U.S. patent application number 17/542150 was filed with the patent office on 2022-09-15 for generation of neurons by reprogramming of oligodendrocytes and oligodendrocyte precursor cells.
The applicant listed for this patent is THE UNIVERSITY OF NORTH CAROLINA AT CHAPEL HILL. Invention is credited to Thomas McCOWN, Marc WEINBERG.
Application Number | 20220288141 17/542150 |
Document ID | / |
Family ID | 1000006351673 |
Filed Date | 2022-09-15 |
United States Patent
Application |
20220288141 |
Kind Code |
A1 |
McCOWN; Thomas ; et
al. |
September 15, 2022 |
GENERATION OF NEURONS BY REPROGRAMMING OF OLIGODENDROCYTES AND
OLIGODENDROCYTE PRECURSOR CELLS
Abstract
The invention relates to products and methods for
transdifferentiating oligodendrocytes and/or oligodendrocyte
precursor cells to neurons. The invention further relates to
methods of treating central nervous system disorders and
conditions.
Inventors: |
McCOWN; Thomas; (Carrboro,
NC) ; WEINBERG; Marc; (Carrboro, NC) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE UNIVERSITY OF NORTH CAROLINA AT CHAPEL HILL |
CHAPEL HILL |
NC |
US |
|
|
Family ID: |
1000006351673 |
Appl. No.: |
17/542150 |
Filed: |
December 3, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16330663 |
Mar 5, 2019 |
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PCT/US2017/050242 |
Sep 6, 2017 |
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17542150 |
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62383802 |
Sep 6, 2016 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2750/14143
20130101; A61K 35/76 20130101; A61P 25/14 20180101; A61K 45/06
20130101; A61P 25/16 20180101; C12N 15/113 20130101; C12N 5/0622
20130101; C12N 2310/111 20130101; C12N 15/86 20130101; A61P 25/28
20180101; A61P 25/00 20180101; C12N 2310/141 20130101; C12N 2310/11
20130101; A61K 31/7105 20130101; C12N 2310/14 20130101; C12N
2330/51 20130101; C12N 2750/14141 20130101; A61K 31/713 20130101;
A61K 9/0019 20130101; C12N 5/0619 20130101; C12N 2310/122
20130101 |
International
Class: |
A61K 35/76 20060101
A61K035/76; A61K 31/7105 20060101 A61K031/7105; C12N 15/113
20060101 C12N015/113; A61P 25/00 20060101 A61P025/00; A61P 25/14
20060101 A61P025/14; A61P 25/16 20060101 A61P025/16; A61P 25/28
20060101 A61P025/28; A61K 45/06 20060101 A61K045/06; A61K 31/713
20060101 A61K031/713; A61K 9/00 20060101 A61K009/00; C12N 5/0793
20060101 C12N005/0793; C12N 5/079 20060101 C12N005/079; C12N 15/86
20060101 C12N015/86 |
Goverment Interests
STATEMENT OF GOVERNMENT SUPPORT
[0002] This invention was made with government support under Grant
No. NS082289 awarded by National Institutes of Health. The
government has certain rights to this invention.
Claims
1. A viral particle comprising an expression cassette comprising a
promoter active in both oligodendrocytes and neurons operably
linked to a polynucleotide encoding an interfering RNA that binds
to a polynucleotide encoding a mammalian polypyrimidine tract
binding protein 1 (PTBP1) and inhibits PTBP1 expression: wherein
the interfering PIA comprises the nucleotide sequence of SEQ ID NO:
3 and the complement thereof or a nucleotide sequence at least 90%
identical thereto or SEQ ID NO: 4 and the complement thereof or a
nucleotide sequence at least 90% thereto; and wherein the virus
particle has a tropism for oligodendrocytes and/or oligodendrocyte
precursor cells.
2. (canceled)
3. The viral particle of claim 1, wherein the viral particle is an
adeno-associated virus (AAV) vector.
4-5. (canceled)
6. The viral particle of claim 1, wherein the interfering RNA is a
shRNA, a siRNA, and/or a miRNA.
7-11. (canceled)
12. A composition comprising the viral particle of claim 1 and a
carrier.
13. A pharmaceutical composition comprising the viral particle of
claim 1 and a pharmaceutically acceptable carrier.
14. A method of attenuating expression of PTBP1 in a cell,
comprising contacting the cell with the viral particle of claim 1,
wherein the expression of PTBP1 is attenuated.
15-17. (canceled)
18. A method of transdifferentiating an oligodendrocyte or a
oligodendrocyte precursor cell to a neuron, comprising contacting
the oligodendrocyte or oligodendrocyte precursor cell with the
viral particle of claim 1, thereby transdifferentiating the
oligodendrocyte or oligodendrocyte precursor cell to a neuron.
19. The method of claim 18, wherein the oligodendrocyte or
oligodendrocyte precursor cell is in vitro or ex vivo.
20. The method of claim 18, wherein the oligodendrocyte or
oligodendrocyte precursor cell is in a mammalian subject.
21. A method of increasing the number of neurons in the brain of a
mammalian subject, comprising delivering to the brain the viral
particle of claim 1, thereby increasing the number of neurons in
the brain of the mammalian subject relative to the number of
neurons prior to the delivery.
22. A method of transdifferentiating an oligodendrocyte or an
oligodendrocyte precursor cell to a neuron in the brain of a
mammalian subject, comprising delivering to the brain the viral
particle of claim 1, thereby transdifferentiating an
oligodendrocyte or an oligodendrocyte precursor cell to a neuron in
the brain of the mammalian subject.
23.-25. (canceled)
26. A method of treating a central nervous system disorder or
condition responsive to an increase in the number of neurons in a
mammalian subject in need thereof, the method comprising delivering
to the brain the viral particle of claim 1, thereby treating the
central nervous system disorder or condition.
27. The method of claim 26, wherein the disorder or condition is a
neurodegenerative disorder.
28. The method of claim 27, wherein the neurodegenerative disorder
is Parkinson's disease, Alzheimer's disease, Huntington's chorea,
and/or amyotrophic lateral sclerosis.
29. The method of claim 26, wherein the disorder or condition is a
traumatic brain injury and/or spinal cord injury and/or stroke.
30. The method of claim 26, wherein the disorder or condition is
aging.
31. The method of claim 26, further comprising delivering to the
oligodendrocyte or oligodendrocyte precursor cell or the brain a
differentiation factor.
32. The method of claim 31, wherein the differentiation factor is
selected from the group consisting of NeuroD1, Asc11, Brn2a, Myt11,
SOX2, and any combination thereof.
33. The method of claim 26, further comprising delivering to the
oligodendrocyte or oligodendrocyte precursor cell or the brain a
neurotrophic factor.
34. (canceled)
35. The method of claim 26, further comprising delivering to the
oligodendrocyte or oligodendrocyte precursor cell or the brain an
inhibitor of expression of a REI silencing transcription factor
complex.
36. (canceled)
Description
STATEMENT OF PRIORITY
[0001] This application is a continuation of and claims priority to
U.S. patent Application Ser. No. 16/330,663, filed Mar. 5, 2019,
which is a 35 USC. .sctn. 371 national phase application of PCT
Application PCT/US2017/050242 filed Sep. 6, 2017, which claims the
benefit of U.S. Provisional Application Ser. No. 62/383,802, filed
Sep. 6, 2016, the entire contents of each of which are incorporated
by reference herein in its entirety.
STATEMENT REGARDING ELECTRONIC FILING OF A SEQUENCE LISTING
[0003] A Sequence Listing in ASCII text format, submitted under 37
C.F.R. .sctn. 1.821, entitled 5470-785CT_ST25.txt, 7,010 bytes in
size, generated on Dec. 3, 2021 and filed via EFS-Web, is provided
in lieu of a paper copy. This Sequence Listing is hereby
incorporated by reference into the specification for its
disclosures.
Field of the Invention
[0004] The invention relates to products and methods for
transdifferentiating oligodendrocytes and/or oligodendrocyte
precursor cells to neurons. The invention further relates to
methods of treating central nervous system disorders and
conditions.
Background of the Invention
[0005] With expanding knowledge of differentiation factors, it has
become possible to reprogram resident cells in vivo (Heinrich et
al., Nat. Cell Bio. 17:204 (2015)). For example, after cortical
injury, the expression of NeuroD1 reprogrammed reactive astrocytes
into NeuN positive cells that fired action potentials and received
synaptic input (Guo et al., Cell Stem Cell 14:188 (2014)).
Similarly astrocytes have been converted into neurons in transgenic
mice that expressed three conversion factors, Asc11, Brn2a and
Myt11 (Torper et al., Proc. Nat. Acad. Sci. 110:7038 (2013)), while
following injury NG2/olig2 positive cells could be
transdifferentiated into neurons by the expression of SOX2 and
Ase11 (Heinrich et al., Stem Cell Reports 3:1000 (2014)). These
studies clearly established that at least in the context of injury,
astrocytes and oligodendrocyte precursor cells (OPCs) can be
induced to transdifferentiate into neurons in the central nervous
system (CNS). However, this proof of principle involved either
transgenic mice or retroviral mediated gene expression where, in
the case of retroviral vectors, the potential for insertional
mutagenesis precludes clinical consideration.
[0006] The present invention overcomes shortcomings in the art by
providing products and methods for conversion of oligodendrocytes
and/or oligodendrocyte precursor cells to functional neurons.
Summary of the Invention
[0007] Given the potential to reprogram cells to neurons in the
CNS, oligodendrocytes and oligodendrocyte precursor cells (OPCs)
provide an excellent endogenous target cell population.
Oligodendrocytes and OPCs comprise a substantial population in the
CNS and in many neurological disorders, the OPC population expands
in areas of neuropathology. For example, a significant increase in
OPCs occurs in clinical samples from ALS patients (Kang et at, Nat.
Neurosci. 16:571 (2013) and in intractable pediatric epileptics
(Sakuma et al., Neurosci. Lett. 566:188 (2014)). Clearly, in the
context of neuropathology this CNS cell population provides a
viable source for neuronal reprogramming.
[0008] The present invention is based, in part, on the development
of vectors and methods for transdifferentiation of oligodendrocytes
and/or OPCs into neurons. The approach relied upon two recent
observations. Xue et a. (Cell 152:82 (2013)) reported that
suppression of polypyrimidine-tract-binding (PTB) protein
expression in cultured fibroblasts caused a portion of the
fibroblasts to differentiate into functional neurons. Thus,
manipulation of a single factor could induce neuronal
reprogramming. Secondly, we recently developed a novel AAV vector
where the chimeric capsid confers a dominant oligodendrocyte
tropism in the rat striatum.
[0009] Thus, one aspect of the invention relates to a expression
cassette comprising a polynucleotide encoding an antisense PNA or
an interfering RNA targeted to a polynucleotide encoding a
mammalian polypyrimidine tract binding protein 1 (PTBP1).
[0010] Another aspect of the invention relates to a virus particle
comprising the expression cassette of the invention and a
composition and pharmaceutical composition comprising the
expression cassette or virus particle of the invention.
[0011] An additional aspect of the invention relates to a method of
attenuating expression of PTBP1 in a cell, comprising contacting
the cell with the expression cassette, virus particle, and/or
composition of the invention, wherein the expression of PTBP1 is
attenuated.
[0012] Another aspect of the invention relates to a method of
transdifferentiating an oligodendrocyte or an oligodendrocyte
precursor cell to a neuron, comprising contacting the
oligodendrocyte or oligodendrocyte precursor cell with the
expression cassette, virus particle, and/or composition of the
invention, thereby transdifferentiating the oligodendrocyte or
oligodendrocyte precursor cell to a neuron.
[0013] A further aspect of the invention relates to a method of
increasing the number of neurons in the brain of a mammalian
subject, comprising delivering to the brain the expression
cassette, virus particle, and/or composition of the invention,
thereby increasing the number of neurons in the brain of the
mammalian subject relative to the number of neurons prior to the
delivery.
[0014] An additional aspect of the invention relates to a method of
transdifferentiating an oligodendrocyte and/or a oligodendrocyte
precursor cell to a neuron in the brain of a mammalian subject,
comprising delivering to the brain the expression cassette, virus
particle, and/or composition the invention, thereby
transdifferentiating an oligodendrocyte and/or oligodendrocyte
precursor cell to a neuron in the brain of the mammalian
subject.
[0015] Another aspect of the invention relates to a method of
treating a central nervous system disorder or condition responsive
to an increase in the number of neurons in a mammalian subject in
need thereof, the method comprising delivering to the brain the
expression cassette, virus particle, and/or composition of the
invention, thereby treating the central nervous system disorder or
condition.
[0016] 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
[0017] FIGS. 1A-IC show in vitro reduction of PTBP1 by siRNAs and
miRNAs and long term selectivity of the Olig001 AAV vector in the
rat striatum. (1A) The first western blot shows that numbers 3 and
4 siRNA completely prevented PTBP1 expression following
transfection of HeLa cells with the siRNA mid a rat ptbp expression
plasmid. The second western blot shows that conversion of numbers 3
and 4 to miRNA packaged in AAV2 virus prevented PTBP1 expression in
HEK293 cells. (1B) The oligodendrocyte preference for the Olig001
AAV-GFP vector in the rat striatum 10 days post-transduction is
shown. GFP positive cells co-localized with Olig2 positive cells
but did not co-localize with NeuN or GFAP positive cells. (I C) The
same transduction pattern from the Olig01-AAV-CFP vector 6 months
after striatal transduction in the rat is shown.
[0018] FIG. 2 shows Olig001 AAV-GFP vectors do not transduce
dividing cells in the rat striatum. Rats received a 100 mg/kg i.p.
dose of BrdU 30 minutes prior to Olig001-AAV-GFP infusion into the
striatum and a second 100 mg/kg i.p. dose of BrdU 30 minutes
post-vector infusion. Two weeks later, as expected very few
dividing cells were found in the striatum, although this labeling
protocol resulted in many BrdU positive cells in the sub-granular
zone of the dentate gyrus. As seen in the confocal image the
dividing cell (white arrow) did not co-localize with the GFP
positive cells. No instances of GFP/BrdU co-localization were found
throughout the areas of transduction for 2 rats. (horizontal
bars=20 microns).
[0019] FIGS. 3A-3C show the progression of oligodendrocyte
transdifferentiation following Olig001AAV-4miRNA-GFP transduction
in the rat striatum. (3A) Confocal images show that 10 days after
Olig001AAV-4miRNA-GFP transduction, the vast majority of the GFP
positive cells exhibit a typical oligodendrocyte morphology
including GFP positive myelin in the striatal patches. Many GFP
positive cells co-localize with Olig2 but do not co-localize with
NeuN. This pattern was present throughout the area of striatal
transduction, in all animals (n=4). (3B) By 6 weeks post
transduction, confocal images show that most of the GFP positive
cells co-localize with NeuN as well as subclasses of striatal
GABAergic cells, such as DARPP32 and parvalbumin (arrows). NeuN/GFP
co-localization was present throughout the area of transduction in
all animals (n=5). (3C) Confocal images show that this neuronal
pattern remains 6 months after vector transduction where GFP
positive cells co-localize with NeuN but not GFAP or Olig2 (n=4).
(Horizontal bars=20 microns).
[0020] FIGS. 4A-4C show electrophysiological and neuroanatomical
evidence that the transdifferentiated oligodendrocytes become
functional striatal neurons. (4A) A voltage clamp recording from a
representative GFP fluorescent striatal cell shows
action-potentials typical of a neuron. (4B) A current-clamp
recording shows spontaneous post-synaptic potentials from the cell
shown in (4A), indicative of synaptic input to the GFP positive
cell. (4C) Confocal images show that 3 months after Olig001
AAV-4miRNA-GFP transduction, GFP positive nerve terminals are
present in both the globus pallidus and the substantia nigra.
Further, when fluorescent latex beads (0.04 .mu.m) are infused into
either the globus pallidus or substantia nigra 3 months after
striatal transduction by OLIG001AAV-4miRNA-GFP, 2 weeks later the
fluorescent beads have been retrogradely transported into GFP
positive cell bodies in the ipsilateral striatum (white arrows).
(Horizontal bars=20 microns).
DETAILED DESCRIPTION OF THE INVENTION
[0021] The present invention is based on the development of
products and methods for transdifferentiating oligodendrocytes into
functional neurons. The methods can be used to treat central
nervous system disorders and conditions.
[0022] 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 four 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.
[0023] 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.
[0024] 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.
[0025] 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.
[0026] 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, NY., 1989); F. M. AUSUBEL et al. CURRENT
PROTOCOLS IN MOLECULAR BIOLOGY (Green Publishing Associates, Inc.
and John Wiley & Sons, Inc., New York).
[0027] All publications, patent applications, patents, nucleotide
sequences, amino acid sequences and other references mentioned
herein are incorporated by reference in their entirety.
[0028] 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 fons as well, unless the context clearly
indicates otherwise.
[0029] 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").
[0030] 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.
[0031] 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 .+-.10%, .+-.5%, .+-.1%, .+-.0.5%, or even
.+-.0.1% of the specified amount.
[0032] 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.
[0033] 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."
[0034] The genomic sequences of various AAV and autonomous
parvoviruses, as well as the sequences of the inverted terminal
repeats (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@
Accession Numbers NC 002077, NC 001401, NC 001729, NC 001863, NC
001829, NC 001862, NC 000883, NC 001701, NC 001510, AF063497,
U189790, AF043303, AF028705, AF028704, 302275, J01901, J02275,
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 al., (1986)J. Virol. 58:921; Gao et al., (2002)
Proc. Nat. Aca. Sci. USA 99:11854; international patent
publications WO 00/28061, WO 99161601. 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 (AA) DNA
replication and integration," Ph.D. Dissertation, University of
Pittsburgh, Pittsburgh, Pa. (incorporated herein it its
entirety).
[0035] 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.
TABLE-US-00001 TABLE 1 GenBank Accession Number Complete Genomes
Adeno-associated virus 1 NC_002077, AF063497 Adeno-associated virus
2 NC_001401 Adeno-associated virus 3 NC_001729 Adeno-associated
virus 3B NC_001863 Adeno-associated virus 4 NC_001829
Adeno-associated virus 5 Y18065, AF085716 Adeno-associated virus 6
NC_001862 Avian AAV ATCC VR-865 AY186198, AY629583, NC_004828 Avian
AAV strain DA-1 NC_006263, AY629583 Bovine AAV NC_005889, AY388617
Clade A AAV1 NC_002077, AF063497 AAV6 NC_001862 Hu.48 AY530611 Hu
43 AY530606 Hu 44 AY530607 Hu 46 AY530609 Clade B Hu. 19 AY530584
Hu. 20 AY530586 Hu 23 AY530589 Hu22 AY530588 Hu24 AY530590 Hu21
AY530587 Hu27 AY530592 Hu28 AY530593 Hu 29 AY530594 Hu63 AY530624
Hu64 AY530625 Hu13 AY530578 Hu56 AY530618 Hu57 AY530619 Hu49
AY530612 Hu58 AY530620 Hu34 AY530598 Hu35 AY530599 AAV2 NC_001401
Hu45 AY530608 Hu47 AY530610 Hu51 AY530613 Hu52 AY530614 Hu T41
AY695378 Hu S17 AY695376 Hu T88 AY695375 Hu T71 AY695374 Hu T70
AY695373 Hu T40 AY695372 Hu T32 AY695371 Hu T17 AY695370 Hu LG15
AY695377 Clade C Hu9 AY530629 Hu10 AY530576 Hu11 AY530577 Hu53
AY530615 Hu55 AY530617 Hu54 AY530616 Hu7 AY530628 Hu18 AY530583
Hu15 AY530580 Hu16 AY530581 Hu25 AY530591 Hu60 AY530622 Ch5
AY243021 Hu3 AY530585 Hu1 AY530575 Hu4 AY530602 Hu2 AY530585 Hu61
AY530623 Clade D Rh62 AY530573 Rh48 AY530561 Rh54 AY530567 Rh55
AY530568 Cy2 AY243020 AAV7 AF513851 Rh35 AY243000 Rh37 AY242998
Rh36 AY242999 Cy6 AY243016 Cy4 AY243018 Cy3 AY243019 Cy5 AY243017
Rh13 AY243013 Clade E Rh38 AY530558 Hu66 AY530626 Hu42 AY530605
Hu67 AY530627 Hu40 AY530603 Hu41 AY530604 Hu37 AY530600 Rh40
AY530559 Rh2 AY243007 Bb1 AY243023 Bb2 AY243022 Rh10 AY243015 Hu17
AY530582 Hu6 AY530621 Rh25 AY530557 Pi2 AY530554 Pi1 AY530553 Pi3
AY530555 Rh57 AY530569 Rh50 AY530563 Rh49 AY530562 Hu39 AY530601
Rh58 AY530570 Rh61 AY530572 Rh52 AY530565 Rh53 AY530566 Rh51
AY530564 Rh64 AY530574 Rh43 AY530560 AAV8 AF513852 Rh8 AY242997 Rh1
AY530556 Clade F Hu14 (AAV9) AY530579 Hu31 AY530596 Hu32 AY530597
Clonal isolate AAV5 Y18065, AF085716 AAV 3 NC_001729 AAV 3B
NC_001863 AAV4 NC_001829 Rh34 AY243001 Rh33 AY243002 Rh32
AY243003
[0036] 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.
[0037] 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.
[0038] The terms "specific for oligodendrocytes and or OPCs" and
"has a tropism for oligodendrocytes and/or OPCs" as used herein
refer to a viral vector that, when administered directly into the
CNS, preferentially transduces oligodendrocytes and/or OPCs over
neurons, astrocytes, and other CNS cell types. In some embodiments,
at least about 80% of the transduced cells are oligodendrocytes
and/or OPCs, e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, 99%
(or more are oligodendrocytes and/or OPCs.
[0039] The term "central nervous system (CNS) disorder or condition
responsive to an increase in the number of neurons" as used herein
refers to a disease, disorder, condition, or injury in which CNS
cells are damaged, lost, or function improperly and which show an
improvement in at least one symptom when the number of neurons in
the CNS (e.g., at the site of tissue damage) is increased. The term
includes diseases, disorders, conditions, and injuries in which CNS
cells are directly affected as well as diseases, disorders,
conditions, and injuries in which CNS cells become dysfunctional
secondary to damage to other cells, tissues, or organs.
[0040] 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.
[0041] 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).
[0042] 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 genu 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.,
oligodendrocytes).
[0043] As used herein, the term "polypeptide" encompasses both
peptides and proteins, unless indicated otherwise.
[0044] A "nucleic acid" or "nucleotide sequence" is a sequence of
nucleotide bases, and may be RNA, DNA or DNA-RA hybrid sequences
(including both naturally occurring and non-naturally occurring
nucleotide), but is preferably either single or double stranded DNA
sequences.
[0045] 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.
[0046] 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.
[0047] 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 anchor 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.
[0048] 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.
[0049] 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.
[0050] 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.
[0051] 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
can package an AAV or rAAV genome or any other nucleic acid
including viral nucleic acids. Alternatively, in some contexts, the
term "vector" "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.
[0052] A "recombinant A-A vector genome" or "rAAV genome" is an NA
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 mid 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.
[0053] 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-AA TR. For example, a non-AAV TR
sequence such as those of other parvoviruses (e.g., canine
parvovirus (CPV), mouse parvovirus (MVM), human parvovuls 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.
[0054] An "AAV inverted terminal repeat" or "AAV ITR" ma y 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, Table 1). A AAV ITR need not have the native terminal repeat
sequence (e.g., a native AAV ITR 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.
[0055] The virus vectors of the invention can further be "targeted"
virus vectors (e.g., having a directed tropism) and/or a "hybrid"
parvovirus (i.e., in which the viral ITRs and viral capsid are from
different parvoviruses) as described in international patent
publication WO 00/28004 and Chao et al, (2000) Mol. Therapy
2:619.
[0056] Further, the viral capsid or genomic elements can contain
other modifications, including insertions, deletions and/or
substitutions.
[0057] The term "template" or "substrate" is used herein to refer
to a polynucleotide sequence that may be replicated to produce the
parvovirus viral DNA. For the purpose of vector production, the
template will typically be embedded within a larger nucleotide
sequence or construct, including but not limited to a plasmid,
naked DNA vector, bacterial artificial chromosome (BAC), yeast
artificial chromosome (YAC) or a viral vector (e.g., adenovirus,
herpesvirus, Epstein-Barr Virus, AAV, baculoviral, retroviral
vectors, and the like). Alternatively, the template may be stably
incorporated into the chromosome of a packaging cell.
[0058] As used herein, parvovirus or AAV "Rep coding sequences"
indicate the nucleic acid sequences that encode the parvoviral or
AAV non-structural proteins that mediate viral replication and the
production of new virus particles. The parvovirus and AAV
replication genes and proteins have been described in, e.g., FIELDS
et. al., VIROLOGY, volume 2, chapters 69 & 70 (4th ed.,
Lippincott-Raven Publishers).
[0059] The "Rep coding sequences" need not encode all of the
parvoviral or AAV Rep proteins. For example, with respect to AAV,
the Rep coding sequences do not need to encode all four AAV Rep
proteins (Rep78, Rep 68, Rep52 and Rep40), in fact, it is believed
that AAV5 only expresses the spliced Rep68 and Rep40 proteins. In
representative embodiments, the Rep coding sequences encode at
least those replication proteins that are necessary for viral
genome replication and packaging into new virions. The Rep coding
sequences will generally encode at least one large Rep protein
(i.e., Rep78/68) and one small Rep protein (i.e. Rep52/40). In
particular embodiments, the Rep coding sequences encode the AAV
Rep78 protein and the AAV Rep52 and/or Rep40 proteins. In other
embodiments, the Rep coding sequences encode the Rep68 and the
Rep52 and/or Rep40 proteins. In a still further embodiment, the Rep
coding sequences encode the Rep68 and Rep52 proteins, Rep68 and
Rep40 proteins, Rep78 and Rep52 proteins, or Rep78 and Rep40
proteins.
[0060] As used herein, the term "large Rep protein" refers to Rep68
and/or Rep78. Large Rep proteins of the claimed invention may be
either wild-type or synthetic. A wild-type large Rep protein may be
from any parvovirus or AAV, including but not limited to serotypes
1, 2, 3a, 3b, 4, 5, 6, 7, 8, 9, 10, 11, or 13, or any other AAV now
known or later discovered (see, e.g., Table 1). A synthetic large
Rep protein may be altered by insertion, deletion, truncation
and/or missense mutations.
[0061] Those skilled in the art will further appreciate that it is
not necessary that the replication proteins be encoded by the same
polynucleotide. For example, for MVM, the NS-1 and NS-2 proteins
(which are splice variants) may be expressed independently of one
another. Likewise, for AAV, the p19 promoter may be inactivated and
the large Rep protein(s) expressed from one polynucleotide and the
small Rep protein(s) expressed from a different polynucleotide.
Typically, however, it will be more convenient to express the
replication proteins from a single construct. In some systems, the
viral promoters (e.g. AAV p19 promoter) may not be recognized by
the cell, and it is therefore necessary to express the large and
small Rep proteins from separate expression cassettes. In other
instances, it may be desirable to express the large Rep and small
Rep proteins separately, i.e., under the control of separate
transcriptional and/or translational control elements. For example,
it may be desirable to control expression of the large Rep
proteins, so as to decrease the ratio of large to small Rep
proteins. In the case of insect cells, it may be advantageous to
down-regulate expression of the large Rep proteins (e.g., Rep78/68)
to avoid toxicity to the cells (see, e.g., Urabe et al., (2002)
Human Gene Therapy 13:1935).
[0062] As used herein, the parvovirus or AAV "cap coding sequences"
encode the structural proteins that form a functional parvovirus or
AAV capsid (i.e. can package DNA and infect target cells).
Typically, the cap coding sequences will encode all of the
parvovirus or AAV capsid subunits, but less than all of the capsid
subunits may be encoded as long as a functional capsid is produced.
Typically, but not necessarily, the cap coding sequences will be
present on a single nucleic acid molecule.
[0063] 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.
[0064] 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).
[0065] 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.
[0066] The terms "transdifferentiation," "transdifferentiating,"
and transdifferentiate" as used herein refer to the conversion of
one cell type to another cell type. The transdifferentiation can be
confirmed by the loss of markers of the first cell type and gain of
markers of the second cell type.
[0067] The term "differentiation factor" as used herein, refers to
a compound, molecule, or polypeptide that promotes the
differentiation of a cell from one cell type or stage to
another.
[0068] The term "antisense nucleotide sequence" or "antisense
oligonucleotide" as used herein, refers to a nucleotide sequence
that is complementary to a specified DNA or RNA sequence. Antisense
oligonucleotides and nucleic acids that express 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 al. The antisense nucleotide sequence can be complementary to
the entire nucleotide sequence encoding the polypeptide or a
portion thereof of at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 75,
100, 150, 200, 300, or 500 contiguous bases or less than 500, 300,
200, 150, 100, 75, 50, 45, 40, 35, 30, 25, 20, 15, or 10 contiguous
bases and will reduce the level of polypeptide production.
[0069] Those skilled in the art will appreciate that it is not
necessary that the antisense nucleotide sequence be fully
complementary to the target sequence as long as the degree of
sequence similarity is sufficient for the antisense nucleotide
sequence to hybridize to its target and reduce production of the
polypeptide. As is known in the art, a higher degree of sequence
similarity is generally required for short antisense nucleotide
sequences, whereas a greater degree of mismatched bases will be
tolerated by longer antisense nucleotide sequences.
[0070] For example, hybridization of such nucleotide sequences can
be carried out under conditions of reduced stringency, medium
stringency or even stringent conditions (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/or 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) to the
nucleotide sequences specifically disclosed herein. See, e.g.,
Sambrook e al. Molecular Cloning: A Laboratory Manual 2nd Ed (Cold
Spring Harbor. NY, 1989).
[0071] In other embodiments, antisense nucleotide sequences of the
invention have at least about 70, 80%, 90%, 95%, 97%, 98% or higher
sequence similarity with the complement of the coding sequences
specifically disclosed herein and will reduce the level of
polypeptide production.
[0072] The length of the antisense nucleotide sequence (i.e., the
number of nucleotides therein) is not critical as long as it binds
selectively to the intended location and reduces transcription
and/or translation of the target sequence, and can be determined in
accordance with routine procedures. In general, the antisense
nucleotide sequence will be from about eight, ten or twelve
nucleotides in length up to about 20, 30, 50, 75 or 100
nucleotides, or longer, in length.
[0073] An antisense nucleotide sequence can be constructed using
chemical synthesis and enzymatic ligation reactions by procedures
known in the art. For example, an antisense nucleotide sequence can
be chemically synthesized using naturally occurring nucleotides or
various modified nucleotides designed to increase the biological
stability of the molecules or to increase the physical stability of
the duplex formed between the antisense and sense nucleotide
sequences, e.g., phosphorothioate derivatives and acridine
substituted nucleotides can be used. Examples of modified
nucleotides which can be used to generate the antisense nucleotide
sequence include 5-fluorouracil, 5-bromouracil, 5-chlorouracil,
5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine,
5-(carboxyhydroxylmethyl) uracil,
5-carboxymethylaminomethyl-2-thiouridine,
5-carboxymethylaminomethyl-halouracil, dihydrouracil,
beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,
1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine,
5-methylcytosine, N6-adenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,
beta-D-mannosylqueosine, 5'-methoxycarboxymethyl-acid,
5-methoxyuracil, 2-methylthio-N6-isopenten-yladenine,
uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine,
2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,
5-methyluracil, uracil-5-oxyacetic acid methylester,
uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil,
3-(3-amino-3-N-2-carboxypropyl) uracil. (acp3)w, and
2,6-diaminopurine. Alternatively, the antisense nucleotide sequence
can be produced using an expression vector into which a nucleic
acid has been cloned in an antisense orientation (i.e., RNA
transcribed from the inserted nucleic acid will be of an antisense
orientation to a target nucleic acid of interest).
[0074] The antisense nucleotide sequences of the invention further
include nucleotide sequences wherein at least one, or all, of the
internucleotide bridging phosphate residues are modified
phosphates, such as methyl phosphonates, methyl phosphonothioates,
phosphoromoipholidates, phosphoropiperazidates and
phosphoramidates. For example, every other one of the
internucleotide bridging phosphate residues can be modified as
described. In another non-limiting example, the antisense
nucleotide sequence is a nucleotide sequence in which one, or all,
of the nucleotides contain a 2' lower alkyl moiety (e.g.,
C.sub.1-C.sub.4, linear or branched, saturated or unsaturated
alkyl, such as methyl, ethyl, ethenyl, propyl, 1-propenyl,
2-propenyl, and isopropyl). For example, every other one of the
nucleotides can be modified as described. See also, Furdon et al,
Nucleic Acids Res. 17:9193 (1989); Agrawal at al, Proc. Natl. Acad.
Sci. USA 87:1401 (1990); Baker et al., Nucleic Acids Res. 18:3537
(1990); Sproat et al, Nucleic Acids Res. 17:3373 (1989); Walder and
Walder, Proc. Natl. Acad. Sci. USA 85:5011 (1988); incorporated by
reference herein in their entireties for their teaching of methods
of making antisense molecules, including those containing modified
nucleotide bases).
[0075] As used herein. "RNAi" or "RNA interference" refers to the
process of sequence-specific post-transcriptional gene silencing,
mediated by an interfering RNA which is a double-stranded RNA
(dsRNA). As used herein, "dsRNA" refers to RNA that is partially or
completely double stranded. Examples of double stranded RINA useful
in RNA interference include small interfering RNA (siRNA), small
interfering nucleic acid (siNA), short hairpin RNA (shRNA),
microRNA (miRNA), and the like. In the RNAi process, dsRNA
comprising a first (antisense) strand that is complementary to a
portion of a target gene and a second (sense) strand that is fully
or partially complementary to the first antisense strand is
introduced into a cell or organism.
[0076] RNAi begins with the cleavage of longer dsRNAs into small
interfering RNAs (siRNAs) by an RNaseIII-like enzyme, dicer. SiRNAs
are dsRNAs that are usually about 19 to about 28 nucleotides, or
about 20 to about 25 nucleotides, or about 21 to about 22
nucleotides in length and often contain 2-nucleotide 3' overhangs,
and 5' phosphate and 3 hydroxyl termini. One strand of the siRNA is
incorporated into a ribonucleoprotein complex known as the
RNA-induced silencing complex (RISC). RISC uses this siRNA strand
to identify mRNA molecules that are at least partially
complementary to the incorporated siRNA strand, and then cleaves
these target mRNAs or inhibits their translation. Therefore, the
siRNA strand that is incorporated into RISC is known as the guide
strand or the antisense strand. The other siRNA strand, known as
the passenger strand or the sense strand, is eliminated from the
siRNA and is at least partially homologous to the target mRNA.
Those of skill in the art will recognize that, in principle, either
strand of an siRNA can be incorporated into RISC and function as a
guide strand. However, siRNA design (e.g., decreased siRNA duplex
stability at the 5' end of the antisense strand) can favor
incorporation of the antisense strand into RISC.
[0077] RISC-mediated cleavage of mRNAs having a sequence at least
partially complementary to the guide strand leads to a decrease in
the steady state level of that mRNA and of the corresponding
protein encoded by this mRNA. Alternatively, RISC can also decrease
expression of the corresponding protein via translational
repression without cleavage of the target mRNA. Other RNA molecules
and RNA-like molecules can also interact with RISC and silence gene
expression. Examples of other RNA molecules that can interact with
RISC include shRNAs, single-stranded siRNAs, miRNAs, and
dicer-substrate 27-mer duplexes. The term "interfering RNAs" as
used herein refers to a double-stranded interfering RNA unless
otherwise noted. Examples of RNA-like molecules that can interact
with RISC include RNA molecules containing one or more chemically
modified nucleotides, one or more deoxyribonucleotides, and/or one
or more non-phosphodiester linkages. For purposes of the present
discussion, all RNA or RNA-like molecules that can interact with
RISC and participate in RISC-mediated changes in gene expression
will be referred to as "interfering RNAs." SiRNAs, shRNAs, miRNAs,
and dicer-substrate 27-mer duplexes are, therefore, subsets of
"interfering RNAs."
[0078] MicroRNAs (miRNAs) are non-protein coding RNAs, generally of
between about IS to about 25 nucleotides in length (commonly about
20-24 nucleotides in length in plants). These miRNAs direct
cleavage in trans of target transcripts, negatively regulating the
expression of genes involved in various regulation and development
pathways (Bartel, Cell 116:281 (2004); Zhang et al., Dev Biol.
289:3 (2006)).
[0079] Genes encoding miRNAs yield primary MiRNAs (termed a
"pri-miRNA") of 70 to 300 bp in length that can form imperfect
stem-loop structures. A single pri-miRNA may contain from one to
several miRNA precursors. In animals, pri-miRNAs are processed in
the nucleus into shorter hairpin RNAs of about 65 nt (pre-miRNAs)
by the RNaseIII enzyme Drosha and its cofactor DGCR8/Pasha. The
pre-miRNA is then exported to the cytoplasm, where it is further
processed by another R-NaseIII enzyme. Dicer, releasing a
miRNA/miRNA* duplex of about 22 nt in size. Many reviews on
microRNA biogenesis and function are available, for example, see,
Bartel, Cell 116:281 (2004), Murchison et al., Curr. Opin. Cell
Biol. 16:223 (2004), Dugas e al., Curr. Opin. Plant Biol. 7:512
(2004) and Kim, Nature Rev. Mol. Cell Biol. 6:376 (2005).
[0080] As used herein, the terms "amount sufficient to inhibit
expression" and "amount sufficient to attenuate expression" refers
to a concentration or amount of the interfering RNA that is
sufficient to reduce levels or stability of mRNA or protein
produced from the PTBP1 gene in a cell. As used herein, "inhibiting
expression" and "attenuating expression" refer to the absence or
observable decrease in the level of protein and/or mRNA product
from the PTBP1 gene.
[0081] As used herein, "complementary" polynucleotides are those
that are capable of base pairing according to the standard
Watson-Crick complementarity rules. Specifically, purines will base
pair with pyrimidines to form a combination of guanine paired with
cytosine (G:C) and adenine paired with either thymine (A:T) in the
case of DNA, or adenine paired with uracil (A:U) in the case of
RNA. For example, the sequence "A-G-T" binds to the complementary
sequence "T-C-A." It is understood that two polynucleotides may
hybridize to each other even if they are not completely
complementary to each other, provided that each has at least one
region that is substantially complementary to the other.
[0082] The terms "complementary" or "complementarity," as used
herein, refer to the natural binding of polynucleotides under
permissive salt and temperature conditions by base-pairing.
Complementarity between two single-stranded molecules may be
"partial," in which only some of the nucleotides bind, or it may be
complete when total complementarity exists between the single
stranded molecules. The degree of complementarity between nucleic
acid strands has significant effects on the efficiency and strength
of hybridization between nucleic acid strands.
[0083] As used herein, the terms "substantially complementary" or
"partially complementary" mean that two nucleic acid sequences are
complementary at least at about 80% of their nucleotides. In some
embodiments, the two nucleic acid sequences can be complementary at
least at 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of their
nucleotides. The terms "substantially complementary" and "partially
complementary" can also mean that two nucleic acid sequences can
hybridize under high stringency conditions and such conditions are
well known in the art.
[0084] As used herein, the terms "contacting," "introducing,"
"delivering," and "administering" are used interchangeably and
refer to a process by which antisense RNA or interfering RNA of the
present invention or a nucleic acid molecule encoding an antisense
RNA or an interfering RINA of this invention is delivered to a cell
or a subject, in order to inhibit or alter or modify expression of
PTBP1 in the cell or subject. The antisense RNA or interfering RA
may be administered in a number of ways, including, but not limited
to, direct introduction into a cell (i.e., intracellularly) and/or
extracellular introduction into a cavity, interstitial space,
regional circulation feeding a particular organ or tissue, or into
a tissue or structure (e.g., the striatum).
[0085] "Introducing" in the context of a cell or a subject means
presenting the nucleic acid molecule to the cell or subject in such
a manner that the nucleic acid molecule gains access to the
interior of a cell. Where more than one nucleic acid molecule is to
be introduced these nucleic acid molecules can be assembled as part
of a single polynucleotide or nucleic acid construct, or as
separate polynucleotide or nucleic acid constructs, and can be
located on the same or different nucleic acid constructs.
Accordingly, these polynucleotides can be introduced into cells in
a single transformation event or in separate transformation events.
Thus, the term "transformation" as used herein refers to the
introduction of a heterologous nucleic acid into a cell.
Transformation of a cell may be stable or transient.
[0086] Embodiments of the invention are directed to expression
cassettes designed to express the nucleic acids of the present
invention. As used herein, "expression cassette" means a nucleic
acid molecule having at least a control sequence operably linked to
a nucleotide sequence of interest. In this manner, for example,
promoters in operable interaction with the nucleotide sequences for
the antisense RNAs or interfering RNAs of the invention are
provided in expression cassettes for expression in a cell.
[0087] As used herein, the term "promoter" refers to a region of a
nucleotide sequence that incorporates the necessary signals for the
efficient expression of a coding sequence. This may include
sequences to which an RNA polymerase binds, but is not limited to
such sequences and can include regions to which other regulatory
proteins bind together with regions involved in the control of
protein translation and can also include coding sequences.
[0088] Furthermore, a "promoter" of this invention is a promoter
capable of initiating transcription in a cell. Such promoters
include those that drive expression of a nucleotide sequence
constitutively, those that drive expression when induced, and those
that drive expression in a tissue- or developmentally-specific
manner, as these various types of promoters are known in the
art.
[0089] For purposes of the invention, the regulatory regions (i.e.
promoters, transcriptional regulatory regions, and translational
termination regions) can be native/analogous to the cell and/or the
regulator regions can be native/analogous to the other regulatory
regions. Alternatively, the regulatory regions may be heterologous
to the cell and/or to each other (i.e., the regulatory regions).
Thus, for example, a promoter can be heterologous when it is
operably linked to a polynucleotide from a species different from
the species from which the polynucleotide was derived.
Alternatively, a promoter can also be heterologous to a selected
nucleotide sequence if the promoter is from the same/analogous
species from which the polynucleotide is derived, but one or both
(i.e., promoter and polynucleotide) are substantially modified from
their original form and/or genomic locus, or the promoter is not
the native promoter for the operably linked polynucleotide.
[0090] The choice of promoters to be used depends upon several
factors, including, but not limited to, cell- or tissue-specific
expression, desired expression level, efficiency, inducibility and
selectability. For example, where expression in a specific tissue
or organ is desired, a tissue-specific or tumor-specific promoter
can be used (e.g., a breast cancer specific promoter). In contrast,
where expression in response to a stimulus is desired, an inducible
promoter can be used. Where continuous expression is desired
throughout the cells of a subject, a constitutive promoter can be
used. It is a routine matter for one of skill in the art to
modulate the expression of a nucleotide sequence by appropriately
selecting and positioning r promoters and other regulatory regions
relative to that sequence.
[0091] Therefore, in some instances, constitutive promoters can be
used. Examples of constitutive promoters include, but are not
limited to, pol III promoters, such as the U6 or H1 promoters, or
pol II promoters, such as the cytomegalovirus promoter and the
SV40-derived initial promoter, and mammalian constitutive protein
gene promoters such as the .beta.-actin gene promoter, the tRNA
promoter, and the like. In some embodiments, the constitutive
promoter is a hybrid chicken beta actin promoter or a truncated
version thereof (CBh, Gray et al., Human Gene Ther. 22:1143
(2011)).
[0092] Moreover, tissue-specific regulated nucleic acids and/or
promoters as well as tumor-specific regulated nucleic acids and/or
promoters have been reported. Thus, in some embodiments,
tissue-specific or tumor-specific promoters can be used. Some
reported tissue-specific nucleic acids include, without limitation,
B29 (B cells), CD14 (monocytic cells), CD43 (leukocytes and
platelets), CD45 (hematopoietic cells), CD68 (macrophages), desmin
(muscle), elastase-1 (pancreatic acinar cells), endoglin
(endothelial cells), fibronectin (differentiating cells and healing
tissues), FLT-1 (endothelial cells), GFAP (astrocytes), GPIIb
(megakaryocytes), ICAM-2 (endothelial cells), INF-f (hematopoietic
cells), Mb (muscle), NPHSI (podocytes), OG-2 (osteoblasts, SP-B
(lungs), SYN1 (neurons), and WASP (hematopoietic cells). Some
reported tumor-specific nucleic acids and promoters include,
without limitation, AFP (hepatocellular carcinoma), CCKAR
(pancreatic cancer), CEA (epithelial cancer), c-erbB2 (breast and
pancreatic cancer). COX-2, CXCR4, E2F-1, HE4, LP, MUC1 (carcinoma),
PRC1 (breast cancer), PSA (prostate cancer), RRM2 (breast cancer),
survivin, TRP1 (melanoma), and TYR (melanoma).
[0093] In some instances, inducible promoters can be used. Examples
of inducible promoters include, but are not limited to,
tetracycline repressor system promoters, Lac repressor system
promoters, copper-inducible system promoters, salicylate-inducible
system promoters (e.g., the PRI a system), glucocorticoid-inducible
promoters, and ecdysone-inducible system promoters.
[0094] In addition to the promoters described above, the expression
cassette also can include other regulatory sequences. As used
herein, "regulatory sequences" means nucleotide sequences located
upstream (5' non-coding sequences), within, or downstream (3'
non-coding sequences) of a coding sequence, and which influence the
transcription, RNA processing or stability, or translation of the
associated coding sequence. Regulatory sequences include, but are
not limited to, enhancers, introns, translation leader sequences,
non-translated leader sequences, and polyadenylation signal
sequences.
[0095] The expression cassette also can optionally include a
transcriptional and/or translational termination region (i.e.,
termination region) that is functional in the cell. A variety of
transcriptional terminators are available for use in expression
cassettes and are responsible for the termination of transcription
beyond the trausgene and correct mRNA polyadenylation. The
termination region may be native to the transcriptional initiation
region, may be native to the operably linked nucleotide sequence of
interest, may be native to the host, or may be derived from another
source (i.e., foreign or heterologous to the promoter, the
nucleotide sequence of interest, the host, or any combination
thereof). In addition, a coding sequence's native transcription
terminator can be used.
[0096] A signal sequence can be operably linked to nucleic acids of
the present invention to direct the nucleotide sequence into a
cellular compartment. In this manner, the expression cassette will
comprise a nucleotide sequence encoding the interfering RNA
operably linked to a nucleic acid sequence for the signal sequence.
The signal sequence may be operably linked at the N- or C-terminus
of the interfering RNA.
[0097] Regardless of the type of regulatory sequence(s) used, they
can be operably linked to the nucleotide sequence of the antisense
RNA or interfering RNA. As used herein, "operably linked" means
that elements of a nucleic acid construct such as an expression
cassette are configured so as to perform their usual function.
Thus, regulatory or control sequences (e.g., promoters) operably
linked to a nucleotide sequence of interest are capable of
effecting expression of the nucleotide sequence of interest. The
control sequences need not be contiguous with the nucleotide
sequence of interest, so long as they function to direct the
expression thereof. Thus, for example, intervening untranslated,
yet transcribed, sequences can be present between a promoter and a
coding sequence, and the promoter sequence can still be considered
"operably linked" to the coding sequence. A nucleotide sequence of
the present invention (i.e., an antisense RNA or interfering RNA)
can be operably linked to a regulatory sequence, thereby allowing
its expression in a cell and/or subject.
[0098] The expression cassette also can include a nucleotide
sequence for a selectable marker, which can be used to select a
transformed cell or subject. As used herein, "selectable marker"
means a nucleic acid that when expressed imparts a distinct
phenotype to the cell or subject expressing the marker and thus
allows such transformed cells or subjects to be distinguished from
those that do not have the marker. Such a nucleic acid may encode
either a selectable or screenable marker, depending on whether the
marker confers a trait that can be selected for by chemical means,
such as by using a selective agent (e.g. an antibiotic or the
like), or on whether the marker is simply a trait that one can
identify through observation or testing, such as by screening. Of
course, many examples of suitable selectable markers are known in
the art and can be used in the expression cassettes described
herein.
[0099] Examples of selectable markers include, but are not limited
to, a nucleic acid encoding neo or nptII, which confers resistance
to kanamycin, G418, and the like (Potrykus e al, Mol. Gen. Genet.
199:183 (1985)); a nucleic acid encoding a nitrilase such as bxn
from Klebsiella ozcaenae that confers resistance to bromoxynil
(Stalker et al, Science 242:419 (1988)); a nucleic acid encoding an
altered acetolactate synthase (ALS) that confers resistance to
imidazolinone, sulfonylurea or other ALS-inhibiting chemicals (EP
Patent Application No. 154204); a nucleic acid encoding a
methotrexate-resistant dihydrofolate reductase (DHFR) (Thillet et
al, J. Biol. Chet. 263:12500 (1988)); a nucleic acid encoding a
dalapon dehalogenase that confers resistance to dalapon; a nucleic
acid encoding a mannose-6-phosphate isomerase (also referred to as
phosphomannose isomerase (PMI) that confers an ability to
metabolize mannose (U.S. Pat. Nos. 5,767,378 and 5,994,629); a
nucleic acid encoding an altered anthranilate synthase that confers
resistance to 5-methyl tryptophan; and/or a nucleic acid encoding
hph that confers resistance to hygromycin. One of skill in the art
is capable of choosing a suitable selectable marker for use in an
expression cassette.
[0100] Additional selectable markers include, but are not limited
to, a nucleic acid encoding .beta.-glucuronidase or uidA (GUS) that
encodes an enzyme for which various chromogenic substrates are
known; a nucleic acid encoding .beta.-lactamase, an enzyme for
which various chromogenic substrates are known (e.g., PADAC, a
chromogenic cephalosporin) (Sutcliffe. Proc. Natl. Acad. Sci. USA
75:3737 (1978)); a nucleic acid encoding xylE that encodes a
catechol dioxygenase (Zukowsky et al., Proc. Natl. Acad. Sci. USA
80:1101 (1983)); a nucleic acid encoding tyrosinase, an enzyme
capable of oxidizing tyrosine to DOPA and dopaquinone, which in
turn condenses to form melanin (Katz et al., J. Gen. Microbiol.
729:2703 (1983)); a nucleic acid encoding i-galactosidase, an
enzyme for which there are chromogenic substrates; a nucleic acid
encoding luciferase (lux) that allows for bioluminescence detection
(Ow et al, Science 234:856 (1986)); a nucleic acid encoding
aequorin which may be employed in calcium-sensitive bioluminescence
detection (Prasher et al. (1985) Biochemn. Biophys. Res. Comm.
126:1259 (1985)); or a nucleic acid encoding green fluorescent
protein (Niedz et al., Plant Cell Reports 14:403 (1995)). One of
skill in the art is capable of choosing a suitable selectable
marker for use in an expression cassette.
[0101] In embodiments of the present invention, the expression
cassette can comprise an expression control sequence operatively
linked to a nucleotide sequence that is a template for one or both
strands of the interfering PNA. The interfering RNA template
comprises (a) a first (antisense) stand having a sequence
complementary to from about 15 to about 30 consecutive nucleotides
of the nucleotide sequence of PTBP1; and (b) a second (sense)
strand having a nucleotide sequence fully complementary or
substantially complementary to the first strand. In further
embodiments, a promoter can flank either end of the template
nucleotide sequence, wherein the promoters drive expression of each
individual DNA strand, thereby generating two complementary (or
substantially complementary) RNAs that hybridize and form the
interfering PNA. In alternative embodiments, the nucleotide
sequence is transcribed into both strands of the interfering RNA on
one transcription unit, wherein the sense strand is transcribed
from the 5' end of the transcription unit and the antisense strand
is transcribed from the 3' end, wherein the two strands are
separated by about 3 to about 500 basepairs, and wherein after
transcription, the RNA transcript folds on itself to form a shRNA
molecule.
[0102] As used herein "sequence identity" refers to the extent to
which two optimally aligned polynucleotide or polypeptide sequences
are invariant throughout a window of alignment of components, e.g.,
nucleotides or amino acids. "Identity" can be readily calculated by
known methods including, but not limited to, those described in:
Computational Molecular Biology (Lesk, A. M., ed.) Oxford
University Press, New York (1988); Biocomputing: Informatics and
Genome Projects (Smith, D. W., ed.) Academic Press, New York
(1993); Computer Analysis of Sequence Data, Part I (Griffin, A. M.,
and Griffin, H. G., eds.) Humana Press, New Jersey (1994); Sequence
Analysis in Molecular Biology (von Heinje, G., ed.) Academic Press
(1987); and Sequence Analysis Primer (Gribskov, M. and Devereux,
J., eds.) Stockton Press, New York (1991).
[0103] As used herein, the term "substantially identical" or
"corresponding to" means that two nucleic acid sequences have at
least about 80% sequence identity. In some embodiments, the two
nucleic acid sequences can have at least about 85%, 90%, 95%, 96%,
97% 98%, 99%- or 100% sequence identity.
[0104] An "identity fraction" for aligned segments of a test
sequence and a reference sequence is the number of identical
components which are shared by the two aligned sequences divided by
the total number of components in reference sequence segment. i.e.,
the entire reference sequence or a smaller defined part of the
reference sequence. As used herein, the term "percent sequence
identity" or "percent identity" refers to the percentage of
identical nucleotides in a linear polynucleotide sequence of a
reference ("query") polynucleotide molecule (or its complementary
strand) as compared to a test ("subject") polynucleotide molecule
(or its complementary strand) when the two sequences are optimally
aligned (with appropriate nucleotide insertions, deletions, or gaps
totaling less than 20 percent of the reference sequence over the
window of comparison). In some embodiments, "percent identity" can
refer to the percentage of identical amino acids in an amino acid
sequence.
[0105] Optimal alignment of sequences for aligning a comparison
window are well known to those skilled in the art and may be
conducted by tools such as the local homology algorithm of Smith
and Waterman, the homology alignment algorithm of Needleman and
Wunsch the search for similarity method of Pearson and Lipman, and
optionally by computerized implementations of these algorithms such
as GAP, BESTFIT, FASTA, and TFASTA available as part of the
GCG.RTM. Wisconsin Package.RTM. (Accelrys Inc., Burlington, Mass.).
An "identity fraction" for aligned segments of a test sequence and
a reference sequence is the number of identical components which
are shared by the two aligned sequences divided by the total number
of components in the reference sequence segment, i.e., the entire
reference sequence or a smaller defined part of the reference
sequence. Percent sequence identity is represented as the identity
fraction multiplied by 100. The comparison of one or more
polynucleotide sequences may be to a full-length polynucleotide
sequence or a portion thereof, or to a longer polynucleotide
sequence. For purposes of this invention "percent identity" may
also be determined using BLASTX version 2.0 for translated
nucleotide sequences and BLASTN version 2.0 for polynucleotide
sequences.
[0106] The percent of sequence identity can be determined using the
"Best Fit" or "Gap" program of the Sequence Analysis Software
Package.TM. (Version 10; Genetics Computer Group, Inc., Madison,
Wis.). "Gap" utilizes the algorithm of Needleman and Wunsch
(Needleman and Wunsch. J Mol. Biol. 48:443-453, 1970) to find the
alignment of two sequences that maximizes the number of matches and
minimizes the number of gaps. "BestFit" performs an optimal
alignment of the best segment of similarity between two sequences
and inserts gaps to maximize the number of matches using the local
homology algorithm of Smith and Waterman (Smith and Waterman, Adv.
Appl. Math. 2:482 (1981); Smith et al., Nucleic Acids Res. 11:2205
(1983)).
[0107] Useful methods for determining sequence identity are also
disclosed in Guide to Huge Computers (Martin J. Bishop, ed.,
Academic Press, San Diego (1994)), and Carillo, H., and Lipton, D.,
Applied Math 48:1073 (1988)). More particularly, preferred computer
programs for determining sequence identity include but are not
limited to the Basic Local Alignment Search Tool (BLAST) programs
which are publicly available from National Center Biotechnology
Information (NCBI) at the National Library of Medicine, National
Institute of Health, Bethesda, Md. 20894; see BLAST Manual.
Altschul et al., NCBI. NLM. NIH; (Altschul et at, J. Mol. Biol.
215:403 (1990)); version 2.0 or higher of BLAST programs allows the
introduction of gaps (deletions and insertions) into alignments;
for peptide sequence BLASTX can be used to determine sequence
identity; and, for polynucleotide sequence BLASTN can be used to
determine sequence identity.
[0108] One aspect of the invention relates to an expression
cassette comprising a polynucleotide encoding an antisense RNA or
an interfering RNA targeted to a polynucleotide encoding a
mammalian polypyrimidine tract binding protein 1 (PTBP1). In some
embodiments, the PTBP1 is a human PTBP1.
[0109] The amino acid sequence of PTBP1 and the nucleotide sequence
encoding PTBP1 are well known in the art and available in sequences
databases such as GenBank. An exemplary cDNA sequence of human
PTBP1 is Accession No. BC002397 (SEQ ID NO:12).
[0110] The expression cassette may be any type of expression
cassette suitable for delivering the polynucleotide to a cell or
subject. The expression cassette may be part of a delivery vector.
The vector can be delivered to cells in vitro or in vivo. In other
embodiments, the vector can be delivered to cells ex vivo, and then
cells containing the vector are delivered to the subject. The
choice of delivery vector can be made based on a number of factors
known in the art, including age and species of the target host, in
vitro versus in vivo delivery, level and persistence of expression
desired, intended purpose (e.g., for therapy or screening), the
target cell or organ, route of delivery, size of the isolated
polynucleotide, safety concerns, and the like.
[0111] Suitable vectors include plasmid vectors, viral vectors
(e.g., retrovirus, alphavirus; vaccinia virus; adenovirus,
adeno-associated virus and other parvoviruses, lentivirus,
poxvirus, or herpes simplex virus), lipid vectors, poly-lysine
vectors, synthetic polyamino polymer vectors, and the like. In one
embodiment, the expression cassette is in an AAV vector.
[0112] In some embodiments, the polynucleotide encodes an antisense
RNA targeted to a mammalian PTBP1. In other embodiments, the
polynucleotide encodes an interfering RNA targeted to an mammalian
PTBP1 protein, e.g., a shRNA, a siRNA, or a miRNA.
[0113] In a particular embodiment, the interfering RNA is a siRNA
comprising the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2
or a nucleotide sequence at least 90% identical thereto, e.g., at
least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
identical.
[0114] In some embodiments, the polynucleotide is operably linked
to a promoter. In one embodiment, the promoter is a constitutive
promoter. A constitutive promoter is advantageous in the present
invention as it ensures that the antisense RNA or interfering RNA
continues to be expressed as the oligodendrocyte or oligodendrocyte
precursor cell transdifferentiates to a neuron. Alternatively, a
cell-type specific promoter may be used, e.g., an
oligodendrocyte-specific promoter.
[0115] Another aspect of the invention relates to a virus particle
comprising the expression cassette of the invention. 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, wherein the AAV
capsid packages an AAV vector genome.
[0116] In some embodiments, the virus particle has a tropism for
oligodendrocytes and/or oligodendrocyte precursor cells. Examples
of virus particles with this tropism are known in the art. In some
embodiments, the virus particle with a tropism for oligodendrocytes
and/or oligodendrocyte precursor cells is an AAV particle, e.g., an
AAV particle having a modified capsid protein that provides the
tropism, e.g., is one described in U.S. Publication No.
2015/0238550 or International Publication No. WO 2016/081811,
incorporated by reference herein in their entirety.
[0117] A further aspect of the invention relates to a composition
comprising the expression cassette and/or the virus particle of the
invention. In some embodiments, the composition is a pharmaceutical
composition comprising the expression cassette and/or the virus
particle of the invention and a pharmaceutically acceptable
carrier.
[0118] 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 virus particles (e.g., the AAV rep and AAV cap
sequences). 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.
[0119] 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 AAV rep and cap coding sequences, 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.
[0120] 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, ag, 293 cells or
other Ela trans-complementing cells.
[0121] 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 pre 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.
[0122] As a further alternative, the rep/cap sequences may be
stably carried (episomal or integrated) within a cell.
[0123] 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.
[0124] 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.
[0125] 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.
[0126] 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.
[0127] 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.
[0128] 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.
[0129] 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.
[0130] 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.
[0131] 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).
[0132] 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.
[0133] 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.
[0134] 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).
[0135] 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 at, (2002) Human Gene Therapy 13:1935-43.
[0136] Other methods of producing AAV use stably transformed
packaging cells (see, e.g., U.S. Pat. No. 5,658,785).
[0137] 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).
[0138] 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' tu/ml, at least about 10' tu/ml,
at least about 10 tu/ml, at least about 10 tu/ml, or at least about
10.sup.10 tu/ml.
[0139] A further aspect of the invention relates to a method of
attenuating expression of PTBP1 in a cell, comprising contacting
the cell with the expression cassette, virus particle, and/or
composition of the invention, wherein the expression of PTBP1 is
attenuated. In some embodiments, the cell is an oligodendrocyte or
oligodendrocyte precursor cell. In certain embodiments, the
expression of PTBP1 is attenuated by at least about 50%, e.g., at
least about 60%, 70% 80%, 90%, or 95%.
[0140] Another aspect of the invention relates to a method of
transdifferentiating an oligodendrocyte or an oligodendrocyte
precursor cell to a neuron, comprising contacting the
oligodendrocyte or oligodendrocyte precursor cell with the
expression cassette, virus particle, and/or composition of the
invention, thereby transdifferentiating the oligodendrocyte or
oligodendrocyte precursor cell to a neuron.
[0141] The transdifferentiation process may be monitored and/or
confirmed by observing the morphological, immunohistochemical,
and/or functional properties of the cell by methods known in the
art and as described herein. For example, the transdifferentiation
process may be monitored and/or confirmed by measuring the
disappearance of oligodendrocyte markers (e.g., olig2) and the
appearance of neuron markers (e.g. NeuN) in the cell. The process
may also be monitored and/or confirmed by detecting the appearance
of electrical activity (e.g., action potentials) in the cell.
[0142] In each of these methods, the cell may be an in vitro cell,
e.g., a cultured cell or a cell line. In certain embodiments, the
cell is an ex vivo cell, e.g., a primary cell isolated from a
subject. In other embodiments, the cell is an in vivo cell, e.g., a
cell in a mammalian subject. The subject may be a laboratory animal
used for research purposes. In some embodiments, the subject is a
patient, e.g., one in need of therapy.
[0143] An additional aspect of the invention relates to a method of
increasing the number of neurons in the brain of a mammalian
subject, comprising delivering to the brain the expression
cassette, virus particle, and/or composition of the invention,
thereby increasing the number of neurons in the brain of the
mammalian subject relative to the number of neurons prior to the
delivery.
[0144] A further aspect of the invention relates to a method of
transdifferentiating an oligodendrocyte or an oligodendrocyte
precursor cell to a neuron in the brain of a mammalian subject,
comprising delivering to the brain the expression cassette, virus
particle, and/or composition of the invention, thereby
transdifferentiating an oligodendrocyte or an oligodendrocyte
precursor cell to a neuron in the brain of the mammalian
subject.
[0145] In some embodiments, the mammalian subject is a laboratory
animal. In some embodiments, the mammalian subject is a human
subject.
[0146] The expression cassette, virus particle, and/or composition
of the invention may be delivered to the brain by any suitable
technique. In some embodiments, the expression cassette, virus
particle, and/or composition is injected directly into the brain,
e.g., into a region of the brain such as the striatum. In other
embodiments, the expression cassette, virus particle, and/or
composition is injected in a manner that provides access to the
brain e.g., intracerebroventricular or intrathecal injection.
[0147] Another aspect of the invention relates to a method of
treating a central nervous system disorder or condition responsive
to an increase in the number of neurons in a mammalian subject in
need thereof, the method comprising delivering to the brain the
expression cassette, virus particle, and/or composition of the
invention, thereby treating the central nervous system disorder or
condition. The subject may be any subject in need of treatment. In
some embodiments, the mammalian subject is a laboratory animal. In
some embodiments, the mammalian subject is a human subject.
[0148] The disorder or condition may be any one in which an
increase in the number of neurons in the brain would be beneficial,
e.g., would improve at least one symptom of the disorder or
condition. In some embodiments, the disorder or condition is a
neurodegenerative disorder, e.g., Parkinson's disease, Alzheimer's
disease, Huntington's chorea, or amyotrophic lateral sclerosis. In
some embodiments, the disorder or condition is a traumatic brain or
spinal cord injury or stroke. In some embodiments, the disorder or
condition is a natural condition such as aging.
[0149] In certain embodiments, the methods of the invention may
further comprise administering an additional compound, molecule, or
agent to enhance transdifferentiation of the cell. In some
embodiments, the methods further comprise delivering to the
oligodendrocyte or oligodendrocyte precursor cell or the brain a
differentiation factor that promotes transdifferentiation to
neurons. The differentiation factor may be, without limitation,
NeuroD1, Asc11, Brn2a, Myt11, SOX2, or any combination thereof.
[0150] In other embodiments, the methods further comprise
delivering to the oligodendrocyte or oligodendrocyte precursor cell
or the brain an inhibitor of expression and/or activity of a
factor, the inhibition of which results in transdifferentiation to
neurons. In one embodiment, the methods further comprise delivering
to the oligodendrocyte or oligodendrocyte precursor cell or the
brain an inhibitor of expression of the REI silencing transcription
factor complex. The inhibitor may be, for example, an antisense
RINA or an interfering RNA targeted to one or more polynucleotides
encoding proteins in the complex.
[0151] In some embodiments, the methods further comprise delivering
to the oligodendrocyte or oligodendrocyte precursor cell or the
brain a factor that promote neuron growth, e.g., a growth factor or
neurotrophic factor such as nerve growth factor, brain-derived
neurotrophic factor, glial cell line-derived neurotrophic factor,
neurotrophin-3, neurotrophin-4, and ciliary neurotrophic
factor.
[0152] In some embodiments, the methods further comprise delivering
to the oligodendrocyte or oligodendrocyte precursor cell or the
brain an additional therapeutic agent for the disorder or condition
be treated. Any suitable therapeutic agent known in the art to be
useful for treating the particular disorder or condition may be
used. Examples of therapeutic agents for neurological disorders
include, without limitation, for Alzheimer's disease: caprylidene,
donepezil, galantamine, tacrine, vitamin E, ergoloid mesylates,
rivastigmine; for Parkinson's disease: nadolol, zonisamide,
amantadine, apomorphine, belladonna, benztropine, biperiden,
bromocriptine, carbidopa, entacapone, levodopa, pergolide mesylate,
pramipexole, procyclidine, rasagiline, ropinirole, rotiotine,
scopolamine, tolcapone, trihexyphenidyl, rivastigmine, seleginline;
for Huntington's disease: baclofen, pregabalin, tetrabenazine,
methylprednisolone, desvenlafaxine, nortriptyline; and for
dementia: haloperidol and ergoloid mesylates; for amyotrophic
lateral sclerosis: riluzole, edaravone; for traumatic brain or
spinal cord injury: diuretics (e.g., mannitol, furosemide,
glycerol, urea), anti-seizure drugs, coma-inducing drugs: for
stroke: anticoagulants/antiplatelets (e.g., aspirin, warfarin),
antihypertensives, tissue plasminogen activator.
[0153] In these embodiments, the additional compound, molecule, or
agent may be delivered by contacting the oligodendrocyte or
oligodendrocyte precursor cell or the brain with the additional
compound molecule, or agent itself or with an expression vector
encoding the additional compound, molecule, or agent.
[0154] Recombinant virus vectors according to the present invention
fid 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, 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 neurodegenerative
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.
[0155] In particular embodiments, the present invention provides a
pharmaceutical composition comprising an expression vector, virus
particle and/or composition 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.
[0156] 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.
[0157] 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.
[0158] 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 a
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.
[0159] 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 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.
[0160] A further aspect of the invention is a method of
administering the virus vectors 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.
[0161] 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.13, 10.sup.14, 10.sup.15, 10.sup.16,
10.sup.17, 10.sup.18 transducing units or more, e.g. about
10.sup.7, 10.sup.8, 10.sup.9, 10.sup.10, 10.sup.11, 10.sup.12,
10.sup.13, 10.sup.14, 10.sup.15, or 10.sup.16 transducing units,
e.g., about 10.sup.12 to 10.sup.14 transducing units.
[0162] 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.
[0163] 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.
[0164] 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.
[0165] Typically, the viral vector will be adminstered 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.
[0166] 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.
[0167] 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 disease, Parkinson's
disease, amyotrophic lateral sclerosis, multiple sclerosis,
epilepsy, CNS tumors, and/or cerebral infarct. In other
embodiments, the BBB compromise can be an induced disruption, e.g.
to promote delivery of agents to the CNS. Temporary BBB compromise
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).
[0168] Delivery to any of these tissues can also be achieved by
delivering a depot comprising the virus vector, which can be
implanted into the 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).
[0169] 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).
[0170] 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.
[0171] The compositions can be presented in unit/dose or multi-dose
container, 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.
[0172] 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.
[0173] 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
Methods
[0174] siRNA and expression plasmid co-transfection: Four unique
ptbp1 siRNAs and a non-targeting control siRNA were tested (Thermo
Scientific ON-TARGETplus Rat ptbp1 gene, Thermo Scientific,
Waltham, Mass.). The prbp1 siRNA sequences were:
1) CGGCAUJCGUCCCAGACAUA (SEQ ID NO: 1),
2) CAAUGGCGGUGUGGUCAAA (SEQ ID NO: 2),
3) CAACUUJGAACCCUGAGAGA (SEQ ID NO: 3), and
[0175] 4) CCAACACUAUGGTUTAACUA (SEQ ID NO: 4). siRNA was
co-transfected with a CMV-promoter containing, DDK-tagged rat ptbp1
expression plasmid (Origene) into HeLa cells in accordance with
published Lipofectamine 2000 plasmid/siRNA co-transfection
protocols (Life Technologies, Carlsbad, Calif.). siRNA was
resuspended in siRNA buffer (Thermo Scientific, Waltham, Mass.).
Transfection was performed into 80% confluent 12-well plates, with
a volume of 1 mL antibiotic-free media. A total of 200 ng plasmid
DNA and 20 pmol dsRNA was mixed with 100 .mu.L optimum and 4 .mu.L
Lipofectamine 2000, and added to each well.
[0176] miRNA plasmid production: The BLOCK-iT.TM. Pol II miR RNAi
expression vector kit (Invitrogen, La Jolla, Calif.) was used to
prepare pol II-based miRNA from the successful siRNA constructs. In
accordance with kit instructions miRNA primers were designed based
on the two most successful siRNA sequences (siRNA 3 (SEQ ID NO: 3)
and 4 (SEQ ID NO: 4)). Sequences designed to match PTBP1 siRNA 3
and 4 were as follows: siRNA 3) Top: TGCTGTCTCT CAGGGTTCAA
GTTGCTGTTT TGGCCACTGA CTGACAGCAA CTTACCCTGA GAGA (SEQ ID NO: 5),
Bottom: CCTGTCTCTC AGGGTAAGTT GCTGTCAGTC AGTGGCCAAA ACAGCAACTT
GAACCCTGAG AGAC (SEQ ID NO: 6); siRNA 4) Top: TGCTGTAGTT AACCA
TAGTG TTGGCAGTTT TGGCCACTGA CTGACTGCCA ACAATGGTTA ACTA (SEQ ID NO:
7). Bottom: CCTGTAGTTA ACCATTGTTG GCAGTCAGTC AGTGGCCAAA ACTGCCAACA
CTATGGTTAA CTAC (SEQ ID NO: 8). Primers were prepared by Integrated
DNA technologies (Coralville, Iowa). Primers were annealed and
ligated with pre-cut pcDNA 6.2-EmGFP plasmid per manufacturer's
instructions. Using high fidelity PCR (Phusion, New England
Biotechnologies, Ipswich, Mass.), sequences were amplified for the
non-specific miRNA (included with the BLOCK-iT kit), and PTBP1
miRNAs 3 and 4, along with their 5' and 3' miR flanking regions
from the pcDNA 6.2-EmGFP plasmid using primers containing a short
overhang and a Not-1 restriction sequence: forward: AGCTGCGGCC
GCAGGGAGGT AGTGAGTCGAC (SEQ ID NO: 9), reverse: TCATGCGGCC
GCGAAAGCTG GGTCTAGATA TC (SEQ ID NO: 10). Amplified products were
gel purified, digested with Not-1 restriction enzyme (NEB), and
ligated immediately after the stop codon of EGFP in the plasmid
TR-CBA-EGFP. Plasmid sequences were verified using a primer within
the EGFP sequence: CGACAACCAC TACCTGAGC (SEQ ID NO: 11).
[0177] Virus production: Virus was produced in HEK-293 cells as
previously described (Greiger et al., Nat. Protoc. 1:1412 (2006)).
Briefly, polyethylenimine max (PEI) was used for the triple
transfection of the pXR2 or pOLIGO001 cap and rep plasmid, the
pXX6-80 helper plasmid, and the TR-EGFP plasmid containing the
nonspecific miRNA, or ptbp1 miRNA-3 or miRNA-4 flanked by inverted
terminal repeats under the chicken beta actin (CBA) promoter. Cells
were harvested between 48 and 72 h post-transfection, and virus was
purified by cesium chloride ultracentrifugation. After identifying
peak fractions by quantitative PCR (qPCR), virus was dialyzed into
phosphate-buffered saline (PBS). Titers were calculated by qPCR
according to established procedures using a LightCycler 480
instrument and SV40 pA primers (Greiger et al., Nat. Protoc. 1:1412
(2006)).
[0178] Transduction/transfection verification of miRNA:
rAAV2-packaged EGFP-miRNA virus was delivered at 1.times.10.sup.5
moi to HEK293 cells immediately upon splitting onto 12 well plates
(2.times.10.sup.5 cells per well). 24 h post-transduction 100 ng of
rat ptbp1 expression plasmid was transfected into cells using PEI
(0.8 .mu.L in 5 .mu.L serum-free RPMI).
[0179] Electrophoresis and Western blot: For both siRNA and
AAV2/miRNA knockdown studies, 48 h post-transfection cells were
harvested by washing in ice-cold PBS and lysed in a buffer
containing 150 mM NaCl, 50 mM Tris-HCl (pH 8), 0.1% NP40, 50 mM
NaF, 30 mM .beta.-glycerophosphate, 1 mM Na.sub.3VO.sub.4 and
1.times. Complete Protease Inhibitor cocktail (Roche). Equal
amounts of proteins were electrophoresed on a 10% SDS-PAGE
denaturing gel, followed by transfer onto a Hybond.TM.-ECL
nitrocellulose membrane (GE Healthcare). The membrane was blocked
with 5% fat-free powdered milk for 1 hour at room temperature,
followed by overnight incubation at 4.degree. C. in anti-DDK
(Origene) in 5% BSA. Tubulin (Cell Signaling) was used as a loading
control. After washing, the membranes were incubated with
anti-rabbit IgG-HRP. Bands were visualized by
chemiluminescence.
[0180] Animals and Stereotactic Infusions: All of the animals were
male Sprague-Dawley rats (Charles River, Morrisville, N.C., USA)
weighing approximately 300 g at time of intracranial injection. The
animals were maintained on a 12-h light-dark cycle and had free
access to water and food. For all animal studies, care and
procedures were in accordance with the National Institutes of
Health Guide for the Care and Use of Laboratory Animals, and all
procedures received prior approval by the University of North
Carolina Institutional Animal Care and Usage Committee.
[0181] Virus vector infusions were performed as previously
described (Haberman et al., Nat. Med. 9:1076 (2003)). First,
animals were anesthetized with 50 mg/kg pentobarbital and placed
into a stereotactic frame. Using a 32 gauge stainless steel
injector and a Sage infusion pump, animals received 2 .mu.l
unilaterally of either OLIG001 AAV-GFP or OLIG001 AAV-4miRNA-GFP
over 10 minutes into the striatum (0.5 mm anterior to Bregma, 3.5
mm lateral, 5.5 nu vertical, according to the atlas of Paxinos and
Watson (Paxinos e al., The Rat Brain in Stereotaxic Coordinates.
4th ed., Academic Press, New York, USA, 1998). The injector was
left in place for 3 minutes post-infusion in order to allow
diffusion from the injector. For the fluorescent head injections, 3
months after the striatal 4miRNA virus infusion, rats were
anesthetized and placed into the stereotactic frame. Subsequently
fluorescent latex beads (0.5 td/5 minutes, Fluospheres 580/605, 1:5
dilution, Molecular Probes Millipore) were infused into the globus
pallidus (1.0 mm posterior to Bregma, 3.0 mm lateral, 7.0 mm
vertical) or the substantia nigra (5.3 mm posterior to Bregma, 2.5
mm lateral, 8.0 mm vertical). The injector was left in place for 3
minutes post-infusion in order to allow diffusion from the
injector.
[0182] Immunohistochemistry and confocal microscopy: Ten days, 3
months or 6 months after the vector infusion animals received an
overdose of pentobarbital (100 mg/kg pentobarbital, ip) and were
perfused transcardially with ice-cold 100 mM sodium
phosphate-buffered saline (PBS) (pH 7.4), followed by 4%
paratbrnaldehyde in PB (pH 7.4). After brains were post-fixed 12-48
h at 4.degree. C. in the paraformaldehyde-PB, 40 .mu.m coronal
sections were cut using a vibrating blade microtome for subsequent
immunofluorescence. The sections were washed 3.times. in PBS and
blocked in 10% goat serum/PBS for 45 minutes. In order to determine
GFP cellular co-localization, tissue sections were incubated in the
blocking solution with one of the following antibody cellular
markers: NeuN (1:500, Chemicon); GFAP (1:2000, Dako); Olig2 (1:500,
Millipore); DARPP32 (1:500, ABCAM); parvalbumin (1:500, Millipore).
Following incubation at 4.degree. C. for 48-72 h in primary
antibodies, the sections were rinsed 3.times. with PBS and blocked
again for 45 minutes at room temperature. Subsequently the tissue
sections were incubated in either Alexafluor 594-conjugated
goat-anti-rabbit IgG or goat-anti mouse (1:500, Invitrogen) for 1
hour at 4.degree. C. Rinsed sections were mounted and fluorescence
was visualized using a Zeiss LM 780 confocal microscope in the UNC
Neuroscience imaging core. GFP co-localization was determined on
the Z axis.
[0183] Patch Clamp Electrophysiology: Electrophysiological
recordings were obtained from GFP fluorescent cells in both
current-clamp and voltage-clamp modes using standard
electrophysiological techniques (Ming et al., Alcohol Clin. Exp.
Res. 30:1400 (2006)). Briefly, 300 t coronal vibrotome sections
were cut in oxygenated (95% O.sub.2/5% CO.sub.2) ice cold,
bicarbonate-buffered artificial CSF. After a 1 h incubation in that
ACSF at room temperature (22.degree. C.), samples were transferred
to a flow chamber containing room temperature ACSF. Whole-cell
patch recordings were obtained from fluorescent cells using a high
Cl.sup.- internal-solution as previously described (Ming et at,
Alcohol Clin. Exp. Res. 30:1400 (2006)). Neurons were clamped to
-60 mV for voltage-clamp recording and either, maintained at their
normal resting potential or forced to -60 mV by current injection
for current-clamp recording. Brief (300 ms) current injections were
administered during voltage clamp for some cells to elicit an
action potential.
Example 2
Transdifferentiation of Oligodendrocytes
[0184] The approach taken to in vivo transdifferentiation of
oligodendrocytes into neurons relied upon two recent observations.
Xue et al. (Cell 152:82 (2013)) reported that suppression of
polypyrimidine-tract-binding (PTB) protein expression in cultured
fibroblasts caused a portion of the fibroblasts to differentiate
into functional neurons. Thus, manipulation of a single factor
could induce neuronal reprogramming. Secondly, a novel AAV vector
was recently developed where the chimeric capsid confers a dominant
oligodendrocyte tropism in the rat striatum. Based upon these two
findings, 2 siRNAs were identified that significantly inhibited
PTBP1 expression in HeLa cells (FIG. 1A) and then these siRNA
sequences were converted into miRNAs using the BLOCK-iT.TM. Pol II
miRNAi expression vector kit. Next the miRNA-GFP construct was
subcloned into an AAV plasmid where the gene expression is driven
by a hybrid chicken-beta actin promoter. Recombinant AAV serotype 2
virus was produced with this construct and the Olig001 AAV vector
(described in WO 2016/081811, incorporated herein by reference in
its entirety) and this virus was used to transduce HEK293 cells in
vitro. Subsequent western blots established that the virus derived
miRNA gene expression substantially reduced the expression of PTBP1
(FIG. 1A).
[0185] As noted above, an advantageous component to this
reprogramming approach involved the ability to target
preferentially oligodendrocytes in vivo where gene expression was
driven by a promoter active in both oligodendrocytes and neurons.
As seen in FIG. 1B, after direct striatal injection recombinant
AAV-GFP vectors containing the oligotropic capsid almost
exclusively transduced oligodendrocytes in the rat striatum. Ten
days post-infusion, the GFP positive cells exhibit the classic
morphology of striatal oligodendrocytes including the dramatic
presence of GFP in striatal patches that were composed primarily of
myelinated projection axons. Moreover, the GFP positive cells did
not co-localize with NeuN, a marker of neurons, or GFAP, a marker
of astrocytes, but did co-localize with Olig2, a marker of
oligodendrocytes (FIG. 1B). Also, this vector did not transduce
dividing cells in the striatum, because 5-bromo-2-deoxyuridine
(BrdU) administration during the initial period of AAV GFP
transduction resulted in a total absence of GFP co-localization
with BrdU-labeled striatal cells 2 weeks later (FIG. 2). Most
importantly, this dominant oligodendrocyte transduction pattern
remained stable over time, given that the same oligodendrocyte
transduction pattern was observed 6 months post-transduction (FIG.
1C). Thus, this AAV vector provided the ability to use a
constitutive promoter to express the PTBP1 miRNA predominantly in
striatal oligodendrocytes, but also subsequently in
transdifferentiated cells.
[0186] The miRNA-GFP construct was packaged into recombinant AAV
vectors using the oligodendrocyte preferring capsid plasmid and
directly injected into the rat striatum. Ten days later the
majority of GFP positive cells exhibited oligodendrocyte morphology
including a substantial GFP presence in the striatal patch,
co-localized with the oligodendrocyte marker, Olig2, but did not
co-localize with NeuN (FIG. 3). However, over time these transduced
oligodendrocytes transdifferentiated into functional striatal
neurons. As seen in FIG. 3B, by 6 weeks post-transduction, the
majority of the GFP positive cells exhibited the typical morphology
of striatal neurons where the GFP co-localized with NeuN. Also,
there was a marked absence of GFP in the striatal patches, and some
of the GFP positive cells co-localized with DARRP32, or
parvalbumin, both markers for subclasses of GABAergic striatal
neurons (FIG. 3B). As importantly, these transdifferentiated
neurons remained present at 3 and 6 months post-striatal
transduction, with little evidence of GFP positive oligodendrocytes
(FIG. 3C.
[0187] Although the transdifferentiated cells exhibited many
morphological and immunohistochemical properties indicative of
striatal neurons, the question remained whether these cells were
functional neurons. To address this question, patch-clamp
recordings were obtained from striatal slices either 6 weeks (3
cells) or 3 months (3 cells) post-AAV miRNA-GFP transduction.
Action potentials were recorded from 6 of 6 fluorescent cells where
these action potentials occurred spontaneously in 4 of 6 cells
during voltage clamp at -60 mV and in 4 of 5 during current clamp
(FIG. 4A). In the 2 cells that did not exhibit spontaneous action
potentials, action-potentials were elicited by a 300 ms current
injection. Spontaneous post-synaptic currents were observed in 5 of
6 cells examined (FIG. 4B). These spontaneous post-synaptic
currents indicate that the recorded neurons were responding to
neuronal inputs. Resting-potential values were determined in 5 of
the 6 neurons and ranged from a low of -26 mV to a high of -71 mV,
with a mean of -47.6 mV.
[0188] Further functional validation was obtained by the presence
and function of GFP positive terminals in two areas of striatal
projection, the globus pallidus and the substantia nigra. Normally,
striatal transduction with the Olig001 AAV GFP vector does not
result in GFP positive terminals in the globus pallidus or
substantia nigra. However, GFP positive axon terminals were present
in the globus pallidus and the substantia nigra 3 months after
striatal transduction by the AAV4 miRNA GFP vectors (FIG. 4C). In
order to test the functional nature of these terminals, 0.04 micron
fluorescent heads were infused into either the globus pallidus or
the substantia nigra, 3 months after striatal AAV-miRNA-GFP vector
administration. Two weeks later the rats were sacrificed and
GFP-fluorescent bead co-localization was determined. Confocal
microscopy identified a number of GFP positive striatal cells that
contained the fluorescent beads from either the globus pallidus or
the substantia nigra (FIG. 4C. Thus, the GFP positive presynaptic
terminals proved capable of internalizing the fluorescent beads and
retrogradely transporting the beads back to the striatal cell body.
The presence of this retrograde axonal transport indicates
functional presynaptic terminals.
[0189] The present results demonstrate that a single non-toxic AAV
vector can induce the transdifferentiation of resident
oligodendrocytes into functional neurons in the rat striatum. This
oligodendrocyte reprogramming produces cells that exhibit
morphological, immunohistochemical and electrophysiological
properties of mature striatal neurons. Olig001 capsid AAV4 miRNA
GFP-transduced striatal cells initially exhibited morphological and
immunohistochemical properties unique to oligodendrocytes, similar
to striatal transduction by control Olig001 capsid AAV GFP vectors.
However, in marked contrast to control Olig001 capsid AAV GFP
vectors, by 6 weeks prost treatment the transduced cells exhibited
morphological, immunohistochemical, and electrophysiological
properties unique to mature striatal neurons. Since Olig001 AAV
GFP-transduced cells did not co-localize with BrdU-labeled cells,
this transition was not due to a differentiation of dividing
progenitor cells. Furthermore, many of the transdifferentiated
neurons expressed cellular markers indicative of subclasses of
striatal neurons, so it is likely that the striatal milieu exerts a
significant influence on the fate of transdifferentiated cells.
Also, the occurrence of inhibitory postsynaptic currents indicates
that these transdifferentiated neurons have integrated into the
local circuitry, while the presence of distal axonal projections
and functional retrograde transport suggest some level of
structure-appropriate, distal integration. These measures strongly
support the presence of functional transdifferentiated neurons.
[0190] An advantageous element to this neuronal replacement
strategy involved the ability to express the gene product over the
course of the initial transduction, as well as in the subsequent
transdifferentiated cell. The vast majority of AAV serotypes
exhibit a predominant neuronal tropism, when gene expression is
driven by a constitutive promoter. However, previous studies have
shown that AAV8 or AAV1/2 serotypes can selectively support in vivo
oligodendrocyte gene expression, but this oligodendrocyte tropism
requires the use of oligodendrocyte-specific promoters, such as
myelin basic protein. Because the oligodendrocyte-specific promoter
is not active in a neuron, as the oligodendrocyte
transdifferentiates into a mature neuron, the resumption of PTB
protein expression likely would reprogram the transdifferentiated
neuron back to an oligodendrocyte or a cellular intermediate. By
employing the Olig001 capsid AAV vector and a constitutive
promoter, striatal gene expression was driven both in the initial
transduced oligodendrocytes and in the subsequent
transdifferentiated neurons. Certainly, the long-term PTB protein
repression proved effective, as evidenced by the presence of
transdifferentiated neurons 6 mouths post-transduction.
[0191] Another property of this vector-derived in vivo
transdifferentiation involved the overall relative efficacy. By 6
weeks post-transduction, substantial numbers of the GFP-positive
cells in the striatum exhibited neuronal properties with very few
remaining GFP-positive oligodendrocytes or GFP-labeled striatal
patches. In comparison, lentiviral-mediated suppression of PTB
protein expression achieved an 8%-15% efficacy for in vitro
embryonic fibroblast re-programming, while retroviral expression of
ND4 and Insm1 induced 40% of cultured astrocytes to differentiate
into neuronal-like cells. One possible explanation for these
differences could be the fact that our viral vector approach
selectively targets endogenous cells within the CNS.
[0192] 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
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19219RNAArtificialptbp1 siRNA 2caauggcggu guggucaaa
19319RNAArtificialptbp1 siRNA 3caacuugaac ccugagaga
19419RNAArtificialptbp1 siRNA 4ccaacacuau gguuaacua
19564DNAArtificialPrimer 5tgctgtctct cagggttcaa gttgctgttt
tggccactga ctgacagcaa cttaccctga 60gaga 64664DNAArtificialPrimer
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60agac 64764DNAArtificialPrimer 7tgctgtagtt aaccatagtg ttggcagttt
tggccactga ctgactgcca acaatggtta 60acta 64864DNAArtificialPrimer
8cctgtagtta accattgttg gcagtcagtc agtggccaaa actgccaaca ctatggttaa
60ctac 64931DNAArtificialPrimer 9agctgcggcc gcagggaggt agtgagtcga c
311032DNAArtificialPrimer 10tcatgcggcc gcgaaagctg ggtctagata tc
321119DNAArtificialPrimer 11cgacaaccac tacctgagc 19123281DNAHomo
sapiens 12ctcggagccg ttgggtcggt tcctgctatt ccggcgcctc cactccgtcc
cccgcgggtc 60tgctctgtgt gccatggacg gcattgtccc agatatagcc gttggtacaa
agcggggatc 120tgacgagctt ttctctactt gtgtcactaa cggaccgttt
atcatgagca gcaactcggc 180ttctgcagca aacggaaatg acagcaagaa
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tccggaagct ccccatcgac gtcacggagg gggaagtcat 300ctccctgggg
ctgccctttg ggaaggtcac caacctcctg atgctgaagg ggaaaaacca
360ggccttcatc gagatgaaca cggaggaggc tgccaacacc atggtgaact
actacacctc 420ggtgacccct gtgctgcgcg gccagcccat ctacatccag
ttctccaacc acaaggagct 480gaagaccgac agctctccca accaggcgcg
ggcccaggcg gccctgcagg cggtgaactc 540ggtccagtcg gggaacctgg
ccttggctgc ctcggcggcg gccgtggacg cagggatggc 600gatggccggg
cagagccccg tgctcaggat catcgtggag aacctcttct accctgtgac
660cctggatgtg ctgcaccaga ttttctccaa gttcggcaca gtgttgaaga
tcatcacctt 720caccaagaac aaccagttcc aggccctgct gcagtatgcg
gaccccgtga gcgcccagca 780cgccaagctg tcgctggacg ggcagaacat
ctacaacgcc tgctgcacgc tgcgcatcga 840cttttccaag ctcaccagcc
tcaacgtcaa gtacaacaat gacaagagcc gtgactacac 900acgcccagac
ctgccttccg gggacagcca gccctcgctg gaccagacca tggccgcggc
960cttcggtgca cctggtataa tctcagcctc tccgtatgca ggagctggtt
tccctcccac 1020ctttgccatt cctcaagctg caggcctttc cgttccgaac
gtccacggcg ccctggcccc 1080cctggccatc ccctcggcgg cggcggcagc
tgcggcggca ggtcggatcg ccatcccggg 1140cctggcgggg gcaggaaatt
ctgtattgct ggtcagcaac ctcaacccag agagagtcac 1200accccaaagc
ctctttattc ttttcggcgt ctacggtgac gtgcagcgcg tgaagatcct
1260gttcaataag aaggagaacg ccctagtgca gatggcggac ggcaaccagg
cccagctggc 1320catgagccac ctgaacgggc acaagctgca cgggaagccc
atccgcatca cgctctcgaa 1380gcaccagaac gtgcagctgc cccgcgaggg
ccaggaggac cagggcctga ccaaggacta 1440cggcaactca cccctgcacc
gcttcaagaa gccgggctcc aagaacttcc agaacatatt 1500cccgccctcg
gccacgctgc acctctccaa catcccgccc tcagtctccg aggaggatct
1560caaggtcctg ttttccagca atgggggcgt cgtcaaagga ttcaagttct
tccagaagga 1620ccgcaagatg gcactgatcc agatgggctc cgtggaggag
gcggtccagg ccctcattga 1680cctgcacaac cacgacctcg gggagaacca
ccacctgcgg gtctccttct ccaagtccac 1740catctagggg cacaggcccc
cacggccggg ccccctggcg acaacttcca tcattccaga 1800gaaaagccac
tttaaaaaca gctgaagtga ccttagcaga ccagagattt tattttttta
1860aagagaaatc agtttacctg tttttaaaaa aattaaatct agttcacctt
gctcaccctg 1920cggtgacagg gacagctcag gctcttggtg actgtggcag
cgggagttcc cggccctcca 1980cacccggggc cagaccctcg gggccatgcc
ttggtggggc ctgtgtcggg cgtggggcct 2040gcaggtgggc gccccgacca
cgacttggct tccttgtgcc ttaaaaaacc tgccttcctg 2100cagccacaca
cccacccggg gtgtcctggg gacccaaggg gtgggggggt cacaccagag
2160agaggcaggg ggcctggccg gctcctgcag gatcatgcag ctggggcgcg
gcggccgcgg 2220ctgcgacacc ccaaccccag ccctctaatc aagtcacgtg
attctccctt caccccgccc 2280ccagggcctt cccttctgcc cccaggcggg
ctccccgctg ctccagctgc ggagctggtc 2340gacataatct ctgtattata
tactttgcag ttgcagacgt ctgtgcctag caatatttcc 2400agttgaccaa
atattctaat cttttttcat ttatatgcaa aagaaatagt tttaagtaac
2460tttttatagc aagatgatac aatggtatga gtgtaatcta aacttccttg
tggtattacc 2520ttgtatgctg ttacttttat tttattcctt gtaattaagt
cacaggcagg acccagtttc 2580cagagagcag gcggggccgc ccagtgggtc
aggcacaggg agccccggtc ctatcttaga 2640gcccctgagc ttcagggaag
gggcgggcgt gtcgccgcct ctggcatcgc ctccggttgc 2700cttacaccac
gccttcacct gcagtcgcct agaaaacttg ctctcaaact tcagggtttt
2760ttcttccttc aaattttgga ccaaagtctc atttctgtgt tttgcctgcc
tctgatgctg 2820ggacccggaa ggcgggcgct cctcctgtct ttgtgctctt
tctaccgccc ccgcgtcctg 2880tcccgggggc tctcctagga tcccctttcc
gtaaaagcgt gtaacaaggg tgtaaatatt 2940tataattttt tatacctgtt
gtgagacccg aggggcggcg gcgcggtttt ttatggtgac 3000acaaatgtat
attttgctaa cagcaattcc aggctcagta ttgtgaccgc ggagccacag
3060gggaccccac gcacattccg ttgccttacc cgatggcttg tgacgcggag
agaaccgatt 3120aaaaccgttt gagaaactcc tcccttgtct agccctgtgt
tcgctgtgga cgctgtagag 3180gcaggttggc cagtctgtac ctggacttcg
aataaatctt ctgtatcctc aaaaaaaaaa 3240aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa a 3281
* * * * *