * *
References