Note: Descriptions are shown in the official language in which they were submitted.
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TAU MODULATORS AND METHODS AND COMPOSITIONS FOR
DELIVERY THEREOF
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims the benefit of U.S. Provisional
Application No. 62/428,871, filed December 1, 2016; U.S. Provisional
Application
No. 62/450,895, filed January 26, 2017; U.S. Provisional Application No.
62/466,198,
filed March 2, 2017; U.S. Provisional Application No. 62/500,807, filed May 3,
2017;
and U.S. Provisional Application No. 62/584,342, filed November 10, 2017, the
disclosures of which are hereby incorporated by reference in their entireties.
TECHNICAL FIELD
[0002] The present disclosure is in the field of diagnostics and
therapeutics for
tauopathies such as Alzheimer's Disease.
BACKGROUND
[0003] Many, perhaps most physiological and pathophysiological
processes
can be associated by the aberrant up or down regulation of gene expression.
Examples include the inappropriate expression of proinflamatory cytokines in
rheumatoid arthritis, under expression of the hepatic LDL receptor in
hypercholesteremia, over expression of proangiogenic factors and under
expression of
antiangiogenic factors in solid tumor growth, to name just a few. In addition,
pathogenic organisms such as viruses, bacteria, fungi, and protozoa could be
controlled by altering gene expression.
[0004] Promoter regions of genes typically comprise proximal, core and
downstream elements, and transcription can be regulated by multiple enhancers.
These sequences contain multiple binding sites for a variety of transcription
factors
and can activate transcription independent of location, distance or
orientation with
.. respect to the promoter sequence. In order to achieve gene expression
regulation,
enhancer-bound transcription factors loop out the intervening sequences and
contact
the promoter region. In addition, activation of eukaryotic genes can require
de-
compaction of the chromatin structure, which can be carried out by recruitment
of
histone modifying enzymes or ATP-dependent chromatin remodeling complexes such
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that chromatin structure is altered and the accessibility of the DNA to other
proteins
involved in gene expression is increased (Ong and Corces (2011) Nat Rev
Genetics
12:283).
[0005] Perturbation of chromatin structure can occur by several
mechanisms-
some which are localized for a specific gene, and others that are genome wide
and
occur during cellular processes such as mitosis where condensation of the
chromatin
is required. Lysine residues on histones may become acetylated, effectively
neutralizing the charge interaction between the histone proteins and the
chromosomal
DNA. This has been observed at the hyperacetylated and highly transcribed 0-
globin
locus which has also been shown to be DNAse sensitive, a hallmark of general
accessibility. Other types of histone modifications that have been observed
include
methylation, phosphorylation, deamination, ADP ribosylation, addition of 13-N-
acetlyglucosamine sugars, ubiquitylation and sumoylation (see Bannister and
Kouzarides (2011) Cell Res 21:381).
[0006] Repression or activation of disease associate genes has been
accomplished through the use of engineered transcription factors. Methods of
designing and using engineered zinc finger transcription factors (ZFP-TF) are
well
documented (see for example U.S. Patent No. 6,534,261), and more recently both
transcription activator like effector transcription factors (TALE-TF) and
clustered
regularly interspaced short palindromic repeat Cas based transcription factors
(CRISPR-Cas-TF) have also been described (see review Kabadi and Gersbach
(2014)
Methods 69(2): 188-197). Non-limiting examples of targeted genes include
phospholamban (Zhang et al., (2012) Mol Ther 20(8): 1508-1515), GDNF
(Langaniere et al., (2010) 1 Neurosci 39(49): 16469) and VEGF (Liu et al.,
(2001)
Biol Chem 276:11323-11334). In addition, activation of genes has been achieved
by
use of a CRIPSR/Cas-acetyltransferase fusion (Hilton et al., (2015) Nat
Biotechnol
33(5):510-517). Engineered TFs that repress gene expression (repressors) have
also
been shown to be effective in treating trinucleotide disorders such as
Huntingtin's
Disease (HD). See, e.g., U.S. Patent No. 8,956,8282 and U.S. Patent
Publication No.
2015/0335708.
[0007] Alzheimer's Disease (AD), is a complex, multifactorial disease
characterized by several distinct disease mechanisms that are not completely
understood, and may interact with each other in as of yet poorly understood
ways. An
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estimated 5.3 million Americans of all ages have AD, making it one of the top
ten
causes of death in America, and it is estimated that by the year 2050, there
will be
106.2 million people worldwide with the disease (van Dijk etal., (2015)Front
Neurosci 9, art. 173). The disease is more prevalent in women (two thirds of
cases)
and also people of African or Hispanic descent are more likely to develop AD
than
people of Caucasian descent. The causes of AD appear to be related to genetics
(especially for early onset, 5% of cases) and environmental and lifestyle
factors.
Typically, the disease is diagnosed in a person's mid-sixties although by the
time a
diagnosis is made, the disease has been progressing for years or even decades.
The
disease continues to be progressive over time, and thus far no therapeutic
interventions have been identified that curtail or reverse the effects of the
disease.
[0008] The hallmark of the disease is a loss of synapses in the brain,
which
leads to cognitive decline. In a healthy brain, synaptic plasticity is thought
to be what
allows learning and memory formation. During the course of AD, synaptic
plasticity
is altered and many of the mechanisms involved in maintaining that plasticity
are
dysregulated, leading to synapse dysfunction and collapse (Spires-Jones and
Hyman
(2014) Neuron 82:756).
[0009] Although it appears that there are many molecular factors that
influence the onset and progression of AD, most of the scientific focus has
been on
two main players in the disease. The first is a 40-42 amino acid fragment of
the
amyloid precursor protein (APP) called amyloid 13 (AP), which is generated via
proteolytic cleavage by beta secretase and gamma secretase (Olsson etal.,
(2014)J
Blot Chem 289(3):1540-1550). Insoluble AP fragments accumulate in 'senile'
plaques in the brain although there does not seem to be a strict correlation
between the
presence of these plaques and neurodegeneration.
[0010] The other actor, Tau, has also received a great deal of
attention. Tau is
a microtubule-associated protein that was originally thought to stabilize
microtubules.
In AD patients, and in the elderly in general but to a lesser extent, tau can
accumulate
in neurofibrillary tangles (NFT). During the course of the disease, tau
becomes
hyperphosphorylated and detaches from microtubules and accumulates in
filaments.
In contrast to the AP plaques, there is a direct correlation between the
presence of
NFT and cognitive decline (Spires-Jones and Hyman, ibid).
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[0011] Interestingly, both tau and AP appear to have important roles
in normal
synaptic function. Tau appears to play an important role in transporting
mitochondria
in the cell to the synapse and it has been shown that overexpression of tau
inhibits this
transport. Impairment of mitochondrial transport is thought to cause synapse
loss due
to the essential roles mitochondria play in ATP production and calcium
buffering. AP
appears to play role in synapse plasticity in a healthy cell. However, the
accumulation
of these two proteins in plaques and tangles is associated with AD
progression. In
fact, it appears that the appearance of the amyloid plaques is important in
the earliest
stages of AD and this leads to the appearance of the NFT, and that the two
proteins
actually act synergistically with each other to speed progression of the
disease (Pooler
et al., (2015) Acta Neuropath Comm 3(14):1). It is becoming more apparent
however
that the soluble forms of these proteins contribute to toxicity (Spires-Jones
and
Hyman, ibid).
[0012] In fact, abnormal levels and/or aggregation of tau has been
implicated
in a number of conditions, collectively referred to as tauopathies. These
include
Alzheimer's Disease AD, Frontotermporal dementia (FTD, see Benussi etal.,
(2015)
Front Ag Neuro 7, art. 171)), Progressive Supranuclear Palsy (PSP),
intractable
genetic epilepsies (e.g. Dravat syndrome, see Gheyara etal., (2014) Ann Neurol
76:443-456) and Corticobasal degeneration (CBD, see Scholz and Bras 2015, Intl
Mo/ Sci 16(10): 24629-24655). Reduction of tau expression in adult mice using
antisense oligonucleotides directly to the cerebral spinal fluid (CSF) caused
a
complete or partial reduction in tau levels and also protected the treated
mice from
chemical induced seizures in terms of seizure severity (DeVos etal., (2013) J
of
NeuroSci 33(31):12887).
[0013] AD has been shown to proceed through the brain in a hierarchical
pattern, starting at the entorhinal cortex and then spreading through the
hippocampal
formation, limbic and association cortices, and finally affecting most brain
areas in
the late stages of the disease. Interestingly, progression of AD is marked by
the
appearance of NFT, implicating tau in the later stages of the disease. It has
even been
suggested that tau may have prion like properties, as work showing that
misfolded,
highly phosphorylated tau protein is more easily taken up by neurons and may
propagate the disease through the brain, and that this misfolded tau isolated
from
brains of AD patients can be readily taken up by mouse neurons (Takeda etal.,
(2015)
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Nat Comm doi:10.1038/ncomms9490; Hyman (2014) Neuron 82:1189). In addition,
work done with a transgenic mouse model showed that expression of a human tau
mutant that is linked to tangle formation only in the entorhinal cortex of the
mouse
brain led to the misfolding of mouse tau and aggregation of that tau in
neurons
without any detectable human tau expression (de Calignon etal., (2012) Neuron
73:685-697), suggesting that the misfolded human protein is able to 'seed'
misfolding
and cause aggregation of the mouse proteins. Further, genetic reduction or
loss of
endogenous mouse tau is protective against neuropathological toxicity caused
by
overexpression of a mutant human tau transgene (Wegmann et al., (2015) EAJBO
J.
34(24):3028-41).
[0014] Thus, there remains a need for methods for the prevention
and/or
treatment of tauopathies, including AD, FTD, PSP CBD and seizures; including
for
modalities that exhibit widespread delivery to the brain.
SUMMARY
[0015] Disclosed herein are methods and compositions for diagnosing,
preventing and/or treating one or more taupathies, such as Alzheimer's Disease
(AD).
In particular, provided herein are methods and compositions for modifying
(e.g.,
modulating expression of) a tau allele so as to treat at least one tauopathy
such as AD,
including engineered transcription factor repressors (that repress tau
expression).
Further, these methods and compositions can be used to modify a tau allele for
the
treatment and/or prevention of other tauopathies, including AD, FTD, PSP CBD
and/or seizures. In particular, provided herein are methods and compositions
for
detecting, reducing and/or eliminating tau aggregates in a subject with a
tauopathy.
[0016] Thus, described herein are genetic modulators of a microtubule
associated protein tau (MAPT) gene, the modulator comprising: a DNA-binding
domain that binds to a target site of at least 12 nucleotides in the MAPT
gene; and
functional domain (e.g., a transcriptional regulatory domain (such as a
repression
domain or an activation domain) or nuclease domain). Any DNA-binding domain
can
be used, including but not limited to, a zinc finger protein (ZFP), a TAL-
effector
domain protein (TALE), a single guide RNA (of a CRISPR systems), an Argonaute
protein and the like. One or more polynucleotides, including viral and non-
viral gene
delivery vehicles (e.g., as mRNA, plasmids, AAV vectors, lentiviral vectors,
Ad
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vectors) encoding the genetic modulators as described herein (or one or more
components thereof on the same or different polynucleotides) are also
provided. In
certain embodiments, the gene delivery vehicle comprises an AAV vector,
including,
but not limited, to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV8, AAV 8.2,
AAV9, AAV rhl 0, pseudotypes of these vectors (e.g., as AAV2/8, AAV2/5,
AAV2/6,
AAV2/9, etc.), including AAV vector variants known in the art (e.g. U.S.
Patent No.
9,585,971 and U.S. Provisional Patent Application No. 62/503,121).
Pharmaceutical
compositions and isolated cells comprising one or more of the genetic
modulators,
one or more polynucleotides, and/or one or more gene delivery vehicles are
also
provided. The invention also provides methods and uses for modulating MAPT
expression in a subject in need thereof, including by providing to the subject
one or
more polynucleotides, one or more gene delivery vehicles, and/or a
pharmaceutical
composition as described herein. In certain embodiments, the compositions
described
herein are used to repress MAPT expression in the subject, including for
treatment
and/or prevention of a tauopathy (e.g., by reducing the amount of tau in the
subject).
The compositions described herein reduce tau levels for sustained periods of
time (6
months to year or more) in the brain (including but not limited to frontal
cortex,
anterior cortex, posterior cortex, hippocampus, brain stem, striatum,
thalamus,
midbrain, cerebellum) and spinal cord (including but not limited to lumbar,
thoracic
and cervical regions). The compositions described herein may be provided to
the
subject by any administration means, including but not limited to,
intracerebroventricular, intrathecal, intracranial, intravenous, orbital
(retro-orbital
(R0)) and/or intracisternal administration. Kits comprising one or more of the
compositions (e.g., genetic modulators, polynucleotides, pharmaceutical
compositions
and/or cells) as described herein as well as instructions for use of these
compositions
are also provided.
[0017] Thus, in one aspect, engineered (non-naturally occurring)
genetic
modulators (e.g., repressors) of one or more tau genes are provided. The
genetic tau
modulators may comprise systems (e.g., zinc finger proteins, TAL effector
(TALE)
proteins or CRISPR/dCas-TF) that modulate (e.g., repress) expression of a tau
allele.
Engineered zinc finger proteins or TALEs are non-naturally occurring zinc
finger or
TALE proteins whose DNA binding domains (e.g., recognition helices or RVDs)
have
been altered (e.g., by selection and/or rational design) to bind to a pre-
selected target
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site. Any of the zinc finger proteins described herein may include 1, 2, 3, 4,
5, 6 or
more zinc fingers, each zinc finger having a recognition helix that binds to a
target
subsite in the selected sequence(s) (e.g., gene(s)). Similarly, any of the
TALE
proteins described herein may include any number of TALE RVDs. In some
embodiments, at least one RVD has non-specific DNA binding characteristics. In
some embodiments, at least one recognition helix (or RVD) is non-naturally
occurring. A CRISPR/Cas-TF includes a single guide RNA that binds to a target
sequence. In certain embodiments, the engineered transcription factor binds to
(e.g.,
via a ZFP, TALE or sgRNA DNA binding domain) to an at least 12 base pair
target
.. site in a tau-encoding gene, for example a target site comprising at least
12 base pairs
(e.g., 12, 13, 14, 15, 16, 17, 18 or more) of Tables 1 through 3 (SEQ ID Nos:1
to 6, 33
and 44-46), including contiguous or non-contiguous sequences within these
target
sites. In certain embodiments, the zinc finger proteins DNA-binding domains
have
the recognition helices in the proteins shown in any of Tables 1 to 3,
including the
ZFPs designated 52288, 52322, 52366, 57890, 57880, 65888, 52364, 52389, 65894,
57930, 65918, 65920, 65887, 57947, 65968, 65976 or 65860 of Tables 1, 2 and 3.
In
certain embodiments, the genetic modulator is a genetic repressor that
comprises a
DNA-binding domain (ZFP, TALE, single guide RNA) as described herein operably
linked to a transcriptional repression domain. In other embodiments, the
genetic
modulator is a genetic repressor comprising a DNA-binding domain (ZFP, TALE,
single guide RNA) as described herein operably linked to at least one nuclease
domain (e.g., one, two or more nuclease domains). The resulting nuclease is
capable
of genetically modifying (by insertions and/or deletions) the target gene, for
example,
within the DNA-binding domain target sequence(s); within the cleavage site(s);
near
(1-50 or more base pairs) from the target sequence(s) and/or cleavage site(s);
and/or
between paired target sites when a pair of nucleases is used for cleavage.
[0018] In certain embodiments, the zinc finger proteins (ZFPs), Cas
protein of
a CRISPR/Cas system or TALE proteins as described herein can be placed in
operative linkage with a regulatory domain (or functional domain) as part of a
fusion
protein. The functional domain can be, for example, a transcriptional
activation
domain, a transcriptional repression domain and/or a nuclease (cleavage)
domain. By
selecting either an activation domain or repression domain for use with the
DNA-
binding domain, such molecules can be used either to activate or to repress
tau
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expression. In certain embodiments, the functional or regulatory domains can
play a
role in histone post-translational modifications. In some instances, the
domain is a
histone acetyltransferase (HAT), a histone deacetylase (HDAC), a histone
methylase,
or an enzyme that sumolyates or biotinylates a histone or other enzyme domain
that
allows post-translation histone modification regulated gene repression
(Kousarides
(2007) Cell 128:693-705). In some embodiments, a molecule comprising a ZFP,
dCas or TALE targeted to a tau gene (e.g., MAPT) as described herein fused to
a
transcriptional repression domain that can be used to down-regulate tau
expression is
provided. In some embodiments, the methods and compositions of the invention
are
useful for treating eukaryotes. In certain embodiments, the activity of the
regulatory
domain is regulated by an exogenous small molecule or ligand such that
interaction
with the cell's transcription machinery will not take place in the absence of
the
exogenous ligand. Such external ligands control the degree of interaction of
the ZFP-
TF, CRISPR/Cas-TF or TALE-TF with the transcription machinery. The regulatory
domain(s) may be operatively linked to any portion(s) of one or more of the
ZFPs,
dCas or TALEs, including between one or more ZFPs, dCas or TALEs, exterior to
one or more ZFPs, dCas or TALEs and any combination thereof In preferred
embodiments, the regulatory domain results in a repression of gene expression
of the
targeted tau gene. Any of the fusion proteins described herein may be
formulated into
a pharmaceutical composition.
[0019] In some embodiments, the methods and compositions of the
invention
include use of two or more fusion proteins as described herein, for instance
two or
more tau modulators (e.g., tau repressors). The two or more fusion proteins
may bind
to different target sites and comprise the same or different functional
domains.
Alternatively, the two or more fusion proteins as described herein may bind to
the
same target site but include different functional domains. In some instances,
three or
more fusion proteins are used, in others, four or more fusion proteins are
used, while
in others, 5 or more fusion proteins are used. In preferred embodiments, the
two or
more, three or more, four or more, or five or more fusion proteins are
delivered to the
cell as nucleic acids. In preferred embodiments, the fusion proteins cause a
repression
of the expression of the targeted gene. In some embodiments, two fusion
proteins are
given at doses where each protein is active on its own but in combination the
repression activity is additive. In preferred embodiments, two fusion proteins
are
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given at doses where neither is active on its own, but in combination, the
repression
activity is synergistic.
[0020] In some embodiments, the engineered DNA binding domains as
described herein can be placed in operative linkage with nuclease (cleavage)
domains
as part of a fusion protein. In some embodiments, the nuclease comprises a
Ttago
nuclease. In other embodiments, nuclease systems such as the CRISPR/Cas system
may be utilized with a specific single guide RNA to target the nuclease to a
target
location in the DNA. In certain embodiments, such nucleases and nuclease
fusions
may be utilized for targeting tau alleles in stem cells such as induced
pluripotent stem
cells (iPSC), human embryonic stem cells (hESC), mesenchymal stem cells (MSC)
or
neuronal stem cells wherein the activity of the nuclease fusion will result in
reduced
expression of a tau allele. In certain embodiments, pharmaceutical
compositions
comprising the modified stem cells are provided.
[0021] In yet another aspect, a polynucleotide encoding any of the DNA
binding proteins, nucleases and/or transcription factors described herein is
provided.
In certain embodiments, the polynucleotide comprises at least one AAV vector
(or
pseudotype or variant thereof), including but not limited to one or more AAV2,
AAV2/9, AAV6, or an AAV9 vector, including but not limited to one or more AAV
vectors as described in U.S. Patent No. 9,585,971 or U.S. Patent No.
7,198,951)
and/or one or more AAV vectors as described in U.S. Provisional Patent
Application
No. 62/503,121.
[0022] In other aspects, the invention comprises delivery of a donor
nucleic
acid to a target cell. The donor may be delivered prior to, after, or along
with the
nucleic acid encoding the nuclease(s). The donor nucleic acid may comprise an
exogenous sequence (transgene) to be integrated into the genome of the cell,
for
example, an endogenous locus. In some embodiments, the donor may comprise a
full-length gene or fragment thereof flanked by regions of hom*ology with the
targeted
cleavage site. In some embodiments, the donor lacks hom*ologous regions and is
integrated into a target locus through hom*ology independent mechanism (i.e.
NHEJ).
The donor may comprise any nucleic acid sequence, for example a nucleic acid
that,
when used as a substrate for hom*ology-directed repair of the nuclease-induced
double-strand break, leads to a donor-specified deletion to be generated at
the
endogenous chromosomal locus or, alternatively (or in addition to), novel
allelic
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forms of (e.g., point mutations that ablate a transcription factor binding
site) the
endogenous locus to be created. In some aspects, the donor nucleic acid is an
oligonucleotide wherein integration leads to a gene correction event, or a
targeted
deletion. In some embodiments, the donor encodes a transcription factor
capable of
repressing Tau expression. In other embodiments, the donor encodes a RNA
molecule that inhibits expression of the Tau protein.
[0023] In some embodiments, the polynucleotide encoding the DNA
binding
protein is an mRNA. In some aspects, the mRNA may be chemically modified (See
e.g. Kormann etal., (2011) Nature Biotechnology 29(2):154-157). In other
aspects,
the mRNA may comprise an ARCA cap (see U.S. Patent Nos. 7,074,596 and
8,153,773). In further embodiments, the mRNA may comprise a mixture of
unmodified and modified nucleotides (see U.S. Patent Publication No.
2012/0195936).
[0024] In yet another aspect, a gene delivery vector comprising any of
the
polynucleotides (e.g., encoding the genetic modulators (repressors)) as
described
herein is provided. In certain embodiments, the vector is an adenovirus vector
(e.g.,
an Ad5/F35 vector), a lentiviral vector (LV) including integration competent
or
integration-defective lentiviral vectors, or an adenovirus associated viral
vector
(AAV). In certain embodiments, the AAV vector is an AAV2, AAV6 or AAV9
vector. In some embodiments, the AAV vector is an AAV variant capable of
crossing
the blood-brain barrier (e.g. U.S. Patent No. 9,585,971 and U.S. Provisional
Patent
Application No. 62/503,121). Also provided herein are adenovirus (Ad) vectors,
LV
or adenovirus associate viral vectors (AAV) comprising a sequence encoding at
least
one nuclease (ZFN or TALEN) and/or a donor sequence for targeted integration
into a
target gene. In certain embodiments, the Ad vector is a chimeric Ad vector,
for
example an Ad5/F35 vector. In certain embodiments, the lentiviral vector is an
integrase-defective lentiviral vector (IDLV) or an integration competent
lentiviral
vector. In certain embodiments, the vector is pseudo-typed with a VSV-G
envelope,
or with other envelopes.
[0025] Additionally, pharmaceutical compositions comprising the nucleic
acids (e.g., delivery (e.g., AAV) vectors comprising sequences encoding the
artificial
transcription factors (tau repressors) described herein) and/or proteins
(e.g., ZFPs, Cas
or TALEs or fusion proteins comprising the ZFPs, Cas or TALEs) are also
provided.
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For example, certain compositions include a nucleic acid comprising a sequence
that
encodes one of the ZFPs, Cas or TALEs described herein operably linked to a
regulatory sequence, combined with a pharmaceutically acceptable carrier or
diluent,
wherein the regulatory sequence allows for expression of the nucleic acid in a
cell. In
certain embodiments, the ZFPs, CRISPR/Cas or TALEs encoded are specific for a
mutant tau allele. In some embodiments, pharmaceutical compositions comprise
ZFPs, CRISPR/Cas or TALEs that modulate a mutant tau allele and ZFPs,
CRISPR/Cas or TALEs that modulate a neurotrophic factor. Protein based
compositions include one of more ZFPs, CRISPR/Cas or TALEs as disclosed herein
and a pharmaceutically acceptable carrier or diluent.
[0026] In yet another aspect also provided is an isolated cell
comprising any
of the proteins, polynucleotides and/or compositions as described herein.
[0027] In another aspect, provided herein are methods for treating
and/or
preventing a tauopathy such as Alzheimer's Disease or seizure using the
methods and
compositions described herein. In some embodiments, the methods involve
compositions where the polynucleotides and/or proteins may be delivered using
a
viral vector, a non-viral vector (e.g., plasmid) and/or combinations thereof
In some
embodiments, the methods involve compositions comprising stem cell populations
comprising a ZFP or TALE, or altered with the ZFNs, TALENs, Ttago or the
CRISPR/Cas nuclease system of the invention. Administration of compositions as
described herein (proteins, polynucleotides, cells and/or pharmaceutical
compositions
comprising these proteins, polynucleotides and/or cells) result in a
therapeutic
(clinical) effect, including, but not limited to, amelioration or elimination
of any the
clinical symptoms associate with AD, tauopathies or seizure as well as an
increase in
function and/or number of CNS cells (e.g., neurons, astrocytes, myelin, etc.).
In
certain embodiments, the compositions and methods described herein reduce tau
expression (as compared to controls not receiving the artificial repressors as
described
herein) by at least 30%, or 40%, preferably by at least 50%, even more
preferably by
at least 70%. In some embodiments, at least 50% reduction is achieved.
[0028] In a still further aspect, described here is a method of delivering
a
repressor of tau to the brain of a subject (e.g., mammalian subject such as a
mouse,
human or non-human primate (NHP)) using a viral or non-viral vector. In
certain
embodiments, the viral vector is an AAV vector, for instance an AAV9 vector,
or an
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AAV vector variant as described in U.S. Patent No. 9,585,971 or U.S.
Provisional
Patent Application No. 62/503,121. Delivery may be to any brain region, for
example, the hippocampus or entorhinal cortex by any suitable means including
via
the use of a cannula or any other delivery technology. Any AAV vector that
provides
widespread delivery of the repressor to brain of the subject, including via
anterograde
and retrograde axonal transport to brain regions not directly administered the
vector
(e.g., delivery to the putamen results in delivery to other structures such as
the cortex,
substantia nigra, thalamus, etc.). In certain embodiments, the subject is a
human and
in other embodiments, the subject is a non-human primate. The administration
may
be in a single dose or in multiple administrations (at any timing between
administrations).
[0029] Thus, in other aspects, described herein is a method of
preventing
and/or treating a tauopathy (e.g., AD) in a subject, the method comprising
administering a repressor of a tau allele to the subject using one or more AAV
vectors. In certain embodiments, the AAV encoding the repressor is
administered to
the CNS (brain and/or CSF) via any delivery method including but not limited
to,
intracerebroventricular, intrathecal, or intracistemal delivery. In other
embodiments,
the AAV encoding the repressor is administered directly into the parenchyma
(e.g.,
hippocampus and/or entorhinal cortex) of the subject. In other embodiments,
the
AAV encoding the repressor is administered intravenously (IV). In any of the
methods described herein, the administering may be done once (single
administration)
or may be done multiple times (with any time between administrations). When
administered multiple times, the same or different dosages and/or delivery
vehicles of
modes of administration may be used (e.g., different AAV vectors administered
IV
and/or ICV). The methods include methods of reducing the aggregation of tau in
the
subject (e.g., reducing NFTs characteristic of tau aggregation) for example in
AD
neurons of a subject with AD; methods of reducing apoptosis in a neuron or
population of neurons (e.g., an AD neuron or population of AD neurons);
methods of
reducing neuronal hyperexcitability; methods of reducing amyloid beta induced
toxicity (e.g. synapse loss and/or neuritic dystrophy); and/or methods of
reduce loss to
one or more cognitive functions in AD subjects, all in comparison with a
subject not
receiving the method, or in comparison to the subject themselves prior to
receiving
the methods. Thus, the methods described herein result in reduction in
biomarkers
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and/or symptoms of tauopathies, including one or more the following:
neurotoxicity,
gliosis, dystrophic neurites, spine loss, excitotoxicity, cortical and
hippocampal
shrinkage, dendritic tau accumulation, cognitive (e.g., the radial arm maze
and the
Morris water maze in rodent models, fear conditioning, etc.), and/or motor
deficits.
[0030] In some aspects, the methods and compositions of the invention for
reducing the amount of a pathogenic tau species in a cell are provided. In
some
embodiments, the methods result in a reduction of hyperphosphorylated tau. In
some
instances, the reduction of hyperphosphorylated tau results in a reduction of
soluble or
granular tau. In other embodiments, the reduction of pathogenic tau species
decreases
tau aggregation and causes a reduction in neurofibrillary tangles (NFTs) as
compared
to a cell or subject that has not been treated following the methods and/or
with the
compositions of the invention. In further embodiments, the methods of
reversing the
amount of NFTs observed in a cell are provided. In still further embodiments,
the
methods and compositions of the invention cause a slowing of the propagation
of
pathogenic tau species (NFTs, hyperphosphorylated tau) within the brain of a
subject.
In some embodiments, propagation of pathogenic tau across the brain is halted,
and in
other embodiments, propagation of pathogenic tau across the brain is reversed.
In
further embodiments, the number of dystrophic neurites associated with
amyloid13
plaques in the brain is reduced. In some embodiments, the number of dystrophic
neurites is reduced to the levels found in an age-matched wild type brain. In
further
embodiments, provided herein are methods and compositions for reducing
hyperphosphorylated tau associated with amyloid [3 plaques in the brain of a
subject.
[0031] In any of the methods described herein, the repressor of the
tau allele
may be a ZFP-TF, for example a fusion protein comprising a ZFP that binds
specifically to a tau allele and a transcriptional repression domain (e.g.,
KOX, KRAB,
etc.). In other embodiments, the repressor of the tau allele may be a TALE-TF,
for
example a fusion protein comprising a TALE polypeptide that binds specifically
to a
tau allele and a transcriptional repression domain (e.g., KOX, KRAB, etc.). In
some
embodiments, the tau allele repressor is a CRISPR/Cas-TF where the nuclease
domains in the Cas protein have been inactivated such that the protein no
longer
cleaves DNA. The resultant Cas RNA-guided DNA binding domain is fused to a
transcription repressor (e.g. KOX, KRAB etc.) to repress the tau allele.
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[0032] In some embodiments, the sequence encoding a genetic modulator
(genetic repressor) as described herein (e.g., ZFP-TF, TALE-TF or CRISPR/Cas-
TF)
is inserted (integrated) into the genome while in other embodiments the
sequence
encoding the repressor is maintained episomally. In some instances, the
nucleic acid
encoding the TF fusion is inserted (e.g., via nuclease-mediated integration)
at a safe
harbor site comprising a promoter such that the endogenous promoter drives
expression. In other embodiments, the repressor (TF) donor sequence is
inserted (via
nuclease-mediated integration) into a safe harbor site and the donor sequence
comprises a promoter that drives expression of the repressor. In some
embodiments,
the sequence encoding the genetic modulator is maintained extrachromosomally
(episomally) after delivery, and may include a heterologous promoter. The
promoter
may be a constitutive or inducible promoter. In some embodiments, the promoter
sequence is broadly expressed while in other embodiments, the promoter is
tissue or
cell/type specific. In preferred embodiments, the promoter sequence is
specific for
.. neuronal cells. In especially preferred embodiments, the promoter chosen is
characterized in that it has low expression. Non-limiting examples of
preferred
promoters include the neural specific promoters NSE, Synapsin, CAMKiia and
MECPs. Non-limiting examples of ubiquitous promoters include CAS and Ubc.
Further embodiments include the use of self-regulating promoters as described
in U.S.
.. Patent Publication No. 2015/0267205.
[0033] In still further embodiments, the repressor may comprise a
nuclease
(e.g., ZFN, TALEN and/or CRISPR/Cas system) that represses the tau allele by
cleaving and thereby inactivating the tau allele. In certain embodiments, the
nuclease
introduces an insertion and/or deletion ("inder) via non-hom*ologous end
joining
(NHEJ) following cleavage by the nuclease. In other embodiments, the nuclease
introduces a donor sequence (by hom*ology or non-hom*ology directed methods), in
which the donor integration inactivates the tau allele.
[0034] In any of the methods described herein, the repressor may be
delivered
to the subject (e.g., brain) as a protein, polynucleotide or any combination
of protein
and polynucleotide. In certain embodiments, the repressor(s) is(are) delivered
using
an AAV vector. In other embodiments, at least one component of the repressor
(e.g.,
sgRNA of a CRISPR/Cas system) is delivered as in RNA form. In other
embodiments, the repressor(s) is(are) delivered using a combination of any of
the
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expression constructs described herein, for example one repressor (or portion
thereof)
on one expression construct (AAV9) and one repressor (or portion thereof) on a
separate expression construct (AAV or other viral or non-viral construct).
[0035] Furthermore, in any of the methods described herein, the
repressors
can be delivered at any concentration (dose) that provides the desired effect.
In
preferred embodiments, the repressor is delivered using an adeno-associated
virus
(AAV) vector at 10,000 - 500,000 vector genome/cell (or any value
therebetween). In
certain embodiments, the repressor is delivered using a lentiviral vector at
MOI
between 250 and 1,000 (or any value therebetween). In other embodiments, the
.. repressor is delivered using a plasmid vector at 0.01-1,000 ng/100,000
cells (or any
value therebetween). In other embodiments, the repressor is delivered as mRNA
at
150-1,500 ng/100,000 cells (or any value therebetween).
[0036] In any of the methods described herein, the method can yield
about
50% or greater, 55% or greater, 60% or greater, 65% or greater, about 70% or
greater,
about 75% or greater, about 85% or greater, about 90% or greater, about 92% or
greater, or about 95% or greater repression of the tau alleles in one or more
AD
neurons of the subject.
[0037] In further aspects, the tau-modulating transcription factors as
described
herein, such as a tau-modulating transcription factors comprising one or more
of a
zinc finger protein (ZFP TFs), a TALEs (TALE-TF), and a CRISPR/Cas-TFs for
example, ZFP-TFs, TALE-TFs or CRISPR/Cas-TFs are used to repress expression of
a mutant or wild type tau allele in of the brain (e.g., neuron) of a subject.
The
repression can be about 50% or greater, 55% or greater, 60% or greater, 65% or
greater, 70% or greater, about 75% or greater, about 85% or greater, about 90%
or
greater, about 92% or greater, or about 95% or greater repression of the tau
alleles in
the one or more neurons of the subject as compared to untreated (wild-type)
neurons
of the subject. In certain embodiments, the tau-modulating transcription
factor can be
used to achieve one or more of the methods described herein.
[0038] Also provided is a kit comprising one or more of the AAV tau-
modulators (e.g., repressors) and/or polynucleotides comprising components of
and/or
encoding the tau-modulators (or components thereof) as described herein. The
kits
may further comprise cells (e.g., neurons), reagents (e.g., for detecting
and/or
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quantifying tau protein, for example in CSF) and/or instructions for use,
including the
methods as described herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0039] Figures 1A through 1D depict MAPT expression following
introduction of engineered MAPT genetic modulators as described herein. Figure
1A
is a schematic showing the target sites within the MAPT gene for the DNA-
binding
molecule of the genetic modulator. The modulator for these experiments was a
repressor. Figure 1B shows graphs showing MAPT expression 24 hours after
administration of the indicated amounts of MAPT repressor (52322, 52364,
52366,
52389, 57880, 57890 and 52288) or GFP or mock controls in mRNA form (at the
indicated doses of 1000 ng, 300 ng, 100 ng, 30, ng, 10 ng or 3 ng, left to
right
respectively) to Neuro 2A cells. Figure 1C depicts graphs showing MAPT
expression
7 days after administration of the MAPT repressor (52322, 52364, 52366, 52389,
57880, 57890 and 52288) or GFP or mock controls using an AAV9 vector (with
CMV promoter) at the indicated doses of 3 x 105 vg/cell, 1 x 105 vg/cell, 3 x
104
vg/cell, 1 x 104 vg/cell to primary mouse cortical neurons (MCNs). Figure 1D
shows
graphs depicting Tau (MAPT) repression and off-target repression by 57880 and
two
exemplary derivatives (65887 and 65888) comprising mutations in the phosphate
.. contacts in the ZFP backbone.
[0040] Figures 2A through 2C depict protein levels (tau, GAPDH and
ZFP)
in MCNs treated with the indicated repressor (52389) carried by an AAV9
vector.
Figure 2A is a Western Blot showing proteins levels of the indicated proteins
at the
indicated AAV9 dosages. Figure 2B and Figure 2C are graphs showing the
proportion of tau/GAPDH proteins (top panels) and ZFP/GAPDH (bottom panels) at
the indicated dosages of AAV9-52389 or mock (M).
[0041] Figure 3 shows results of microarray analysis results showing
specificity of the indicated repressors (52322, 52364, 52366, 52389, 57880,
57890 or
52288) for the MAPT gene. Analysis was performed 24 hours after administration
to
Neuro2A cells of the repressors in mRNA form at 300 ng. Results are discussed
in
Example 3. The numbers above each graph represent the number of genes whose
expression increased (up arrow) or decreased (repression) (down arrow).
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[0042] Figure 4 shows results of microarray analysis results of the
indicated
repressors (52322, 52364, 52366, 52389, 57880, 57890, 52288) 24 hours after
administration to primary human fibroblasts of the repressors in mRNA form at
300
ng. Results are discussed in Example 3. The numbers above each graph represent
the
.. number of genes whose expression increased (up arrow) or decreased
(repression)
(down arrow).
[0043] Figures 5A and 5B show results of microarray analysis results
of the
indicated repressors (52322, 52364, 52366, 52389, 57880, 57890). Figure 5A
shows
results 7 days after administration to mouse cortical neurons of the
repressors carried
.. by an AAV vector (AAV) at 1 x 105 vg/cell. Results are discussed in Example
3. The
numbers above each graph represent the number of genes whose expression
increased
(up arrow) or decreased (repression) (down arrow). Figure 5B shows the
microarray
analysis results comparing two repressors 57880 and 65888 with identical
helices.
The data for the 57880 protein is shown on the top row while the data for the
65888
protein is shown on the bottom row. 65888 has some potential phosphate
contacts
removed from the zinc finger backbone (see Table 2) and the data demonstrates
a
substantial increase in specificity in the 65888 protein (57880 upregulated 75
genes
and repressed 110 gene in these experimental conditions while 65888
upregulated 3
genes and repressed 4, including tau) while maintaining similar tau repression
activity
in mouse cortical neurons.
[0044] Figures 6A and 6B are graphs depicting mRNA expression in cells
using genetic repressors carried by AAV vector (AAV9) where expression of the
repressor is driven by the indicated promoter (CMV, synapsin (SYN1), alpha-
calcium/calmodulin-dependent protein kinase promoter (CamKII), and a 229 bp
fragment of the methyl CpG-binding protein 2 promoter (MeCP2). Figure 6A shows
expression in MCNs. Figure 6B shows expression in primary hippocampal neurons.
[0045] Figures 7A through 7D show mRNA (human tau and ZFP) levels in
human iPSC-derived neurons. Figure 7A is a schematic showing partial sequence
of
human and mouse MAPT target sites (SEQ ID NO:31 and 32). Figure 7B shows
graphs showing mRNA expression in the iPSCs 18 days after administration of
the
indicated repressors carried by an AAV vector at the indicated dosages. Figure
7C
shows graphs showing mRNA expression in the iPSCs 18 days after administration
of
the indicated repressor (52366) carried by an AAV vector with the indicated
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promoters. Figure 7D is a series of microarray plots using exemplary ZFP TFs
in the
human iPSC-derived neurons, and indicates that the ZFP TFs are highly
specific. The
cells were exposed to 1E5 of AAV6 comprising the ZFP-TF donor for 19 days
prior
to analysis.
[0046] Figures 8A through 8H show efficient neuronal transduction of ZFP-
TFs and in vivo effects on mRNA (tau and ZFP) expression in mouse subjects
using
various AAV vectors to deliver the tau repressors. See, Example 6. Figure 8A
shows
efficient transduction of the motor cortex (top panels), hippocampus (middle
panels)
and cerebellum (bottom panels) following AAV administration either IV (right
panels) or ICV (left panels). Figure 8B shows potent tau reduction (-50-70%
reduction) via both ICV and IV administration of key tauopathy regions,
including
motor cortex (left panel, top row), brainstem (right panel, top row) and
hippocampus
(bottom left panel). Figure 8C shows tau mRNA reduction (-40-70% reduction)
throughout the brain and spinal cord following IV or ICV administration of AAV-
ZFP-TF vectors. Figure 8D shows levels of tau (MAPT) mRNA and Figure 8E shows
levels of the repressor (ZFP) mRNA for each hippocampal section. Figure 8F are
graphs depicting robust repression of tau throughout the CNS and spinal cord
following administration of the tau repressors described herein (left panel
shows
52389 repressor and right panel shows 57890 repressor) using AAV vectors.
Figure
8G are graphs depicting tau repression is rapid and sustained over months in
cortex
(left panels) and hippocampus (right panels) of animals administered the
indicated
compositions ("GFP" refers to the GFP control; "172" refers to the irrelevant
control
ZFP-TF that does not bind to MAPT; "52389" refers to the 52389 repressor
carried on
an AAV construct; "57890" refers to the 57890 repressor described herein
carried on
an AAV construct; and "PBS" refers to the control of animals receiving only
PBS).
The top row in Figure 8G depicts Tau mRNA expression and the bottom row
depicts
normalized Tau protein levels. Figure 8H are graphs showing total mouse tau
protein
levels in CSF (top row, left panel), cortex (top row, right panel) and
hippocampus
(top row, far right graph) of animals receiving the indicated compositions
("VEH"
refers to control animals administered vehicle only; "172V" refers to animals
receiving the irrelevant control ZFP-TF that does not bind to MAPT carried on
AAV
vector; "389V" refers to the 52389 repressor carried on an AAV construct; and
"890V" refers to the 57890 repressor described herein carried on an AAV
construct).
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Also shown (bottom row) is the statistically analysis showing the correlation
between
CSF and the levels in the cortex (right panel) and hippocampus (left panel).
[0047] Figures 9A through 9D depict further analysis of in vivo
modulation
(repression) of MAPT using statistical analysis as described in Example 6.
Figure 9A
.. shows ANOVA followed by Sidak's post-test analyses for all slices (top
panel shows
tau, bottom panel shows ZFP). Figure 9B shows average maximal tau reduction
(top
panel shows tau, bottom panel shows ZFP). Figure 9C shows a high correlation
between MAPT and ZFP-TF levels in animals treated with the 52322 MAPT
repressor. Figure 9D shows a high correlation between MAPT and ZFP-TF levels
in
animals treated with the 52389 MAPT repressor.
[0048] Figures 10A through 10D are graphs depicting MAPT (tau) mRNA
repression following treatment with the tau-specific ZFP-TF in vivo following
injection into the hippocampus. In this experiment, four different promoters
(CMV,
SNY1, CAMKII or MeCP2) were used to drive the expression of the ZFP-TF-venus
construct. Figure 10A shows the expression of tau 6 weeks after injection and
demonstrates a decrease in all animals that received the ZFP-TF, regardless of
promoter chosen. Asterisks indicate significance of the signal as compared to
the
PBS control (p<0.0001 for all promoters). Figure 10B shows the expression of
the
ZFP-TF from the various promoters where highest ZFP-TF expression was detected
in the animals treated with the CAMKII driven vector. The presence of
activated
astrocytes was measured for each treatment group (Figure 10C) through
detection of
GFAP. In this experiment, the MeCP2 promoter resulted in no GFAP elevation
compared to PBS-injected animals, SYN1 resulted in 4.0-fold higher GFAP levels
(P<0.0001), CMV resulting in 3.2-fold higher levels (P<0.01), and CAMKII
resulted
in levels 2.4-fold higher (P<0.05). Similarly, the presence of microglia was
also
determined (Figure 10D) in the treatment groups where the MeCP2 promoter
resulted
in no significant changes in IBA1 levels compared to PBS-injected animals,
SYN1
resulted in 4.7-fold higher IBA1 levels (P<0.0001), CMV resulting in 3.0-fold
higher
levels (P<0.05), and CAMKII resulted in levels 3.2-fold higher (P<0.001).
[0049] Figure 11 is a graph showing the reduction in tau protein expression
in
the groups described in Figure 10. Compared to PBS-injected control levels
(269
ng/ml), the rAAV9-SYN1-52389V construct resulted in 23% of control levels (61
ng/ml, P<0.0001), the rAAV9-CAMKII-52389V construct yielded 18% of control
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levels (49 ng/ml, P<0.0001), the rAAV9-SYN1-52389V construct had 12% of
control
levels (32 ng/ml, P<0.0001). Thus, neuronal promoters are capable of driving
>80%
tau mRNA and protein reduction throughout the mouse hippocampus.
[0050] Figures 12A through 12F depict the histological demonstrations
of
tau reduction in the hippocampus and connected brain regions. AAVs comprising
the
tau-specific ZFP-TF driven by the CMV promoter were delivered to the
hippocampus
to valuate endogenous tau protein reduction by immunofluorescent staining. The
ZFP-TFs were linked to a fluorescent protein Venus for detection, and animals
were
sacrificed 6 weeks following injection. Figure 12A is a cross section of the
hippocampus and delineates 4 boxed regions for closer study. Figure 12B is a
close
up of Box 1 from Figure 12A and shows GFP/DAPI staining (left panel);
GFP/tau/DAPI (middle panel) and tau (right panel). Figure 12C is a graph of
fluorescence intensity across Box 1, demonstrating a reduction in tau in the
ipsilateral
side of the section (right panel). Figure 12D shows a close up of Box 2, while
Figure
12E is a close up of Box 3, demonstrating a decrease in tau staining on the
ipsilateral
side (right panel). Figure 12F shows Box 4 where the top panel shows GFP
(indication of ZFP-TF) signal in the ipsilateral side of the injection with a
concomitant decrease in tau staining (lower panel). The lower middle panel
also
shows a graphical depiction of the decrease in tau staining. The lower left
and right
panels show staining in ipsilateral (left) and contralateral (right) sections.
The data
are discussed in greater detail in the Examples.
[0051] Figures 13A through 13J demonstrate the safety and sustained
expression of the ZFP-TF for six months in the hippocampus in vivo. Figure 13A
depicts the signal for transduced cells in the hippocampus as a whole at the 6-
week
time point, indicating the expression of the ZFP-TF and GFP alone vectors are
nearly
the same. Figure 13B shows similar results specifically for astrocytes while
Figure
13C shows the results for the microglia. Figure 13D shows the transduction
data
similar to Figure 13A, except for the 6-month time point. Similarly, Figure13E
shows
the astrocyte data for the 6-month time point and Figure 13F shows the
microglia data
for the 6-month time point. Figure 13G demonstrates that the mean thickness of
a
region of the hippocampus is maintained in the various treatment groups,
demonstrating no overt neuronal toxicity from long-term expression of the ZFP-
TF.
Figure 13H shows the coverage of the injections in the anterior (left panel)
and
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posterior (right panel) hippocampus. The data are discussed in greater detail
in the
Examples. Figure 131 are graphs showing normalized expression levels in
subjects
treated with the indicated ZFs (tau in top left, ZFs in top middle, VG/cell in
top right
GFPA (glial fibrillary acid protein, a marker for astrocytes) in bottom left,
IBA1
.. (ionized calcium-binding adapter molecule 1, a marker of microglia) in
bottom
middle, and NeuN (a well-recognized marker of post-mitotic neurons that is
highly
conserved among different species, (Wang et al., (2015) Sci Reports 5:17383,
doi
10.1038/5pre17383)) in bottom right). Figure 13J are graphs showing normalized
expression levels of the indicated proteins in subjects treated with the
indicated
compositions ("PBS" refers to no tau modulator; "89V 6 weeks" refers to mice
treated
with the 52389 Venus construct sampled at 6 weeks, and "89V llm" refers to
52389
Venus construct sampled at 11 months (tau, ZFP, GFAP, IBA1 from left to right
in
top panels; NeuN, MAP1, MAFIA, MAP2 from left to right in bottom panels).
[0052] Figures 14A through 14F are micrographs of brain sections of
.. APP/PS1 mice treated with the ZFP-TFs. In these images, GFP is green in
color, RFP
is red in color and antibodies specific for AP are labeled with a secondary
antibody
(blue). Figures 14A and 14C are images of a wild type cortex (CTX) where the
left
CTX was treated with the irrelevant ZFP-TF (Figure 14A) and the right CTX was
treated with AAV encoding the fluorescent protein tRFP (Figure 14C). Figures
14B
and 14D are images of brain cortexes from APP/PS1 mice, where the left CTX was
treated with the AAV encoding the tau-specific ZFP-TF ("389dV", Figure 14B) or
AAV encoding the tRFP (Figure 14D). The images illustrate a reduction in
dystrophic neurites (identifiable as punctate staining around the AP plaques
and
indicated by arrows) in the CTX treated with the tau-specific ZFP-TF. Figures
14E
and 14F are higher magnification images of two examples of the APP/PS1 CTX
sections treated either with the tau-specific ZFP-TF (Figure 14E) or the tRFP
(Figure
14F) where there are more dystrophic neurites (indicated by arrows) in the
tRFP
treated section than the 389 ZFP-TF treated section.
[0053] Figure 15 is a graph showing a quantitative representation of
the
number of dystrophic neurites per AP plaque in the APP/PS1 mice. Each dot
represents the average number of dystrophic neurites per plaque for all
plaques in one
cortical section; 3-5 sections were analyzed per hemisphere per mouse. As can
be
seen, there was a statistically significant reduction in dystrophic neurites
in the CTX
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treated with the 389dV ZFP-TF as compared to the CTX treated with the tRFP
(left-
most (gray) bar is 389dV treated, second from left (white) bar is tRFP
treated). Also
shown is a comparison of the CTX in mice treated with the irrelevant ZFP-TF
("172dV", gray striped bar, 2nd from right bar) or tRFP (white striped bar,
right-most
bar). As can be seen, there was no difference in the number of dystrophic
neurites/plaque between these samples.
[0054] Figure 16 is a graph depicting a quantitative comparison of the
data
represented in Figure 15 accounting for the variation amongst animals in
baseline
dystrophic neurites per plaque. The number of dystrophies per AP plaque were
averaged for each ZFP-TF treated hemisphere or the contralateral tRFP-treated
hemisphere and compared using a paired two-tailed T-test. The values for each
cohort were scaled to the mean of the tRFP-treated side (set to 100%). Using
this
paired analysis, dystrophic neurites were found to be significantly reduced
(mean 34%
reduction) for the 52389V-treated cohort (left most (gray) bar is 389dV
treated,
.. second from left (white) bar is tRFP treated), but not for the cohort
treated with an
irrelevant ZFP-TF (172dV, second from right (gray striped) bar; tRFP, right
most
(white striped) bar).
[0055] Figure 17 depicts exemplary results of a microarray analysis on
hippocampal tissue derived from mice treated with ZFP-TFs. The ZFP-TFs were
delivered to the animals using AAVs described herein (2.5 e12 vg/animal) via
retro-
orbital delivery. The mice were treated and sacrificed at ten weeks, and the
results
depicted are data compared to an irrelevant ZFP-TF control.
DETAILED DESCRIPTION
[0056] Disclosed herein are compositions and methods for the prevention
and/or treatment of tauopathies. In particular, the compositions and methods
described herein are used to repress the expression of a MAPT (tau) protein to
prevent
or treat tauopathies such as Alzheimer's Disease (AD), Frontotermporal
Dementia,
Progressive Supranuclear Palsy, seizure disorders and/or Corticobasal
Degeneration.
The MAPT repressors (e.g., MAPT-modulating transcription factors, such as MAPT-
modulating transcription factors comprising zinc finger proteins (ZFP TFs),
TALEs
(TALE-TF), and/or CRISPR/Cas-TFs), modify the CNS such that the effects and/or
symptoms of the tauopathy is reduced or eliminated, for example by reducing
the
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aggregation of tau in the brain of a subject with a tauopathy (e.g., AD) and
reducing
the occurrence of neural tangles. In preferred embodiments, the MAPT-
modulating
transcription factors are delivered to the brain by a viral vector such as an
AAV.
AAV has been shown to be well suited for brain delivery, so use of these viral
vectors
to deliver MAPT modulating transcription factors is especially useful for the
treatment of diseases such as Alzheimer's Disease associated with the
inappropriate
expression and thereby aggregation of tau protein.
General
[0057] Practice of the methods, as well as preparation and use of the
compositions disclosed herein employ, unless otherwise indicated, conventional
techniques in molecular biology, biochemistry, chromatin structure and
analysis,
computational chemistry, cell culture, recombinant DNA and related fields as
are
within the skill of the art. These techniques are fully explained in the
literature. See,
for example, Sambrook etal., MOLECULAR CLONING: A LABORATORY
MANUAL, Second edition, Cold Spring Harbor Laboratory Press, 1989 and Third
edition, 2001; Ausubel etal., CURRENT PROTOCOLS IN MOLECULAR
BIOLOGY, John Wiley & Sons, New York, 1987 and periodic updates; the series
METHODS IN ENZYMOLOGY, Academic Press, San Diego; Wolfe,
CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San
Diego, 1998; METHODS IN ENZYMOLOGY, Vol. 304, "Chromatin" (P.M.
Wassarman and A. P. Wolffe, eds.), Academic Press, San Diego, 1999; and
METHODS IN MOLECULAR BIOLOGY, Vol. 119, "Chromatin Protocols" (P.B.
Becker, ed.) Humana Press, Totowa, 1999.
Definitions
[0058] The terms "nucleic acid," "polynucleotide," and
"oligonucleotide" are used
interchangeably and refer to a deoxyribonucleotide or ribonucleotide polymer,
in linear or
circular conformation, and in either single- or double-stranded form. For the
purposes of
the present disclosure, these terms are not to be construed as limiting with
respect to the
length of a polymer. The terms can encompass known analogues of natural
nucleotides, as
well as nucleotides that are modified in the base, sugar and/or phosphate
moieties (e.g.,
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phosphorothioate backbones). In general, an analogue of a particular
nucleotide has the
same base-pairing specificity; i.e., an analogue of A will base-pair with T.
[0059] The terms "polypeptide," "peptide" and "protein" are used
interchangeably
to refer to a polymer of amino acid residues. The term also applies to amino
acid
polymers in which one or more amino acids are chemical analogues or modified
derivatives of a corresponding naturally-occurring amino acid.
[0060] "Binding" refers to a sequence-specific, non-covalent
interaction
between macromolecules (e.g., between a protein and a nucleic acid). Not all
components of a binding interaction need be sequence-specific (e.g., contacts
with
phosphate residues in a DNA backbone), as long as the interaction as a whole
is
sequence-specific. Such interactions are generally characterized by a
dissociation
constant (Ka) of 10-6 M-1 or lower. "Affinity" refers to the strength of
binding:
increased binding affinity being correlated with a lower K.
[0061] A "binding protein" is a protein that is able to bind non-
covalently to
another molecule. A binding protein can bind to, for example, a DNA molecule
(a DNA-
binding protein), an RNA molecule (an RNA-binding protein) and/or a protein
molecule (a
protein-binding protein). In the case of a protein-binding protein, it can
bind to itself (to
form hom*odimers, hom*otrimers, etc.) and/or it can bind to one or more
molecules of a
different protein or proteins. A binding protein can have more than one type
of binding
activity. For example, zinc finger proteins have DNA-binding, RNA-binding and
protein-
binding activity.
[0062] A "zinc finger DNA binding protein" (or binding domain) is a
protein, or a
domain within a larger protein, that binds DNA in a sequence-specific manner
through one
or more zinc fingers, which are regions of amino acid sequence within the
binding domain
.. whose structure is stabilized through coordination of a zinc ion. The term
zinc finger
DNA binding protein is often abbreviated as zinc finger protein or ZFP.
[0063] A "TALE DNA binding domain" or "TALE" is a polypeptide
comprising
one or more TALE repeat domains/units. The repeat domains are involved in
binding of
the TALE to its cognate target DNA sequence. A single "repeat unit" (also
referred to as a
"repeat") is typically 33-35 amino acids in length and exhibits at least some
sequence
hom*ology with other TALE repeat sequences within a naturally occurring TALE
protein.
See, e.g., U.S. Patent No. 8,586,526.
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[0064] "TtAgo" is a prokaryotic Argonaute protein thought to be
involved in
gene silencing. TtAgo is derived from the bacteria Therms thermophilus. See,
e.g.,
Swarts etal., (2014) Nature 507(7491):258-261, G. Sheng etal., (2013) Proc.
Natl.
Acad. Sci. USA. 111, 652). A "TtAgo system" is all the components required
including, for example, guide DNAs for cleavage by a TtAgo enzyme.
"Recombination" refers to a process of exchange of genetic information between
two
polynucleotides, including but not limited to, donor capture by non-hom*ologous
end
joining (NHEJ) and hom*ologous recombination. For the purposes of this
disclosure,
"hom*ologous recombination (HR)" refers to the specialized form of such
exchange
that takes place, for example, during repair of double-strand breaks in cells
via
hom*ology-directed repair mechanisms. This process requires nucleotide sequence
hom*ology, uses a "donor" molecule to template repair of a "target" molecule
(i.e., the
one that experienced the double-strand break), and is variously known as "non-
crossover gene conversion" or "short tract gene conversion," because it leads
to the
transfer of genetic information from the donor to the target. Without wishing
to be
bound by any particular theory, such transfer can involve mismatch correction
of
heteroduplex DNA that forms between the broken target and the donor, and/or
"synthesis-dependent strand annealing," in which the donor is used to
resynthesize
genetic information that will become part of the target, and/or related
processes. Such
specialized HR often results in an alteration of the sequence of the target
molecule
such that part or all of the sequence of the donor polynucleotide is
incorporated into
the target polynucleotide.
[0065] DNA-binding domains such as sgRNAs, zinc finger binding domains
or TALE DNA binding domains can be "engineered" to bind to a predetermined
nucleotide sequence, for example via design of a sgRNA that binds to a
selected
target site or by engineering (altering one or more amino acids) of the
recognition
helix region of a naturally occurring zinc finger protein or by engineering
the RVDs
of a TALE protein. Therefore, engineered zinc finger proteins or TALEs are
proteins
that are non-naturally occurring. Non-limiting examples of methods for
engineering
DNA-binding domains are design and selection. A "designed" zinc finger protein
or
TALE is a protein not occurring in nature whose design/composition results
principally from rational criteria. Rational criteria for design include
application of
substitution rules and computerized algorithms for processing information in a
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database storing information of existing ZFP designs and binding data. A
"selected"
zinc finger protein or TALE is a protein not found in nature whose production
results
primarily from an empirical process such as phage display, interaction trap or
hybrid
selection. See, for example, U.S. Patent Nos. 8,586,526; 6,140,081; 6,453,242;
6,746,838; 7,241,573; 6,866,997; 7,241,574; and 6,534,261; see also
International
Patent Publication No. WO 03/016496.
[0066] The term "sequence" refers to a nucleotide sequence of any
length,
which can be DNA or RNA; can be linear, circular or branched and can be either
single-stranded or double stranded. The term "donor sequence" refers to a
nucleotide
sequence that is inserted into a genome. A donor sequence can be of any
length, for
example between 2 and 10,000 nucleotides in length (or any integer value
therebetween or thereabove), preferably between about 100 and 1,000
nucleotides in
length (or any integer therebetween), more preferably between about 200 and
500
nucleotides in length.
[0067] A "target site" or "target sequence" is a nucleic acid sequence that
defines a portion of a nucleic acid to which a binding molecule will bind,
provided
sufficient conditions for binding exist.
[0068] An "exogenous" molecule is a molecule that is not normally
present in
a cell, but can be introduced into a cell by one or more genetic, biochemical
or other
methods. "Normal presence in the cell" is determined with respect to the
particular
developmental stage and environmental conditions of the cell. Thus, for
example, a
molecule that is present only during embryonic development of muscle is an
exogenous molecule with respect to an adult muscle cell. Similarly, a molecule
induced by heat shock is an exogenous molecule with respect to a non-heat-
shocked
cell. An exogenous molecule can comprise, for example, a functioning version
of a
malfunctioning endogenous molecule or a malfunctioning version of a normally-
functioning endogenous molecule.
[0069] An exogenous molecule can be, among other things, a small
molecule,
such as is generated by a combinatorial chemistry process, or a macromolecule
such
.. as a protein, nucleic acid, carbohydrate, lipid, glycoprotein, lipoprotein,
polysaccharide, any modified derivative of the above molecules, or any complex
comprising one or more of the above molecules. Nucleic acids include DNA and
RNA, can be single- or double-stranded; can be linear, branched or circular;
and can
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be of any length. Nucleic acids include those capable of forming duplexes, as
well as
triplex-forming nucleic acids. See, for example, U.S. Patent Nos. 5,176,996
and
5,422,251. Proteins include, but are not limited to, DNA-binding proteins,
transcription factors, chromatin remodeling factors, methylated DNA binding
proteins, polymerases, methylases, demethylases, acetylases, deacetylases,
kinases,
phosphatases, integrases, recombinases, ligases, topoisomerases, gyrases and
helicases.
[0070] An exogenous molecule can be the same type of molecule as an
endogenous molecule, e.g., an exogenous protein or nucleic acid. For example,
an
exogenous nucleic acid can comprise an infecting viral genome, a plasmid or
episome
introduced into a cell, or a chromosome that is not normally present in the
cell.
Methods for the introduction of exogenous molecules into cells are known to
those of
skill in the art and include, but are not limited to, lipid-mediated transfer
(i.e.,
liposomes, including neutral and cationic lipids), electroporation, direct
injection, cell
fusion, particle bombardment, calcium phosphate co-precipitation, DEAE-dextran-
mediated transfer and viral vector-mediated transfer. An exogenous molecule
can also
be the same type of molecule as an endogenous molecule but derived from a
different
species than the cell is derived from. For example, a human nucleic acid
sequence
may be introduced into a cell line originally derived from a mouse or hamster.
[0071] By contrast, an "endogenous" molecule is one that is normally
present
in a particular cell at a particular developmental stage under particular
environmental
conditions. For example, an endogenous nucleic acid can comprise a chromosome,
the genome of a mitochondrion, chloroplast or other organelle, or a naturally-
occurring episomal nucleic acid. Additional endogenous molecules can include
proteins, for example, transcription factors and enzymes.
[0072] A "fusion" molecule is a molecule in which two or more subunit
molecules are linked, preferably covalently. The subunit molecules can be the
same
chemical type of molecule, or can be different chemical types of molecules.
Examples of the first type of fusion molecule include, but are not limited to,
fusion
.. proteins (for example, a fusion between a ZFP or TALE DNA-binding domain
and
one or more activation domains) and fusion nucleic acids (for example, a
nucleic acid
encoding the fusion protein described supra). Examples of the second type of
fusion
molecule include, but are not limited to, a fusion between a triplex-forming
nucleic
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acid and a polypeptide, and a fusion between a minor groove binder and a
nucleic
acid. The term also includes systems in which a polynucleotide component
associates
with a polypeptide component to form a functional molecule (e.g., a CRISPR/Cas
system in which a single guide RNA associates with a functional domain to
modulate
gene expression).
[0073] Expression of a fusion protein in a cell can result from
delivery of the
fusion protein to the cell or by delivery of a polynucleotide encoding the
fusion
protein to a cell, wherein the polynucleotide is transcribed, and the
transcript is
translated, to generate the fusion protein. Trans-splicing, polypeptide
cleavage and
polypeptide ligation can also be involved in expression of a protein in a
cell. Methods
for polynucleotide and polypeptide delivery to cells are presented elsewhere
in this
disclosure.
[0074] A "multimerization domain", (also referred to as a
"dimerization
domain" or "protein interaction domain") is a domain incorporated at the
amino,
carboxy or amino and carboxy terminal regions of a ZFP TF or TALE TF. These
domains allow for multimerization of multiple ZFP TF or TALE TF units such
that
larger tracts of trinucleotide repeat domains become preferentially bound by
multimerized ZFP TFs or TALE TFs relative to shorter tracts with wild-type
numbers
of lengths. Examples of multimerization domains include leucine zippers.
Multimerization domains may also be regulated by small molecules wherein the
multimerization domain assumes a proper conformation to allow for interaction
with
another multimerization domain only in the presence of a small molecule or
external
ligand. In this way, exogenous ligands can be used to regulate the activity of
these
domains.
[0075] A "gene," for the purposes of the present disclosure, includes a DNA
region encoding a gene product (see infra), as well as all DNA regions which
regulate
the production of the gene product, whether or not such regulatory sequences
are
adjacent to coding and/or transcribed sequences. Accordingly, a gene includes,
but is
not necessarily limited to, promoter sequences, terminators, translational
regulatory
sequences such as ribosome binding sites and internal ribosome entry sites,
enhancers,
silencers, insulators, boundary elements, replication origins, matrix
attachment sites
and locus control regions.
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[0076] "Gene expression" refers to the conversion of the information,
contained in a gene, into a gene product. A gene product can be the direct
transcriptional product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA,
ribozyme, structural RNA or any other type of RNA) or a protein produced by
translation of an mRNA. Gene products also include RNAs which are modified, by
processes such as capping, polyadenylation, methylation, and editing, and
proteins
modified by, for example, methylation, acetylation, phosphorylation,
ubiquitination,
ADP-ribosylation, myristilation, and glycosylation.
[0077] "Modulation" of gene expression refers to a change in the
activity of a
gene. Modulation of expression can include, but is not limited to, gene
activation and
gene repression. Genome editing (e.g., cleavage, alteration, inactivation,
random
mutation) can be used to modulate expression. Gene inactivation refers to any
reduction in gene expression as compared to a cell that does not include a ZFP
or
TALE protein as described herein. Thus, gene inactivation may be partial or
complete.
[0078] A "genetic modulator" refers to any molecule that alters the
expression
and/or sequence of one or more genes. Non-limiting examples of genetic
modulators
include transcription factors (such as artificial transcription factors as
described
herein) that bind to the target gene and alter its expression and nucleases
that modify
the sequence of the target gene, which in turn alters its expression (e.g.,
inactivation
of the target via insertions and/or deletions). Thus, a genetic modulator may
be
a genetic repressor (that represses and/or inactivates gene expression) or a
genetic activator.
[0079] A "region of interest" is any region of cellular chromatin,
such as, for
example, a gene or a non-coding sequence within or adjacent to a gene, in
which it is
desirable to bind an exogenous molecule. Binding can be for the purposes of
targeted
DNA cleavage and/or targeted recombination. A region of interest can be
present in a
chromosome, an episome, an organellar genome (e.g., mitochondrial,
chloroplast), or
an infecting viral genome, for example. A region of interest can be within the
coding
region of a gene, within transcribed non-coding regions such as, for example,
leader
sequences, trailer sequences or introns, or within non-transcribed regions,
either
upstream or downstream of the coding region. A region of interest can be as
small as
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a single nucleotide pair or up to 2,000 nucleotide pairs in length, or any
integral value
of nucleotide pairs.
[0080] "Eukaryotic" cells include, but are not limited to, fungal
cells (such as
yeast), plant cells, animal cells, mammalian cells and human cells (e.g., T-
cells).
[0081] The terms "operative linkage" and "operatively linked" (or "operably
linked") are used interchangeably with reference to a juxtaposition of two or
more
components (such as sequence elements), in which the components are arranged
such
that both components function normally and allow the possibility that at least
one of
the components can mediate a function that is exerted upon at least one of the
other
components. By way of illustration, a transcriptional regulatory sequence,
such as a
promoter, is operatively linked to a coding sequence if the transcriptional
regulatory
sequence controls the level of transcription of the coding sequence in
response to the
presence or absence of one or more transcriptional regulatory factors. A
transcriptional regulatory sequence is generally operatively linked in cis
with a coding
sequence, but need not be directly adjacent to it. For example, an enhancer is
a
transcriptional regulatory sequence that is operatively linked to a coding
sequence,
even though they are not contiguous.
[0082] With respect to fusion polypeptides, the term "operatively
linked" can
refer to the fact that each of the components performs the same function in
linkage to
the other component as it would if it were not so linked. For example, with
respect to
a fusion molecule in which a ZFP or TALE DNA-binding domain is fused to an
activation domain, the ZFP or TALE DNA-binding domain and the activation
domain
are in operative linkage if, in the fusion polypeptide, the ZFP or TALE DNA-
binding
domain portion is able to bind its target site and/or its binding site, while
the
activation domain is able to upregulate gene expression. ZFPs fused to domains
capable of regulating gene expression are collectively referred to as "ZFP-
TFs" or
"zinc finger transcription factors", while TALEs fused to domains capable of
regulating gene expression are collectively referred to as "TALE-TFs" or "TALE
transcription factors." When a fusion polypeptide in which a ZFP DNA-binding
domain is fused to a cleavage domain (a "ZFN" or "zinc finger nuclease"), the
ZFP
DNA-binding domain and the cleavage domain are in operative linkage if, in the
fusion polypeptide, the ZFP DNA-binding domain portion is able to bind its
target site
and/or its binding site, while the cleavage domain is able to cleave DNA in
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vicinity of the target site. When a fusion polypeptide in which a TALE DNA-
binding
domain is fused to a cleavage domain (a "TALEN" or "TALE nuclease"), the TALE
DNA-binding domain and the cleavage domain are in operative linkage if, in the
fusion polypeptide, the TALE DNA-binding domain portion is able to bind its
target
site and/or its binding site, while the cleavage domain is able to cleave DNA
in the
vicinity of the target site. With respect to a fusion molecule in which a Cas
DNA-
binding domain (e.g., single guide RNA) is fused to an activation domain, the
Cas
DNA-binding domain and the activation domain are in operative linkage if, in
the
fusion polypeptide, the Cos DNA-binding domain portion is able to bind its
target site
and/or its binding site, while the activation domain is able to up-regulate
gene
expression. When a fusion polypeptide in which a Cos DNA-binding domain is
fused
to a cleavage domain, the Cos DNA-binding domain and the cleavage domain are
in
operative linkage if, in the fusion polypeptide, the Cas DNA-binding domain
portion
is able to bind its target site and/or its binding site, while the cleavage
domain is able
.. to cleave DNA in the vicinity of the target site.
[0083] A "functional fragment" of a protein, polypeptide or nucleic
acid is a
protein, polypeptide or nucleic acid whose sequence is not identical to the
full-length
protein, polypeptide or nucleic acid, yet retains the same function as the
full-length
protein, polypeptide or nucleic acid. A functional fragment can possess more,
fewer,
or the same number of residues as the corresponding native molecule, and/or
can
contain one or more amino acid or nucleotide substitutions. Methods for
determining
the function of a nucleic acid (e.g., coding function, ability to hybridize to
another
nucleic acid) are well-known in the art. Similarly, methods for determining
protein
function are well-known. For example, the DNA-binding function of a
polypeptide
can be determined, for example, by filter-binding, electrophoretic mobility-
shift, or
immunoprecipitation assays. DNA cleavage can be assayed by gel
electrophoresis.
See Ausubel etal., supra. The ability of a protein to interact with another
protein can
be determined, for example, by co-immunoprecipitation, two-hybrid assays or
complementation, both genetic and biochemical. See, for example, Fields etal.,
(1989) Nature 340:245-246; U.S. Patent No. 5,585,245 and International Patent
Publication No. WO 98/44350.
[0084] A "vector" is capable of transferring gene sequences to target
cells.
Typically, "vector construct," "expression vector," and "gene transfer
vector," mean
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any nucleic acid construct capable of directing the expression of a gene of
interest and
which can transfer gene sequences to target cells. Thus, the term includes
cloning, and
expression vehicles, as well as integrating vectors.
[0085] A "reporter gene" or "reporter sequence" refers to any sequence
that
produces a protein product that is easily measured, preferably although not
necessarily
in a routine assay. Suitable reporter genes include, but are not limited to,
sequences
encoding proteins that mediate antibiotic resistance (e.g., ampicillin
resistance,
neomycin resistance, G418 resistance, puromycin resistance), sequences
encoding
colored or fluorescent or luminescent proteins (e.g., green fluorescent
protein,
enhanced green fluorescent protein, red fluorescent protein, luciferase), and
proteins
which mediate enhanced cell growth and/or gene amplification (e.g.,
dihydrofolate
reductase). Epitope tags include, for example, one or more copies of FLAG,
His,
myc, Tap, HA or any detectable amino acid sequence. "Expression tags" include
sequences that encode reporters that may be operably linked to a desired gene
sequence in order to monitor expression of the gene of interest.
Tau and Alzheimer's Disease
[0086] The tau protein is encoded by the MAPT gene which comprises 16
exons. Interestingly, exons 1, 4, 5, 7, 9, 11 and 12 and constitutively
expressed
whereas exons 2, 3, and 10 can be present in tau protein species derived from
alternatively spliced variants, leading to the presence of six different tau
protein
isoforms in the adult brain. Tau binds to microtubules via 3 or 4 repeated
tubulin-
binding motifs in the C-terminal half of the protein, and is thought to
stabilize the
tubules where tau4R (4 tubulin binding motifs) is thought to interact more
strongly
.. with microtubules that tau3R. The ratio of 3R to 4R is generally stable but
can be
affected in pathological states. The tau form that interacts with microtubules
is
phosphorylated and it appears that hyperphosphorylation causes the tau to
detach
from microtubules. Hyperphosphoylated tau can be sequestered in the cell,
which
then leads to conformational changes in the protein and to aggregation. These
.. aggregates may be the initial step in the formation of pathogenic
neurofibrillary
tangles (NFTs), however, hyperphosphorylated tau may be pathogenic in a
soluble
form as well as when present in the tangles (Bodea etal., (2016)J of Neurochem
138
(Suppl 1): 71-94). NFTs are restricted to the entorhinal cortex and medial
temporal
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lobe in the early stages of AD, and by the time of severe clinical symptoms of
the
disease present, NFTs are widespread throughout the brain. Coincident with the
presence of abundant NFTs, widespread distribution of amyloid plaques also
occurs.
In fact, it appears that the amyloid deposition in the cortex leads to an
increase in the
speed of tau propagation and the spread of NFT to distal regions of the brain.
As tau
tangles spread, there is a concomitant increase in neuronal loss (Pooler
etal., (2015)
Acta Neuropathol Commun 3:14, doi:10.1186/s40478-015-0199-x).
[0087] Tau has 95 amino acid residues that are capable of being
phosphorylated, and several kinases have been identified that may be
responsible for
tau phosphorylation, which may be possible target candidates for new
therapeutics,
including glycogen synthase-3, cyclin-dependent kinase 5, members of the MAPK
family, extracellular-regulated kinase, c-Jun N-terminal kinase and
microtubule-
affinity regulating kinase (Bodea (2016) ibid).
[0088] Amyloid (3 protein (AP) is the major constituent of senile
plaques,
which together with NFTs, are the hallmarks of a neuropathological
confirmation of
Alzheimer's Disease. AP is a peptide that has between 39 and 42 amino acid
chains;
the 42 amino acids form aggregates more avidly and is thought to be implicated
in the
pathogenesis of the disease and is the basis of the amyloid hypothesis (the
proposal
that accumulation of AP in the brain is the primary cause of AD, see review
Hardy
and Selkoe (2002) Science 297:353). Ar3s are products of the proteolytic
cleavage of
amyloid precursor protein (APP), a ubiquitous, glycosylated, sulfated, and
phosphorylated integral membrane protein (Sorrentino et al., (2014) FEBS Lett
588:641-652). However, it is becoming clear that the pathogenesis leading to
AD is
extremely complex and that the pathogenesis of AP accumulation may play a role
in
abnormal tau behavior (Ando etal., (2016) PLoS Genet 12(3):e1005917.
[0089] Reduction of tau in the brain has been shown to improve the
pathology
of AD. Regulated suppression of a tau transgene expression in a murine AD
model
demonstrated a reduction of transgene associate tau aggregates and a decrease
in the
concentration of hyperphosphorylated tau and NFT. In fact, this work also
showed a
loss in overall NFT, indicating that the accumulation of NFT may be reversible
(Polydoro et al., (2013) J of Neurosci 33(33):13300-13311). Additionally,
studies
performed with an intracellular anti-tau antibody delivered via AAV directly
through
intrahippocampal administration demonstrated a reduction in insoluble tau
species,
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NFT and a rescue of the hippocampal atrophy that is observed in the untreated
mouse
model (Liu et al., (2016)J Neurosci 36(49):12425-12435).
DNA-binding domains
[0090] The methods described herein make use of compositions, for example
tau-modulating transcription factors, comprising a DNA-binding domain that
specifically binds to a target sequence in a tau (MATP) gene. Any
polynucleotide or
polypeptide DNA-binding domain can be used in the compositions and methods
disclosed herein, for example DNA-binding proteins (e.g., ZFPs or TALEs) or
DNA-
binding polynucleotides (e.g., single guide RNAs). Thus, genetic modulators
(repressors) of tau genes are described.
[0091] In certain embodiments, the tau-repressor, or DNA binding
domain
therein, comprises a zinc finger protein. Selection of target sites; ZFPs and
methods
for design and construction of fusion proteins (and polynucleotides encoding
same)
are known to those of skill in the art and described in detail in U.S. Patent
Nos. 6,140,081; 5,789,538; 6,453,242; 6,534,261; 5,925,523; 6,007,988;
6,013,453;
6,200,759; and International Patent Publication Nos. WO 95/19431; WO 96/06166;
WO 98/53057; WO 98/54311; WO 00/27878; WO 01/60970; WO 01/88197;
WO 02/099084; WO 98/53058; WO 98/53059; WO 98/53060; WO 02/016536; and
WO 03/016496.
[0092] Tau target sites typically include at least one zinc finger but
can
include a plurality of zinc fingers (e.g., 2, 3, 4, 5, 6 or more fingers).
Usually, the
ZFPs include at least three fingers. Certain of the ZFPs include four, five or
six
fingers, while some ZFPs include 8, 9, 10, 11 or 12 fingers. The ZFPs that
include
three fingers typically recognize a target site that includes 9 or 10
nucleotides; ZFPs
that include four fingers typically recognize a target site that includes 12
to 14
nucleotides; while ZFPs having six fingers can recognize target sites that
include 18
to 21 nucleotides. The ZFPs can also be fusion proteins that include one or
more
regulatory domains, which domains can be transcriptional activation or
repression
domains. In some embodiments, the fusion protein comprises two ZFP DNA binding
domains linked together. These zinc finger proteins can thus comprise 8, 9,
10, 11, 12
or more fingers. In some embodiments, the two DNA binding domains are linked
via
an extendable flexible linker such that one DNA binding domain comprises 4, 5,
or 6
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zinc fingers and the second DNA binding domain comprises an additional 4, 5,
or 5
zinc fingers. In some embodiments, the linker is a standard inter-finger
linker such
that the finger array comprises one DNA binding domain comprising 8, 9, 10, 11
or
12 or more fingers. In other embodiments, the linker is an atypical linker
such as a
flexible linker. The DNA binding domains are fused to at least one regulatory
domain
and can be thought of as a `ZFP-ZFP-TF' architecture. Specific examples of
these
embodiments can be referred to as "ZFP-ZFP-KOX" which comprises two DNA
binding domains linked with a flexible linker and fused to a KOX repressor and
"ZFP-KOX-ZFP-KOX" where two ZFP-KOX fusion proteins are fused together via a
linker.
[0093] An engineered zinc finger binding domain can have a novel
binding
specificity, compared to a naturally-occurring zinc finger protein.
Engineering
methods include, but are not limited to, rational design and various types of
selection.
Rational design includes, for example, using databases comprising triplet (or
quadruplet) nucleotide sequences and individual zinc finger amino acid
sequences, in
which each triplet or quadruplet nucleotide sequence is associated with one or
more
amino acid sequences of zinc fingers which bind the particular triplet or
quadruplet
sequence. See, for example, co-owned U.S. Patent Nos. 6,453,242 and 6,534,261,
incorporated by reference herein in their entireties.
[0094] In addition, as disclosed in these and other references, zinc finger
domains and/or multi-fingered zinc finger proteins may be linked together
using any
suitable linker sequences, including for example, linkers of 5 or more amino
acids in
length. See, also, U.S. Patent Nos. 6,479,626; 6,903,185; and 7,153,949 for
exemplary linker sequences 6 or more amino acids in length. The proteins
described
herein may include any combination of suitable linkers between the individual
zinc
fingers of the protein.
[0095] A ZFP can be operably associated (linked) to one or more
transcriptional regulatory (e.g., repression domains) to form a ZF-TF (e.g.,
repressor).
Methods and compositions can also be used to increase the specificity of a ZFP
for its
intended target relative to other unintended cleavage sites, known as off-
target sites
for example by mutations to the ZFP backbone as described in U.S. Patent
Application No. 15/685,580. Thus, tau repressors described herein can comprise
mutations in one or more of their DNA binding domain backbone regions and/or
one
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or more mutations in their transcriptional regulatory domains. These ZFPs can
include mutations to amino acid within the ZFP DNA binding domain (`ZFP
backbone') that can interact non-specifically with phosphates on the DNA
backbone,
but they do not comprise changes in the DNA recognition helices. Thus, the
invention
includes mutations of cationic amino acid residues in the ZFP backbone that
are not
required for nucleotide target specificity. In some embodiments, these
mutations in
the ZFP backbone comprise mutating a cationic amino acid residue to a neutral
or
anionic amino acid residue. In some embodiments, these mutations in the ZFP
backbone comprise mutating a polar amino acid residue to a neutral or non-
polar
amino acid residue. In preferred embodiments, mutations at made at position (-
5), (-
9) and/or position (-14) relative to the DNA binding helix. In some
embodiments, a
zinc finger may comprise one or more mutations at (-5), (-9) and/or (-14). In
further
embodiments, one or more zinc finger in a multi-finger zinc finger protein may
comprise mutations in (-5), (-9) and/or (-14). In some embodiments, the amino
acids
at (-5), (-9) and/or (-14) (e.g. an arginine (R) or lysine (K)) are mutated to
an alanine
(A), leucine (L), Ser (S), Asp (N), Glu (E), Tyr (Y) and/or glutamine (Q).
[0096] Alternatively, the DNA-binding domain may be derived from a
nuclease. For example, the recognition sequences of homing endonucleases and
meganucleases such as I-SceI,I-CeuI,PI-PspI,PI-Sce,I-SceIV ,I-CsmI,I-PanI, I-
Sce11,I-PpoI, I-SceIII, I-CreI,I-TevI, I-TevII and I-TevIII are known. See
also U.S.
Patent No. 5,420,032; U.S. Patent No. 6,833,252; Belfort et al., (1997)
Nucleic Acids
Res. 25:3379-3388; Dujon etal., (1989) Gene 82:115-118; Perler etal., (1994)
Nucleic Acids Res. 22, 1125-1127; Jasin (1996) Trends Genet. 12:224-228;
Gimble
etal., (1996)1 Mol. Biol. 263:163-180; Argast etal., (1998)1 Mol. Biol.
280:345-
353 and the New England Biolabs catalogue. In addition, the DNA-binding
specificity of homing endonucleases and meganucleases can be engineered to
bind
non-natural target sites. See, for example, Chevalier et al., (2002)Molec.
Cell
10:895-905; Epinat etal., (2003) Nucleic Acids Res. 31:2952-2962; Ashworth
etal.,
(2006) Nature 441:656-659; Paques etal., (2007) Current Gene Therapy 7:49-66;
U.S. Patent Publication No. 2007/0117128.
[0097] "Two handed" zinc finger proteins are those proteins in which
two
clusters of zinc finger DNA binding domains are separated by intervening amino
acids so that the two zinc finger domains bind to two discontinuous target
sites. An
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example of a two handed type of zinc finger binding protein is SIP1, where a
cluster
of four zinc fingers is located at the amino terminus of the protein and a
cluster of
three fingers is located at the carboxyl terminus (see Remade et al., (1999)
MIRO
Journal 18 (18): 5073-5084). Each cluster of zinc fingers in these proteins is
able to
bind to a unique target sequence and the spacing between the two target
sequences
can comprise many nucleotides. Two-handed ZFPs may include a functional
domain, for example fused to one or both of the ZFPs. Thus, it will be
apparent that
the functional domain may be attached to the exterior of one or both ZFPs or
may be
positioned between the ZFPs (attached to both ZFPs).
[0098] In certain embodiments, the DNA-binding domain comprises a
naturally occurring or engineered (non-naturally occurring) TAL effector
(TALE)
DNA binding domain. See, e.g., U .S . Patent No. 8,586,526, incorporated by
reference
in its entirety herein. In certain embodiments, the TALE DNA-binding protein
comprises binds to 12, 13, 14, 15, 16, 17, 18, 19, 20 or more contiguous
nucleotides
of a tau target site as shown in Table 1 or 2 (SEQ ID NO:1 to 6 or 33). The
RVDs of
the TALE DNA-binding protein that binds to a tau target site may be naturally
occurring or non-naturally occurring RVDs. See, U.S. Patent Nos. 8,586,526 and
9,458,205.
[0099] The plant pathogenic bacteria of the genus Xanthom*onas are
known to
cause many diseases in important crop plants. Pathogenicity of Xanthom*onas
depends
on a conserved type III secretion (T35) system which injects more than 25
different
effector proteins into the plant cell. Among these injected proteins are
transcription
activator-like effectors (TALE) which mimic plant transcriptional activators
and
manipulate the plant transcriptome (see Kay et al., (2007) Science 318:648-
651).
.. These proteins contain a DNA binding domain and a transcriptional
activation
domain. One of the most well characterized TALEs is AvrBs3 from Xanthom*onas
campestgris pv. Vesicatoria (see Bonas et al., (1989) Mol Gen Genet 218: 127-
136
and W02010079430). TALEs contain a centralized domain of tandem repeats, each
repeat containing approximately 34 amino acids, which are key to the DNA
binding
specificity of these proteins. In addition, they contain a nuclear
localization sequence
and an acidic transcriptional activation domain (for a review see Schornack S,
et al.,
(2006) J Plant Physiol 163(3): 256-272). In addition, in the phytopathogenic
bacteria
Ralstonia solanacearum two genes, designated brgll and hpx17 have been found
that
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are hom*ologous to the AvrBs3 family of Xanthom*onas in the R. solanacearum
biovar
1 strain GMI1000 and in the biovar 4 strain RS1000 (See Heuer etal., (2007)
App!
and Envir Micro 73(13): 4379-4384). These genes are 98.9% identical in
nucleotide
sequence to each other but differ by a deletion of 1,575 bp in the repeat
domain of
hpx17. However, both gene products have less than 40% sequence identity with
AvrBs3 family proteins of Xanthom*onas
[0100] Specificity of these TALEs depends on the sequences found in
the
tandem repeats. The repeated sequence comprises approximately 102 bp and the
repeats are typically 91-100% hom*ologous with each other (Bonas etal., ibid).
.. Polymorphism of the repeats is usually located at positions 12 and 13 and
there
appears to be a one-to-one correspondence between the identity of the
hypervariable
diresidues at positions 12 and 13 with the identity of the contiguous
nucleotides in the
TALE's target sequence (see Moscou and Bogdanove (2009) Science 326:1501 and
Boch etal., (2009) Science 326:1509-1512). Experimentally, the code for DNA
recognition of these TALEs has been determined such that an HD sequence at
positions 12 and 13 leads to a binding to cytosine (C), NG binds to T, NI to
A, C, G
or T, NN binds to A or G, and NG binds to T. These DNA binding repeats have
been
assembled into proteins with new combinations and numbers of repeats, to make
artificial transcription factors that are able to interact with new sequences.
In
addition, U.S. Patent No. 8,586,526 and U.S. Patent Publication No.
2013/0196373,
incorporated by reference in their entireties herein, describe TALEs with N-
cap
polypeptides, C-cap polypeptides (e.g., +63, +231 or +278) and/or novel
(atypical)
RVDs.
[0101] Exemplary TALE are described in U.S. Patent No. 8,586,526 and
.. 9,458,205, incorporated by reference in their entireties.
[0102] In certain embodiments, the DNA binding domains include a
dimerization and/or multimerization domain, for example a coiled-coil (CC) and
dimerizing zinc finger (DZ). See, U.S. Patent Publication No. 2013/0253040.
[0103] In still further embodiments, the DNA-binding domain comprises
a
single-guide RNA of a CRISPR/Cas system, for example sgRNAs as disclosed in
20150056705.
[0104] Compelling evidence has recently emerged for the existence of
an
RNA-mediated genome defense pathway in archaea and many bacteria that has been
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hypothesized to parallel the eukaryotic RNAi pathway (for reviews, see Godde
and
Bickerton, 2006. Mol. Evol. 62: 718-729; Lillestol etal., 2006. Archaea 2: 59-
72;
Makarova et al., 2006. Biol. Direct 1: 7.; Sorek etal., 2008. Nat. Rev.
Microbiol. 6:
181-186). Known as the CRISPR-Cas system or prokaryotic RNAi (pRNAi), the
pathway is proposed to arise from two evolutionarily and often physically
linked gene
loci: the CRISPR (clustered regularly interspaced short palindromic repeats)
locus,
which encodes RNA components of the system, and the cas (CRISPR-associated)
locus, which encodes proteins (Jansen etal., 2002. Mol. Microbiol. 43: 1565-
1575;
Makarova et al., 2002. Nucleic Acids Res. 30: 482-496; Makarova etal., 2006.
Biol.
Direct 1: 7; Haft etal., 2005. PLoS Comput Biol. 1: e60). CRISPR loci in
microbial
hosts contain a combination of CRISPR-associated (Cas) genes as well as non-
coding
RNA elements capable of programming the specificity of the CRISPR-mediated
nucleic acid cleavage. The individual Cas proteins do not share significant
sequence
similarity with protein components of the eukaryotic RNAi machinery, but have
.. analogous predicted functions (e.g., RNA binding, nuclease, helicase, etc.)
(Makarova
etal., 2006. Biol. Direct 1: 7). The CRISPR-associated (cas) genes are often
associated with CRISPR repeat-spacer arrays. More than forty different Cos
protein
families have been described. Of these protein families, Cosi appears to be
ubiquitous
among different CRISPR/Cas systems. Particular combinations of cas genes and
repeat structures have been used to define 8 CRISPR subtypes (Ecoli, Ypest,
Nmeni,
Dvulg, Tneap, Hmari, Apem, and Mtube), some of which are associated with an
additional gene module encoding repeat-associated mysterious proteins (RAMPs).
More than one CRISPR subtype may occur in a single genome. The sporadic
distribution of the CRISPR/Cas subtypes suggests that the system is subject to
horizontal gene transfer during microbial evolution.
[0105] The Type II CRISPR, initially described in S. pyogenes, is one
of the
most well characterized systems and carries out targeted DNA double-strand
break in
four sequential steps. First, two non-coding RNA, the pre-crRNA array and
tracrRNA, are transcribed from the CRISPR locus. Second, tracrRNA hybridizes
to
the repeat regions of the pre-crRNA and mediates the processing of pre-crRNA
into
mature crRNAs containing individual spacer sequences where processing occurs
by a
double strand-specific RNase III in the presence of the Cas9 protein. Third,
the
mature crRNA:tracrRNA complex directs Cas9 to the target DNA via Watson-Crick
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base-pairing between the spacer on the crRNA and the protospacer on the target
DNA
next to the protospacer adjacent motif (PAM), an additional requirement for
target
recognition. In addition, the tracrRNA must also be present as it base pairs
with the
crRNA at its 3' end, and this association triggers Cas9 activity. Finally,
Cas9
mediates cleavage of target DNA to create a double-stranded break within the
protospacer. Activity of the CRISPR/Cas system comprises of three steps: (i)
insertion of alien DNA sequences into the CRISPR array to prevent future
attacks, in
a process called 'adaptation,' (ii) expression of the relevant proteins, as
well as
expression and processing of the array, followed by (iii) RNA-mediated
interference
with the alien nucleic acid. Thus, in the bacterial cell, several of the so-
called `Cas'
proteins are involved with the natural function of the CRISPR/Cas system.
[0106] Type II CRISPR systems have been found in many different
bacteria.
BLAST searches on publically available genomes by Fonfara etal., ((2013)Nuc
Acid
Res 42(4):2377-2590) found Cas9 orthologs in 347 species of bacteria.
Additionally,
.. this group demonstrated in vitro CRISPR/Cas cleavage of a DNA target using
Cas9
orthologs from S. pyo genes, S. mutans, S. therophilus, C. jejuni, N.
meningitides, P.
multocida and E novicida. Thus, the term "Cas9" refers to an RNA guided DNA
nuclease comprising a DNA binding domain and two nuclease domains, where the
gene encoding the Cas9 may be derived from any suitable bacteria.
[0107] The Cas9 protein has at least two nuclease domains: one nuclease
domain is similar to a HNH endonuclease, while the other resembles a Ruv
endonuclease domain. The HNH-type domain appears to be responsible for
cleaving
the DNA strand that is complementary to the crRNA while the Ruv domain cleaves
the non-complementary strand. The Cos 9 nuclease can be engineered such that
only
one of the nuclease domains is functional, creating a Cas nickase (see Jinek
et al.,
(2012) Science 337:816). Nickases can be generated by specific mutation of
amino
acids in the catalytic domain of the enzyme, or by truncation of part or all
of the
domain such that it is no longer functional. Since Cas 9 comprises two
nuclease
domains, this approach may be taken on either domain. A double strand break
can be
achieved in the target DNA by the use of two such Cas 9 nickases. The nickases
will
each cleave one strand of the DNA and the use of two will create a double
strand
break.
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[0108] The requirement of the crRNA-tracrRNA complex can be avoided by
use of an engineered "single-guide RNA" (sgRNA) that comprises the hairpin
normally formed by the annealing of the crRNA and the tracrRNA (see Jinek et
al.,
ibid and Cong et al., (2013) Sciencexpress/10.1126/science.1231143). In S.
pyrogenes, the engineered tracrRNA: crRNA fusion, or the sgRNA, guides Cas9 to
cleave the target DNA when a double strand RNA:DNA heterodimer forms between
the Cas associated RNAs and the target DNA. This system comprising the Cas9
protein and an engineered sgRNA containing a PAM sequence has been used for
RNA guided genome editing (see Ramalingam et al., Stem Cells and Development
22(4):595-610 (2013)) and has been useful for zebrafish embryo genomic editing
in
vivo (see Hwang et al., (2013) Nature Biotechnology 31 (3):227) with editing
efficiencies similar to ZFNs and TALENs.
[0109] The primary products of the CRISPR loci appear to be short RNAs
that
contain the invader targeting sequences, and are termed guide RNAs or
prokaryotic
silencing RNAs (psiRNAs) based on their hypothesized role in the pathway
(Makarova et al., 2006. Biol. Direct 1: 7; Hale et al., 2008. RNA, 14: 2572-
2579).
RNA analysis indicates that CRISPR locus transcripts are cleaved within the
repeat
sequences to release -60- to 70-nt RNA intermediates that contain individual
invader
targeting sequences and flanking repeat fragments (Tang et al., 2002. Proc.
Natl.
Acad. Sci. 99: 7536-7541; Tang et al., 2005. Mol. Microbiol. 55: 469-481;
Lillestol et
al., 2006. Archaea 2: 59-72; Brouns et al., 2008. Science 321: 960-964; Hale
et al.,
2008. RNA, 14: 2572-2579). In the archaeon Pyrococcus furiosus, these
intermediate
RNAs are further processed to abundant, stable -35- to 45-nt mature psiRNAs
(Hale et
al., 2008. RNA, 14: 2572-2579).
[0110] The requirement of the crRNA-tracrRNA complex can be avoided by
use of an engineered "single-guide RNA" (sgRNA) that comprises the hairpin
normally formed by the annealing of the crRNA and the tracrRNA (see Jinek et
al.,
(2012) Science 337:816 and Cong et al., (2013)
Sciencexpress/10.1126/science.1231143). In S. pyrogenes, the engineered
tracrRNA:crRNA fusion, or the sgRNA, guides Cas9 to cleave the target DNA when
a double strand RNA:DNA heterodimer forms between the Cas associated RNAs and
the target DNA. This system comprising the Cas9 protein and an engineered
sgRNA
containing a PAM sequence has been used for RNA guided genome editing (see
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Ramalingam, ibid) and has been useful for zebrafish embryo genomic editing in
vivo
(see Hwang etal., (2013) Nature Biotechnology 31 (3):227) with editing
efficiencies
similar to ZFNs and TALENs.
101111 Chimeric or sgRNAs can be engineered to comprise a sequence
complementary to any desired target. In some embodiments, a guide sequence is
about or more than about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,
22, 23, 24,
25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotides in length. In
some
embodiments, a guide sequence is less than about 75, 50, 45, 40, 35, 30, 25,
20, 15,
12, or fewer nucleotides in length. In certain embodiments, the sgRNA
comprises a
sequence that binds to 12, 13, 14, 15, 16, 17, 18, 19, 20 or more contiguous
nucleotides of a tau target site as shown in Table 1 or 2 (SEQ ID NO:1 to 6 or
33). In
some embodiments, the RNAs comprise 22 bases of complementarity to a target
and
of the form G[n191, followed by a protospacer-adjacent motif (PAM) of the form
NGG or NAG for use with a S. pyogenes CRISPR/Cas system. Thus, in one method,
sgRNAs can be designed by utilization of a known ZFN target in a gene of
interest by
(i) aligning the recognition sequence of the ZFN heterodimer with the
reference
sequence of the relevant genome (human, mouse, or of a particular plant
species); (ii)
identifying the spacer region between the ZFN half-sites; (iii) identifying
the location
of the motif G[N201GG that is closest to the spacer region (when more than one
such
motif overlaps the spacer, the motif that is centered relative to the spacer
is chosen);
(iv) using that motif as the core of the sgRNA. This method advantageously
relies on
proven nuclease targets. Alternatively, sgRNAs can be designed to target any
region
of interest simply by identifying a suitable target sequence the conforms to
the
G[n201GG formula. Along with the complementarity region, an sgRNA may
comprise additional nucleotides to extend to tail region of the tracrRNA
portion of the
sgRNA (see Hsu etal., (2013) Nature Biotech doi:10.1038/nbt.2647). Tails may
be
of +67 to +85 nucleotides, or any number therebetween with a preferred length
of
+85 nucleotides. Truncated sgRNAs may also be used, "tru-gRNAs" (see Fu et
al.,
(2014) Nature Biotech 32(3): 279). In tru-gRNAs, the complementarity region is
diminished to 17 or 18 nucleotides in length.
[0112] Further, alternative PAM sequences may also be utilized, where
a
PAM sequence can be NAG as an alternative to NGG (Hsu 2013, ibid) using a S
pyo genes Cas9. Additional PAM sequences may also include those lacking the
initial
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G (Sander and Joung (2014) Nature Biotech 32(4):347). In addition to the S.
pyogenes encoded Cas9 PAM sequences, other PAM sequences can be used that are
specific for Cas9 proteins from other bacterial sources. For example, the PAM
sequences shown below (adapted from Sander and Joung, ibid, and Esvelt etal.,
(2013) Nat Meth 10(11):1116) are specific for these Cas9 proteins:
Species PAM
S. pyogenes NGG
S. pyogenes NAG
S. mutans NGG
S. thermophilius NGGNG
S. thermophilius NNAAAW
S. thermophilius NNAGAA
S. thermophilius NNNGATT
C. jejuni NNNNACA
N. meningitides NNNNGATT
P. multocida GNNNCNNA
F. novicida NG
[0113] Thus, a suitable target sequence for use with a S. pyogenes
CRISPR/Cas system can be chosen according to the following guideline: [n17,
n18,
n19, or n201(G/A)G. Alternatively the PAM sequence can follow the guideline
G[n17, n18, n19, n201(G/A)G. For Cas9 proteins derived from non-S. pyogenes
bacteria, the same guidelines may be used where the alternate PAMs are
substituted in
for the S. pyogenes PAM sequences.
[0114] Most preferred is to choose a target sequence with the highest
likelihood of specificity that avoids potential off target sequences. These
undesired
off target sequences can be identified by considering the following
attributes: i)
similarity in the target sequence that is followed by a PAM sequence known to
function with the Cas9 protein being utilized; ii) a similar target sequence
with fewer
than three mismatches from the desired target sequence; iii) a similar target
sequence
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as in ii), where the mismatches are all located in the PAM distal region
rather than the
PAM proximal region (there is some evidence that nucleotides 1-5 immediately
adjacent or proximal to the PAM, sometimes referred to as the 'seed' region
(Wu et
al., (2014) Nature Biotech doi:10.1038/nbt2889) are the most critical for
recognition,
so putative off target sites with mismatches located in the seed region may be
the least
likely be recognized by the sg RNA); and iv) a similar target sequence where
the
mismatches are not consecutively spaced or are spaced greater than four
nucleotides
apart (Hsu 2014, ibid). Thus, by performing an analysis of the number of
potential off
target sites in a genome for whichever CRIPSR/Cas system is being employed,
using
these criteria above, a suitable target sequence for the sgRNA may be
identified.
[0115] In certain embodiments, the Cas protein may be a "functional
derivative" of a naturally occurring Cos protein. A "functional derivative" of
a native
sequence polypeptide is a compound having a qualitative biological property in
common with a native sequence polypeptide. "Functional derivatives" include,
but are
not limited to, fragments of a native sequence and derivatives of a native
sequence
polypeptide and its fragments, provided that they have a biological activity
in
common with a corresponding native sequence polypeptide. A biological activity
contemplated herein is the ability of the functional derivative to hydrolyze a
DNA
substrate into fragments. The term "derivative" encompasses both amino acid
sequence variants of polypeptide, covalent modifications, and fusions thereof
In
some aspects, a functional derivative may comprise a single biological
property of a
naturally occurring Cas protein. In other aspects, a function derivative may
comprise
a subset of biological properties of a naturally occurring Cas protein.
Suitable
derivatives of a Cas polypeptide or a fragment thereof include but are not
limited to
mutants, fusions, covalent modifications of Cos protein or a fragment thereof
Cas
protein, which includes Cas protein or a fragment thereof, as well as
derivatives of
Cas protein or a fragment thereof, may be obtainable from a cell or
synthesized
chemically or by a combination of these two procedures. The cell may be a cell
that
naturally produces Cas protein, or a cell that naturally produces Cas protein
and is
genetically engineered to produce the endogenous Cas protein at a higher
expression
level or to produce a Cas protein from an exogenously introduced nucleic acid,
which
nucleic acid encodes a Cas that is same or different from the endogenous Cas.
In some
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case, the cell does not naturally produce Cas protein and is genetically
engineered to
produce a Cas protein.
[0116] Exemplary CRISPR/Cas nuclease systems targeted to specific
genes
(including safe harbor genes) are disclosed for example, in U.S. Publication
No.
2015/0056705.
[0117] Thus, the nuclease comprises a DNA-binding domain in that
specifically binds to a target site in any gene into which it is desired to
insert a donor
(transgene) in combination with a nuclease domain that cleaves DNA.
Tau Gene Modulators
[0118] The tau DNA-binding domains may be fused to or otherwise
associate
with any additional molecules (e.g., polypeptides) for use in the methods
described
herein. In certain embodiments, the methods employ fusion molecules comprising
at
least one DNA-binding molecule (e.g., ZFP, TALE or single guide RNA) and a
heterologous regulatory (functional) domain (or functional fragment thereof).
[0119] In certain embodiments, the functional domain of the tau
modulator
comprises a transcriptional regulatory domain. Common domains include, e.g.,
transcription factor domains (activators, repressors, co-activators, co-
repressors),
silencers, oncogenes (e.g., myc, jun, fos, myb, max, mad, rel, ets, bcl, myb,
mos
family members etc.); DNA repair enzymes and their associated factors and
modifiers; DNA rearrangement enzymes and their associated factors and
modifiers;
chromatin associated proteins and their modifiers (e.g. kinases, acetylases
and
deacetylases); and DNA modifying enzymes (e.g., methyltransferases,
topoisomerases, helicases, ligases, kinases, phosphatases, polymerases,
endonucleases) and their associated factors and modifiers. See, e.g., U.S.
Publication
No. 2013/0253040, incorporated by reference in its entirety herein.
[0120] Suitable domains for achieving activation include the HSV VP16
activation domain (see, e.g., Hagmann etal., I Virol. 71, 5952-5962 (1997))
nuclear
hormone receptors (see, e.g., Torchia etal., Curr. Opin. Cell. Biol. 10:373-
383
(1998)); the p65 subunit of nuclear factor kappa B (Bitko & Bank, I Virol.
72:5610-
5618 (1998) and Doyle & Hunt, Neuroreport 8:2937-2942 (1997)); Liu et al.,
Cancer
Gene Ther. 5:3-28 (1998)), or artificial chimeric functional domains such as
VP64
(Beerli etal., (1998) Proc. Natl. Acad. Sci. USA 95:14623-33), and degron
(Molinari
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etal., (1999) EAJBO J. 18, 6439-6447). Additional exemplary activation domains
include, Oct 1, Oct-2A, Spl, AP-2, and CTF1 (Seipel etal., EMBO J. 11,4961-
4968
(1992) as well as p300, CBP, PCAF, SRC1 PvALF, AtHD2A and ERF-2. See, for
example, Robyr et al., (2000)Mol. Endocrinol. 14:329-347; Collingwood et al.,
(1999)1 Mol. Endocrinol. 23:255-275; Leo etal., (2000) Gene 245:1-11;
Manteuffel-
Cymborowska (1999)Acta Biochim. Pol. 46:77-89; McKenna et al., (1999)1 Steroid
Biochem. Mol. Biol. 69:3-12; Malik etal., (2000) Trends Biochem. Sci. 25:277-
283;
and Lemon etal., (1999) Curr. Opin. Genet. Dev. 9:499-504. Additional
exemplary
activation domains include, but are not limited to, OsGAI, HALF-1, Cl, AP',
ARF-
5,-6,-7, and -8, CPRF1, CPRF4, MYC-RP/GP, and TRABl. See, for example, Ogawa
etal., (2000) Gene 245:21-29; Okanami etal., (1996) Genes Cells 1:87-99; Goff
et
al., (1991) Genes Dev. 5:298-309; Cho etal., (1999) Plant Mol. Biol. 40:419-
429;
Ulmason etal., (1999) Proc. Natl. Acad. Sci. USA 96:5844-5849; Sprenger-
Haussels
etal., (2000) Plant J. 22:1-8; Gong etal., (1999) Plant Mol. Biol. 41:33-44;
and Hobo
etal., (1999) Proc. Natl. Acad. Sci. USA 96:15,348-15,353.
[0121] Exemplary repression domains that can be used to make tau
repressors
include, but are not limited to, KRAB A/B, KOX, TGF-beta-inducible early gene
(TIEG), v-erbA, SID, MBD2, MBD3, members of the DNMT family (e.g., DNMT1,
DNMT3A, DNMT3B), Rb, and MeCP2. See, for example, Bird etal., (1999) Cell
99:451-454; Tyler etal., (1999) Cell 99:443-446; Knoepfler etal., (1999) Cell
99:447-450; and Robertson etal., (2000) Nature Genet. 25:338-342. Additional
exemplary repression domains include, but are not limited to, ROM2 and AtHD2A.
See, for example, Chem etal., (1996) Plant Cell 8:305-321; and Wu etal.,
(2000)
Plant 22:19-27.
[0122] In some instances, the domain is involved in epigenetic regulation
of a
chromosome. In some embodiments, the domain is a histone acetyltransferase
(HAT), e.g. type-A, nuclear localized such as MYST family members MOZ,
Ybf2/Sas3, MOF, and Tip60, GNAT family members Gcn5 or pCAF, the p300
family members CBP, p300 or Rtt109 (Berndsen and Denu (2008) Curr Opin Struct
Biol 18(6):682-689). In other instances the domain is a histone deacetylase
(HDAC)
such as the class I (HDAC-1, 2, 3, and 8), class II (HDAC IIA (HDAC-4, 5, 7
and 9),
HDAC JIB (HDAC 6 and 10)), class IV (HDAC-11), class III (also known as
sirtuins
(SIRTs); SIRT1-7) (see Mottamal etal., (2015)Molecules 20(3):3898-3941).
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Another domain that is used in some embodiments is a histone phosphorylase or
kinase, where examples include MSK1, MSK2, ATR, ATM, DNA-PK, Bubl, VprBP,
IKK-a, PKC131, Dik/Zip, JAK2, PKC5, WSTF and CK2. In some embodiments, a
methylation domain is used and may be chosen from groups such as Ezh2,
PRMT1/6,
PRMT5/7, PRMT 2/6, CARM1, set7/9, MLL, ALL-1, Suv 39h, G9a, SETDB1, Ezh2,
Set2, Doti, PRMT 1/6, PRMT 5/7, PR-Set7 and Suv4-20h. Domains involved in
sumoylation and biotinylation (Lys9, 13, 4, 18 and 12) may also be used in
some
embodiments (review see Kousarides (2007) Cell 128:693-705).
[0123] Fusion molecules are constructed by methods of cloning and
biochemical conjugation that are well known to those of skill in the art.
Fusion
molecules comprise a DNA-binding domain and a functional domain (e.g., a
transcriptional activation or repression domain). Fusion molecules also
optionally
comprise nuclear localization signals (such as, for example, that from the
SV40
medium T-antigen) and epitope tags (such as, for example, FLAG and
hemagglutinin). Fusion proteins (and nucleic acids encoding them) are designed
such
that the translational reading frame is preserved among the components of the
fusion.
[0124] Fusions between a polypeptide component of a functional domain
(or a
functional fragment thereof) on the one hand, and a non-protein DNA-binding
domain
(e.g., antibiotic, intercalator, minor groove binder, nucleic acid) on the
other, are
.. constructed by methods of biochemical conjugation known to those of skill
in the art.
See, for example, the Pierce Chemical Company (Rockford, IL) Catalogue.
Methods
and compositions for making fusions between a minor groove binder and a
polypeptide have been described. Mapp et al., (2000) Proc. Natl. Acad. Sci.
USA
97:3930-3935. Likewise, CRISPR/Cas TFs and nucleases comprising a sgRNA
nucleic acid component in association with a polypeptide component function
domain
are also known to those of skill in the art and detailed herein.
[0125] The fusion molecule may be formulated with a pharmaceutically
acceptable carrier, as is known to those of skill in the art. See, for
example,
Remington's Pharmaceutical Sciences, 17th ed., 1985; and co-owned
International
.. Patent Publication No. WO 00/42219.
[0126] The functional component/domain of a fusion molecule can be
selected
from any of a variety of different components capable of influencing
transcription of a
gene once the fusion molecule binds to a target sequence via its DNA binding
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domain. Hence, the functional component can include, but is not limited to,
various
transcription factor domains, such as activators, repressors, co-activators,
co-
repressors, and silencers.
[0127] In certain embodiments, the fusion molecule comprises a DNA-
binding domain and a nuclease domain to create functional entities that are
able to
recognize their intended nucleic acid target through their engineered (ZFP or
TALE or
sgRNA) DNA binding domains and create nucleases (e.g., zinc finger nuclease or
TALE nucleases or CRISPR/Cas nucleases) cause the DNA to be cut near the DNA
binding site via the nuclease activity. This cleavage results in inactivation
(repression) of a tau gene. Thus, tau repressors also include tau nucleases.
[0128] Thus, the methods and compositions described herein are broadly
applicable and may involve any nuclease of interest. Non-limiting examples of
nucleases include meganucleases, TALENs and zinc finger nucleases. The
nuclease
may comprise heterologous DNA-binding and cleavage domains (e.g., zinc finger
nucleases; TALENs; meganuclease DNA-binding domains with heterologous
cleavage domains, sgRNAs in association with nuclease domains) or,
alternatively,
the DNA-binding domain of a naturally-occurring nuclease may be altered to
bind to
a selected target site (e.g., a meganuclease that has been engineered to bind
to site
different than the cognate binding site).
[0129] The nuclease domain may be derived from any nuclease, for example
any endonuclease or exonuclease. Non-limiting examples of suitable nuclease
(cleavage) domains that may be fused to tau DNA-binding domains as described
herein include domains from any restriction enzyme, for example a Type ITS
Restriction Enzyme (e.g., FokI). In certain embodiments, the cleavage domains
are
.. cleavage half-domains that require dimerization for cleavage activity. See,
e.g., U.S.
Patent Nos. 8,586,526; 8,409,861; and 7,888,121, incorporated by reference in
their
entireties herein. In general, two fusion proteins are required for cleavage
if the
fusion proteins comprise cleavage half-domains. Alternatively, a single
protein
comprising two cleavage half-domains can be used. The two cleavage half-
domains
can be derived from the same endonuclease (or functional fragments thereof),
or each
cleavage half-domain can be derived from a different endonuclease (or
functional
fragments thereof). In addition, the target sites for the two fusion proteins
are
preferably disposed, with respect to each other, such that binding of the two
fusion
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proteins to their respective target sites places the cleavage half-domains in
a spatial
orientation to each other that allows the cleavage half-domains to form a
functional
cleavage domain, e.g., by dimerizing.
[0130] The nuclease domain may also be derived any meganuclease
(homing
endonuclease) domain with cleavage activity may also be used with the
nucleases
described herein, including but not limited to I-SceI,I-CeuI,PI-PspI,PI-Sce,I-
SceIV ,
I-CsmI,I-PanI,I-SceII,I-PpoI, I-SceIII, I-CreI,I-TevI, I-TevII and I-TevIII.
[0131] In certain embodiments, the nuclease comprises a compact TALEN
(cTALEN). These are single chain fusion proteins linking a TALE DNA binding
domain to a TevI nuclease domain. The fusion protein can act as either a
nickase
localized by the TALE region, or can create a double strand break, depending
upon
where the TALE DNA binding domain is located with respect to the meganuclease
(e.g., Tevl) nuclease domain (see Beurdeley etal., (2013) Nat Comm: 1-8 DOT:
10.1038/ncomms2782).
[0132] In other embodiments, the TALE-nuclease is a mega TAL. These
mega TAL nucleases are fusion proteins comprising a TALE DNA binding domain
and a meganuclease cleavage domain. The meganuclease cleavage domain is active
as a monomer and does not require dimerization for activity. (See Boissel
etal.,
(2013) Nucl Acid Res: 1-13, doi: 10.1093/nar/gkt1224).
[0133] In addition, the nuclease domain of the meganuclease may also
exhibit
DNA-binding functionality. Any TALENs may be used in combination with
additional TALENs (e.g., one or more TALENs (cTALENs or FokI-TALENs) with
one or more mega-TALs) and/or ZFNs.
[0134] In addition, cleavage domains may include one or more
alterations as
compared to wild-type, for example for the formation of obligate heterodimers
that
reduce or eliminate off-target cleavage effects. See, e.g., U.S. Patent Nos.
7,914,796;
8,034,598; and 8,623,618, incorporated by reference in their entireties
herein.
[0135] Nucleases as described herein may generate double- or single-
stranded
breaks in a double-stranded target (e.g., gene). The generation of single-
stranded
breaks ("nicks") is described, for example in U.S. Patent Nos. 8,703,489 and
9,200,266, incorporated herein by reference which describes how mutation of
the
catalytic domain of one of the nucleases domains results in a nickase.
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[0136] Thus, a nuclease (cleavage) domain or cleavage half-domain can
be
any portion of a protein that retains cleavage activity, or that retains the
ability to
multimerize (e.g., dimerize) to form a functional cleavage domain.
[0137] Alternatively, nucleases may be assembled in vivo at the
nucleic acid
target site using so-called "split-enzyme" technology (see e.g. U.S. Patent
Publication
No. 2009/0068164). Components of such split enzymes may be expressed either on
separate expression constructs, or can be linked in one open reading frame
where the
individual components are separated, for example, by a self-cleaving 2A
peptide or
IRES sequence. Components may be individual zinc finger binding domains or
domains of a meganuclease nucleic acid binding domain.
[0138] Nucleases can be screened for activity prior to use, for
example in a
yeast-based chromosomal system as described in U.S. Publication No.
2009/0111119.
Nuclease expression constructs can be readily designed using methods known in
the
art.
[0139] Expression of the fusion proteins (or component thereof) may be
under
the control of a constitutive promoter or an inducible promoter, for example
the
galactokinase promoter which is activated (de-repressed) in the presence of
raffinose
and/or galactose and repressed in presence of glucose. Non-limiting examples
of
preferred promoters include the neural specific promoters NSE, Synapsin,
CAMKiia
and MECPs. Non-limiting examples of ubiquitous promoters include CAS and Ubc.
Further embodiments include the use of self-regulating promoters (via the
inclusion of
high affinity binding sites for the tau DNA-binding domain) as described in
U.S.
Patent Publication No. 2015/0267205).
Delivery
[0140] The proteins and/or polynucleotides (e.g., tau modulators) and
compositions comprising the proteins and/or polynucleotides described herein
may be
delivered to a target cell by any suitable means including, for example, by
injection of
proteins, via mRNA and/or using an expression construct (e.g., plasmid,
lentiviral
vector, AAV vector, Ad vector, etc.). In preferred embodiments, the repressor
is
delivered using an AAV vector, including but not limited to AAV9 (see U.S.
Patent
No. 7,198,951), an AAV vector as described in U.S. Patent No. 9,585,971 and/or
an
AAV vector as described in U.S. Provisional Patent Application No. 62/503,121.
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[0141] Methods of delivering proteins comprising zinc finger proteins
as
described herein are described, for example, in U.S. Patent Nos. 6,453,242;
6,503,717; 6,534,261; 6,599,692; 6,607,882; 6,689,558; 6,824,978; 6,933,113;
6,979,539; 7,013,219; and 7,163,824, the disclosures of all of which are
incorporated
by reference herein in their entireties.
[0142] Any vector systems may be used including, but not limited to,
plasmid
vectors, retroviral vectors, lentiviral vectors, adenovirus vectors, poxvirus
vectors;
herpesvirus vectors and adeno-associated virus vectors, etc. See, also, U.S.
Patent
Nos. 8,586,526; 6,534,261; 6,607,882; 6,824,978; 6,933,113; 6,979,539;
7,013,219;
and 7,163,824, incorporated by reference herein in their entireties.
Furthermore, it
will be apparent that any of these vectors may comprise one or more DNA-
binding
protein-encoding sequences. Thus, when one or more tau modulators (e.g.,
repressors) are introduced into the cell, the sequences encoding the protein
components and/or polynucleotide components may be carried on the same vector
or
on different vectors. When multiple vectors are used, each vector may comprise
a
sequence encoding one or multiple tau modulators (e.g., repressors) or
components
thereof In preferred embodiments, the vector system is an AAV vector, for
example
AAV9 or an AAV variant described in U.S. Patent No. 9,585,971 and/or U.S.
Provisional Patent Application No. 62/503,121.
[0143] Conventional viral and non-viral based gene transfer methods can be
used to introduce nucleic acids encoding engineered tau modulators in cells
(e.g.,
mammalian cells) and target tissues. Such methods can also be used to
administer
nucleic acids encoding such repressors (or components thereof) to cells in
vitro. In
certain embodiments, nucleic acids encoding the repressors are administered
for in
vivo or ex vivo gene therapy uses. Non-viral vector delivery systems include
DNA
plasmids, naked nucleic acid, and nucleic acid complexed with a delivery
vehicle such
as a liposome or poloxamer. Viral vector delivery systems include DNA and RNA
viruses, which have either episomal or integrated genomes after delivery to
the cell.
For a review of gene therapy procedures, see Anderson, Science 256:808-813
(1992);
Nabel & Felgner, TIBTECH 11:211-217 (1993); Mitani & Caskey, TIB TECH 11:162-
166 (1993); Dillon, TIB TECH 11:167-175 (1993); Miller, Nature 357:455-460
(1992); Van Brunt, Biotechnology 6(10):1149-1154 (1988); Vigne, Restorative
Neurology and Neuroscience 8:35-36 (1995); Kremer & Perricaudet, British
Medical
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Bulletin 51(1):31-44 (1995); Haddada et al., in Current Topics in Microbiology
and
Immunology Doerfler and Bohm (eds.) (1995); and Yu etal., Gene Therapy 1:13-26
(1994).
[0144] Methods of non-viral delivery of nucleic acids include
electroporation,
lipofection, microinjection, biolistics, virosomes, liposomes,
immunoliposomes,
polycation or lipid:nucleic acid conjugates, naked DNA, naked RNA, artificial
virions, and agent-enhanced uptake of DNA. Sonoporation using, e.g., the
Sonitron
2000 system (Rich-Mar) can also be used for delivery of nucleic acids. In a
preferred
embodiment, one or more nucleic acids are delivered as mRNA. Also preferred is
the
use of capped mRNAs to increase translational efficiency and/or mRNA
stability.
Especially preferred are ARCA (anti-reverse cap analog) caps or variants
thereof See
U.S. Patent Nos. 7,074,596 and 8,153,773, incorporated by reference herein.
[0145] Additional exemplary nucleic acid delivery systems include
those
provided by Amaxa Biosystems (Cologne, Germany), Maxcyte, Inc. (Rockville,
Maryland), BTX Molecular Delivery Systems (Holliston, MA) and Copernicus
Therapeutics Inc, (see for example U.S. Patent No. 6,008,336). Lipofection is
described in e.g., U.S. Patent Nos. 5,049,386; 4,946,787; and 4,897,355) and
lipofection reagents are sold commercially (e.g., TransfectamTm and
LipofectinTM and
LipofectamineTM RNAiMAX). Cationic and neutral lipids that are suitable for
efficient receptor-recognition lipofection of polynucleotides include those of
Felgner,
International Patent Publication Nos. WO 91/17424 and WO 91/16024. Delivery
can
be to cells (ex vivo administration) or target tissues (in vivo
administration).
[0146] The preparation of lipid:nucleic acid complexes, including
targeted
liposomes such as immunolipid complexes, is well known to one of skill in the
art
(see, e.g., Crystal, Science 270:404-410 (1995); Blaese et al., Cancer Gene
Ther.
2:291-297 (1995); Behr et al., Bioconjugate Chem. 5:382-389 (1994); Remy et
al.,
Bioconjugate Chem. 5:647-654 (1994); Gao et al., Gene Therapy 2:710-722
(1995);
Ahmad et al., Cancer Res. 52:4817-4820 (1992); U.S. Patent Nos. 4,186,183;
4,217,344; 4,235,871; 4,261,975; 4,485,054; 4,501,728; 4,774,085; 4,837,028;
and
4,946,787).
[0147] Additional methods of delivery include the use of packaging the
nucleic acids to be delivered into EnGeneIC delivery vehicles (EDVs). These
EDVs
are specifically delivered to target tissues using bispecific antibodies where
one arm
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of the antibody has specificity for the target tissue and the other has
specificity for the
EDV. The antibody brings the EDVs to the target cell surface and then the EDV
is
brought into the cell by endocytosis. Once in the cell, the contents are
released (see
MacDiarmid etal., (2009) Nature Biotechnology 27(7):643).
[0148] The use of RNA or DNA viral based systems for the delivery of
nucleic acids encoding engineered ZFPs, TALEs or CRISPR/Cas systems take
advantage of highly evolved processes for targeting a virus to specific cells
in the
body and trafficking the viral payload to the nucleus. Viral vectors can be
administered directly to patients (in vivo) or they can be used to treat cells
in vitro and
the modified cells are administered to patients (ex vivo). Conventional viral
based
systems for the delivery of ZFPs, TALEs or CRISPR/Cas systems include, but are
not
limited to, retroviral, lentivirus, adenoviral, adeno-associated, vaccinia and
herpes
simplex virus vectors for gene transfer. Integration in the host genome is
possible
with the retrovirus, lentivirus, and adeno-associated virus gene transfer
methods, often
resulting in long term expression of the inserted transgene. Additionally,
high
transduction efficiencies have been observed in many different cell types and
target
tissues.
[0149] The tropism of a retrovirus can be altered by incorporating
foreign
envelope proteins, expanding the potential target population of target cells.
Lentiviral
vectors are retroviral vectors that are able to transduce or infect non-
dividing cells and
typically produce high viral titers. Selection of a retroviral gene transfer
system
depends on the target tissue. Retroviral vectors are comprised of cis-acting
long
terminal repeats with packaging capacity for up to 6-10 kb of foreign
sequence. The
minimum cis-acting LTRs are sufficient for replication and packaging of the
vectors,
which are then used to integrate the therapeutic gene into the target cell to
provide
permanent transgene expression. Widely used retroviral vectors include those
based
upon mouse leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), Simian
Immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and
combinations thereof (see, e.g., Buchscher etal., I Virol. 66:2731-2739
(1992);
Johann etal., I Virol. 66:1635-1640 (1992); Sommerfelt etal., Virol. 176:58-59
(1990); Wilson etal., I Virol. 63:2374-2378 (1989); Miller etal., I Virol.
65:2220-
2224 (1991); International Patent Publication NO. WO 1994/026877).
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[0150] In applications in which transient expression is preferred,
adenoviral
based systems can be used. Adenoviral based vectors are capable of very high
transduction efficiency in many cell types and do not require cell division.
With such
vectors, high titer and high levels of expression have been obtained. This
vector can
be produced in large quantities in a relatively simple system. Adeno-
associated virus
("AAV") vectors are also used to transduce cells with target nucleic acids,
e.g., in the
in vitro production of nucleic acids and peptides, and for in vivo and ex vivo
gene
therapy procedures (see, e.g., West et al., Virology 160:38-47 (1987); U.S.
Patent No.
4,797,368; International Patent Publication No. WO 93/24641; Kotin, Human Gene
Therapy 5:793-801 (1994); Muzyczka,i Clin. Invest. 94:1351 (1994).
Construction
of recombinant AAV vectors are described in a number of publications,
including
U.S. Patent No. 5,173,414; Tratschin et al., Mol. Cell. Biol. 5:3251-3260
(1985);
Tratschin et al., Mol. Cell. Biol. 4:2072-2081 (1984); Hermonat & Muzyczka,
PNAS
81:6466-6470 (1984); and Samulski et al., I Virol. 63:03822-3828 (1989).
[0151] At least six viral vector approaches are currently available for
gene
transfer in clinical trials, which utilize approaches that involve
complementation of
defective vectors by genes inserted into helper cell lines to generate the
transducing
agent.
[0152] pLASN and MFG-S are examples of retroviral vectors that have
been
used in clinical trials (Dunbar et al., Blood 85:3048-305 (1995); Kohn et al.,
Nat.
Med. 1:1017-102 (1995); Malech et al., PNAS 94:22 12133-12138 (1997)).
PA317/pLASN was the first therapeutic vector used in a gene therapy trial.
(Blaese et
al., Science 270:475-480 (1995)). Transduction efficiencies of 50% or greater
have
been observed for MFG-S packaged vectors. (Ellem et al., Immunol Immunother
44(1):10-20 (1997); Dranoff et al., Hum. Gene Ther. 1:111-2 (1997).
[0153] Recombinant adeno-associated virus vectors (rAAV) are a
promising
alternative gene delivery systems based on the defective and nonpathogenic
parvovirus adeno-associated type 2 virus. All vectors are derived from a
plasmid that
retains only the AAV 145 bp inverted terminal repeats flanking the transgene
expression cassette. Efficient gene transfer and stable transgene delivery due
to
integration into the genomes of the transduced cell are key features for this
vector
system. (Wagner et al., Lancet 351:9117 1702-3 (1998), Kearns et al., Gene
Ther.
9:748-55 (1996)). Other AAV serotypes, including AAV1, AAV3, AAV4, AAV5,
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AAV6, AAV8, AAV 8.2, AAV9, and AAV rh10 and pseudotyped AAV such as
AAV2/8, AAV2/5, AAV2/9 and AAV2/6 can also be used in accordance with the
present invention. Novel AAV serotypes capable of crossing the blood-brain
barrier
can also be used in accordance with the present invention (see e.g. U.S.
Patent No.
9,585,971). In preferred embodiments, an AAV9 vector (including variants and
pseudotypes of AAV9) is used.
[0154] Replication-deficient recombinant adenoviral vectors (Ad) can
be
produced at high titer and readily infect a number of different cell types.
Most
adenovirus vectors are engineered such that a transgene replaces the Ad El a,
El b,
and/or E3 genes; subsequently the replication defective vector is propagated
in human
293 cells that supply deleted gene function in trans. Ad vectors can transduce
multiple types of tissues in vivo, including nondividing, differentiated cells
such as
those found in liver, kidney and muscle. Conventional Ad vectors have a large
carrying capacity. An example of the use of an Ad vector in a clinical trial
involved
polynucleotide therapy for antitumor immunization with intramuscular injection
(Sterman etal., Hum. Gene Ther. 7:1083-9 (1998)). Additional examples of the
use
of adenovirus vectors for gene transfer in clinical trials include Rosenecker
et al.,
Infection 24:1 5-10 (1996); Sterman etal., Hum. Gene Ther. 9:7 1083-1089
(1998);
Welsh etal., Hum. Gene Ther. 2:205-18 (1995); Alvarez etal., Hum. Gene Ther.
5:597-613 (1997); Topf et al., Gene Ther. 5:507-513 (1998); Sterman etal.,
Hum.
Gene Ther. 7:1083-1089 (1998).
[0155] Packaging cells are used to form virus particles that are
capable of
infecting a host cell. Such cells include 293 cells, which package adenovirus,
and kv2
cells or PA317 cells, which package retrovirus. Viral vectors used in gene
therapy are
usually generated by a producer cell line that packages a nucleic acid vector
into a
viral particle. The vectors typically contain the minimal viral sequences
required for
packaging and subsequent integration into a host (if applicable), other viral
sequences
being replaced by an expression cassette encoding the protein to be expressed.
The
missing viral functions are supplied in trans by the packaging cell line. For
example,
AAV vectors used in gene therapy typically only possess inverted terminal
repeat
(ITR) sequences from the AAV genome which are required for packaging and
integration into the host genome. Viral DNA is packaged in a cell line, which
contains a helper plasmid encoding the other AAV genes, namely rep and cap,
but
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lacking ITR sequences. The cell line is also infected with adenovirus as a
helper. The
helper virus promotes replication of the AAV vector and expression of AAV
genes
from the helper plasmid. The helper plasmid is not packaged in significant
amounts
due to a lack of ITR sequences. Contamination with adenovirus can be reduced
by,
e.g., heat treatment to which adenovirus is more sensitive than AAV.
[0156] In many gene therapy applications, it is desirable that the
gene therapy
vector be delivered with a high degree of specificity to a particular tissue
type.
Accordingly, a viral vector can be modified to have specificity for a given
cell type by
expressing a ligand as a fusion protein with a viral coat protein on the outer
surface of
the virus. The ligand is chosen to have affinity for a receptor known to be
present on
the cell type of interest. For example, Han et al., Proc. Natl. Acad. Sci. USA
92:9747-
9751 (1995), reported that Moloney mouse leukemia virus can be modified to
express
human heregulin fused to gp70, and the recombinant virus infects certain human
breast cancer cells expressing human epidermal growth factor receptor. This
principle
can be extended to other virus-target cell pairs, in which the target cell
expresses a
receptor and the virus expresses a fusion protein comprising a ligand for the
cell-
surface receptor. For example, filamentous phage can be engineered to display
antibody fragments (e.g., FAB or Fv) having specific binding affinity for
virtually any
chosen cellular receptor. Although the above description applies primarily to
viral
vectors, the same principles can be applied to nonviral vectors. Such vectors
can be
engineered to contain specific uptake sequences which favor uptake by specific
target
cells.
[0157] Gene therapy vectors can be delivered in vivo by administration
to an
individual patient, typically by systemic administration (e.g., intravenous,
intraperitoneal, intramuscular, subdermal, intrathecal, intracisternal,
intracerebroventricular, or intracranial infusion, including direct injection
into the
brain including into any region of the brain such as the hippocampus, cortex,
striatum,
etc.) or topical application, as described below. Alternatively, vectors can
be
delivered to cells ex vivo, such as cells explanted from an individual patient
(e.g.,
lymphocytes, bone marrow aspirates, tissue biopsy) or universal donor
hematopoietic
stem cells, followed by reimplantation of the cells into a patient, usually
after
selection for cells which have incorporated the vector.
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[0158] In certain embodiments, the compositions as described herein
(e.g.,
polynucleotides and/or proteins) are delivered directly in vivo. The
compositions
(cells, polynucleotides and/or proteins) may be administered directly into the
central
nervous system (CNS), including but not limited to direct injection into the
brain or
spinal cord. One or more areas of the brain may be targeted, including but not
limited
to, the hippocampus, the substantia nigra, the nucleus basalis of Meynert
(NBM), the
striatum and/or the cortex. Alternatively or in addition to CNS delivery, the
compositions may be administered systemically (e.g., intravenous,
intraperitoneal,
intracardial, intramuscular, subdermal, intrathecal, intracisternal,
intracerebroventricular and/or intracranial infusion). Methods and
compositions for
delivery of compositions as described herein directly to a subject (including
directly
into the CNS) include but are not limited to direct injection (e.g.,
stereotactic
injection) via needle assemblies. Such methods are described, for example, in
U.S.
Patent Nos. 7,837,668 and 8,092,429, relating to delivery of compositions
(including
expression vectors) to the brain and U.S. Patent Publication No. 2006/0239966,
incorporated herein by reference in their entireties.
[0159] The effective amount to be administered will vary from patient
to
patient and according to the mode of administration and site of
administration.
Accordingly, effective amounts are best determined by the physician
administering
the compositions and appropriate dosages can be determined readily by one of
ordinary skill in the art. After allowing sufficient time for integration and
expression
(typically 4-15 days, for example), analysis of the serum or other tissue
levels of the
therapeutic polypeptide and comparison to the initial level prior to
administration will
determine whether the amount being administered is too low, within the right
range or
too high. Suitable regimes for initial and subsequent administrations are also
variable,
but are typified by an initial administration followed by subsequent
administrations if
necessary. Subsequent administrations may be administered at variable
intervals,
ranging from daily to annually to every several years. In certain embodiments,
[0160] To deliver ZFPs using adeno-associated viral (AAV) vectors
directly
to the human brain, a dose range of lx101 -5x101-5 (or any value therebetween)
vector
genome per striatum can be applied. As noted, dosages may be varied for other
brain
structures and for different delivery protocols. Methods of delivering AAV
vectors
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directly to the brain are known in the art. See, e.g., U.S. Patent Nos.
9,089,667;
9,050,299; 8,337,458; 8,309,355; 7,182,944; 6,953,575; and 6,309,634.
[0161] Ex vivo cell transfection for diagnostics, research, or for
gene therapy
(e.g., via re-infusion of the transfected cells into the host organism) is
well known to
those of skill in the art. In a preferred embodiment, cells are isolated from
the subject
organism, transfected with at least one tau modulator (e.g., repressor) or
component
thereof and re-infused back into the subject organism (e.g., patient). In a
preferred
embodiment, one or more nucleic acids of the tau modulator (e.g., repressor)
are
delivered using AAV9. In other embodiments, one or more nucleic acids of the
tau
modulator (e.g., repressor) are delivered as mRNA. Also preferred is the use
of
capped mRNAs to increase translational efficiency and/or mRNA stability.
Especially preferred are ARCA (anti-reverse cap analog) caps or variants
thereof See
U.S. Patent Nos. 7,074,596 and 8,153,773, incorporated by reference herein in
their
entireties. Various cell types suitable for ex vivo transfection are well
known to those
of skill in the art (see, e.g., Freshney etal., Culture of Animal Cells, A
Manual of
Basic Technique (3rd ed. 1994)) and the references cited therein for a
discussion of
how to isolate and culture cells from patients).
[0162] In one embodiment, stem cells are used in ex vivo procedures
for cell
transfection and gene therapy. The advantage to using stem cells is that they
can be
differentiated into other cell types in vitro, or can be introduced into a
mammal (such
as the donor of the cells) where they will engraft in the bone marrow. Methods
for
differentiating CD34+ cells in vitro into clinically important immune cell
types using
cytokines such a GM-CSF, IFN-y and TNF-a are known (see Inaba et al., I Exp.
Med. 176:1693-1702 (1992)).
[0163] Stem cells are isolated for transduction and differentiation using
known methods. For example, stem cells are isolated from bone marrow cells by
panning the bone marrow cells with antibodies which bind unwanted cells, such
as
CD4+ and CD8+ (T cells), CD45+ (panB cells), GR-1 (granulocytes), and lad
(differentiated antigen presenting cells) (see Inaba etal., I Exp. Med.
176:1693-1702
(1992)).
[0164] Stem cells that have been modified may also be used in some
embodiments. For example, neuronal stem cells that have been made resistant to
apoptosis may be used as therapeutic compositions where the stem cells also
contain
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the ZFP TFs of the invention. Resistance to apoptosis may come about, for
example,
by knocking out BAX and/or BAK using BAX- or BAK-specific TALENs or ZFNs
(see, U.S. Patent No. 8,597,912) in the stem cells, or those that are
disrupted in a
caspase, again using caspase-6 specific ZFNs for example. These cells can be
transfected with the ZFP TFs or TALE TFs that are known to regulate a tau
gene.
[0165] Vectors (e.g., retroviruses, adenoviruses, liposomes, etc.)
containing
therapeutic ZFP nucleic acids can also be administered directly to an organism
for
transduction of cells in vivo. Alternatively, naked DNA can be administered.
Administration is by any of the routes normally used for introducing a
molecule into
ultimate contact with blood or tissue cells including, but not limited to,
injection,
infusion, topical application and electroporation. Suitable methods of
administering
such nucleic acids are available and well known to those of skill in the art,
and,
although more than one route can be used to administer a particular
composition, a
particular route can often provide a more immediate and more effective
reaction than
another route.
[0166] Methods for introduction of DNA into hematopoietic stem cells
are
disclosed, for example, in U.S. Patent No. 5,928,638. Vectors useful for
introduction
of transgenes into hematopoietic stem cells, e.g., CD34+ cells, include
adenovirus
Type 35.
[0167] Vectors suitable for introduction of transgenes into immune cells
(e.g.,
T-cells) include non-integrating lentivirus vectors. See, for example, Ory
etal.,
(1996) Proc. Natl. Acad. Sci. USA 93:11382-11388; Dull etal., (1998)1 Virol.
72:8463-8471; Zuffery etal., (1998) J Virol. 72:9873-9880; Follenzi etal.,
(2000)
Nature Genetics 25:217-222.
[0168] Pharmaceutically acceptable carriers are determined in part by the
particular composition being administered, as well as by the particular method
used to
administer the composition. Accordingly, there is a wide variety of suitable
formulations of pharmaceutical compositions available, as described below
(see, e.g.,
Remington's Pharmaceutical Sciences, 17th ed., 1989).
[0169] As noted above, the disclosed methods and compositions can be used
in any type of cell including, but not limited to, prokaryotic cells, fungal
cells,
Archaeal cells, plant cells, insect cells, animal cells, vertebrate cells,
mammalian cells
and human cells. Suitable cell lines for protein expression are known to those
of skill
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in the art and include, but are not limited to COS, CHO (e.g., CHO-S, CHO-K1,
CHO-DG44, CHO-DUXB11), VERO, MDCK, WI38, V79, B14AF28-G3, BHK,
HaK, NSO, SP2/0-Ag14, HeLa, HEK293 (e.g., HEK293-F, HEK293-H, HEK293-T),
perC6, insect cells such as Spodoptera fugiperda (Sf), and fungal cells such
as
Saccharomyces, Pischia and Schizosaccharomyces. Progeny, variants and
derivatives
of these cell lines can also be used. In a preferred embodiment, the methods
and
composition are delivered directly to a brain cell, for example in the
striatum.
Models of CNS disorders
[0170] Studies of CNS disorders can be carried out in animal model systems
such as non-human primates (e.g., Parkinson's Disease (Johnston and Fox (2015)
Curr Top Behav Neurosci 22: 221-35); Amyotrophic lateral sclerosis (Jackson
etal.,
(2015) J Med Primatol: 44(2):66-75), Huntington's Disease (Yang etal., (2008)
Nature 453(7197):921-4); Alzheimer's Disease (Park etal., (2015) Int J Mol Sci
16(2):2386-402); Seizure (Hsiao etal., (2016) E Bio Med 9:257-77), canines
(e.g.
MPS VII (Gurda etal., (2016)Mol Ther 24(2):206-216); Alzheimer's Disease
(Schutt
et al., (2016) J Alzheimers Dis 52(2):433-49); Seizure (Varatharajah etal.,
(2017) Int
J Neural Syst 27(1):1650046) and mice (e.g. Seizure (Kadiyala etal., (2015)
Epilepsy
Res 109:183-96); Alzheimer's Disease (Li etal., (2015) J Alzheimers Dis Parkin
5(3)
doi 10:4172/2161-0460), (review: Webster etal., (2014)Front Genet 5 art 88,
doi:10.3389f/gene.2014.00088). These models may be used even when there is no
animal model that completely recapitulates a CNS disease as they may be useful
for
investigating specific symptom sets of a disease. The models may be helpful in
determining efficacy and safety profiles of a therapeutic methods and
compositions
(genetic repressors) described herein.
Applications
[0171] Tau modulators (e.g., tau repressors) as described herein
comprising
MAPT-binding molecules (e.g., ZFPs, TALEs, CRISPR/Cas systems, Ttago, etc.) as
described herein, and the nucleic acids encoding them, can be used for a
variety of
applications. These applications include therapeutic methods in which a MAPT-
binding molecule (including a nucleic acid encoding a DNA-binding protein) is
administered to a subject using a viral (e.g., AAV) or non-viral vector and
used to
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modulate the expression of a target gene within the subject. The modulation
can be in
the form of repression, for example, repression of tau expression that is
contributing
to an AD disease state. Alternatively, the modulation can be in the form of
activation
when activation of expression or increased expression of an endogenous
cellular gene
can ameliorate a diseased state. In still further embodiments, the modulation
can be
repression via cleavage (e.g., by one or more nucleases), for example, for
inactivation
of a MAPT gene. As noted above, for such applications, the MAPT-binding
molecules, or more typically, nucleic acids encoding them are formulated with
a
pharmaceutically acceptable carrier as a pharmaceutical composition.
[0172] The MAPT-binding molecules, or vectors encoding them, alone or in
combination with other suitable components (e.g. liposomes, nanoparticles or
other
components known in the art), can be made into aerosol formulations (i.e.,
they can be
"nebulized") to be administered via inhalation. Aerosol formulations can be
placed
into pressurized acceptable propellants, such as dichlorodifluoromethane,
propane,
nitrogen, and the like. Formulations suitable for parenteral administration,
such as,
for example, by intravenous, intramuscular, intradermal, and subcutaneous
routes,
include aqueous and non-aqueous, isotonic sterile injection solutions, which
can
contain antioxidants, buffers, bacteriostats, and solutes that render the
formulation
isotonic with the blood of the intended recipient, and aqueous and non-aqueous
sterile
suspensions that can include suspending agents, solubilizers, thickening
agents,
stabilizers, and preservatives. Compositions can be administered, for example,
by
intravenous infusion, orally, topically, intraperitoneally, intravesically,
retro-orbitally
(RO), intracranially (e.g., to any area of the brain including but not limited
to the
hippocampus and/or cortex) or intrathecally. The formulations of compounds can
be
presented in unit-dose or multi-dose sealed containers, such as ampules and
vials.
Injection solutions and suspensions can be prepared from sterile powders,
granules,
and tablets of the kind previously described.
[0173] The dose administered to a patient should be sufficient to
effect a
beneficial therapeutic response in the patient over time. The dose is
determined by
the efficacy and Ka of the particular MAPT-binding molecule employed, the
target
cell, and the condition of the patient, as well as the body weight or surface
area of the
patient to be treated. The size of the dose also is determined by the
existence, nature,
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and extent of any adverse side-effects that accompany the administration of a
particular compound or vector in a particular patient
[0174] The following Examples relate to exemplary embodiments of the
present disclosure in which the MAPT-modulator comprises a zinc finger
protein. It
will be appreciated that this is for purposes of exemplification only and that
other
MAPT-modulators (e.g., repressors) can be used, including, but not limited to,
TALE-
TFs, a CRISPR/Cas system, additional ZFPs, ZFNs, TALENs, additional
CRISPR/Cas systems, homing endonucleases (meganucleases) with engineered DNA-
binding domains. It will be apparent that these modulators can be readily
obtained
using methods known to the skilled artisan to bind to the target sites as
exemplified
below. Similarly, the following Examples relate to exemplary embodiments in
which
the delivery vehicle is any AAV vector but it will apparent that any viral
(Ad, LV,
etc.) or non-viral (plasmid, mRNA, etc.) can be used to deliver the tau
repressors
described herein.
EXAMPLES
Example 1: MAPT repressors
[0175] A screen of approximately 185 zinc finger proteins engineered
essentially as described in U.S. Patent No. 6,534,261; U.S. Patent Publication
Nos.
2015/0056705; 2011/0082093; 2013/0253040; and 2015/0335708 was performed and
the ZFPs bound to their MAPT target sites. Zinc finger proteins 52288, 52322,
52364, 52366, 52389, 57880, 57890 and 65888 (see Tables 1 through 3 below)
targeted to mouse MAPT were selected for further study. The phosphate contact
mutants listed for 65888 are as previously described (see e.g. U.S. Patent
Application
No. 15/685,580). Table 1 shows the recognition helices of the DNA binding
domain
of these ZFPs, and the target sequences of these ZFPs. A set of ZFPs were also
made
to target MAPT sequences shared between the mouse and human genes. These are
shown in Table 2. Table 3 shows parent and derivative ZFP TFs where the ZFP
backbone has been mutated at the indicated locations to remove potential non-
specific
phosphate contacts. The ZFPs were evaluated by standard SELEX analysis and
shown to bind to their target sites.
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Table 1: mouse MAPT-specific repressor designs
SBS #,Target Design
Fl F2 F3 F4 F5 F6
SBS#52364
RSDNLAR DRSHLAR QSGNLAR QSNTRIM
cgACAGAAGGCGAG
(SEQ ID (SEQ ID (SEQ ID (SEQ ID N/A N/A
gacagaagaggaca
NO:7) NO:8) NO:9) NO:10)
(SEQ ID NO:1)
SBS#52389
ccGTTGCGCCTGAT ERGTLAR TSANLSR TSGNLTR HRTSLTD RSHSLLR HPSARKR
tGATGCCcagctcc (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID
(SEQ ID NO:2) NO:11) NO:12) NO:13) NO:14) NO:15) NO:16)
SBS#52322
RSANLTR DSSHLEL DRSNLTR DRSHLTR DRSHLAR
gtGGCGGAGACTGA
(SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID N/A
GAGcgcgcgcggcc
NO:17) NO:18) NO:19) NO:20) NO:8)
(SEQ ID NO:3)
SBS# 57930
cgGCAGAAGGTGG DRSHLTR LKQHLTR RSAHLSR TSGHLSR QSGNLAR QSGDLTR
GcGGTGGCggcggc (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID
g (SEQ ID NO:20) NO:37) NO:25) NO:26) NO:9) NO:35)
NO :44)
SBS# 57947
gcGGCGGCgGCAG RSAHLSR TSGHLSR QSGNLAR QSGDLTR DRSHLSR DRSHLAR
AAGGTGGGcggtgg (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID
C (SEQ ID NO:25) NO:26) NO:9) NO:35) NO:38) NO:8)
NO :45)
Table 2: human/mouse MAPT-specific repressor designs
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SBS #,Target Design
Fl F2 F3 F4 F5 F6
SBS# 52366
RSDNLSE TSSNRKT TSGNLTR DRSALAR RNSDRTK
ctTCTGTCGATTAT
(SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID N/A
CAGgtaagcgccgc
NO:21) NO:22) NO:13) NO:23) NO:24)
(SEQ ID NO:4)
SBS# 57880
DSSHLEL DRSNLTR DRSHLTR DRSHLAR RSAHLSR TSGHLSR
ctGGTGGGtGGCGG
(SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID
AGACTGAgagcgcg
NO:18) NO:19) NO:20) NO:8) NO:25) NO:26)
(SEQ ID NO:5)
SBS# 57890
tgGTGCTGGAGCT LRHHLTR RRFTLSK RSDVLSE KHSTRRV RSDVLSE RLYTLHK
GGTGGGTggcggag (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID
a (SEQ ID NO:27) NO:28) NO:29) NO:30) NO:29) NO:30)
NO: 6)
SBS# 52288
RSADLTR QSGDLTR RSDHLSE RSAHLSR
agGGGCGGGCAGCG
(SEQ ID (SEQ ID (SEQ ID (SEQ ID N/A N/A
aggcctgggcgggc
NO:34) NO:35) NO:36) NO:25)
(SEQ ID NO:33)
SBS# 65976
DRSNLSR LRQNLIM TSANLTV RSDHLSR QSGNLAR QRNDRKS
ctCCAGAAGGGGAT
(SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID
CATGACctcctcac
NO:39) NO:40) NO:41) NO:42) NO:9) NO:43)
(SEQ ID NO:46)
65976 Phos
contact Qm5 none Qm5 none Qm5 none
mutants
101761 ZFPs including one or more mutations in the backbone regions as
described in U.S. Patent Application No. 15/685,580 (e.g., mutations at made
at
position (-5), (-9) and/or position (-14) relative to the DNA binding helix
for example
R or K to A, L, S, N, E, Y or Q) were also prepared (see, e.g., 65888, 57930,
65918,
65920, 65894, 57947, 65,968, 65887, 65860 and 65976 in Tables 1-3). All ZFPs
described herein were operably linked to a KRAB repression domain to form ZFP-
TFs and all repressed MAPT expression.
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[0177] Exemplary ZFP TFs were transfected into mouse Neuro2a cells or
primary mouse cortical neurons. After 24 hours, total RNA was extracted and
the
expression of MAPT and two reference genes (ATP5b, RPL38) was monitored using
real-time RT-qPCR. As shown in Figure 1, the ZFP-TFs were found to be
effective in
repressing MAPT expression with a diversity of dose-response and MAPT
repression
activity (see Figure 1B).
[0178] ZFP-TF tau repressors were engineered that included mutations
to the
backbone region. Table 3 depicts exemplary ZFP-TF repressors (and their parent
compounds). The results with these optimized ZFP TFs (e.g. exemplary results
shown comparing 65888 to 57880) demonstrated dramatically improved specificity
(by over 10-fold) without affecting activity in primary neurons (tau
repression, see
Figure 1B). Further, two derivatives of the 57880 parent, 65887 and 65888,
were
tested in Neuro2A cells for repression activity for the BOD1 and MOSPD1 off
targets
(see Figure 1D), where off-target repression by the proteins comprising
phosphate
contact mutations in the ZFP backbone were reduced. See, also, U.S. Patent
Application No. 15/685,580.
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Table 3: Optimized ZFP-TF designs
SBS #,Target Design
Fl F2 F3 F4 F5 F6
SBS# 57930
cgGCAGAAGGTGG
DRSHLTR LKQHLTR RSAHLSR TSGHLSR QSGNLAR QSGDLTR
GcGGTGGCggcggc
(SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID
g (SEQ ID
NO 44) NO:20) NO:37) NO:25) NO:26) NO:9) NO:35)
:
[Parent]
SBS# 65918
cgGCAGAAGGTGG DRSHLTR LKQHLTR RSAHLSR TSGHLSR QSGNLAR QSGDLTR
GcGGTGGCggcggc (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID
g (SEQ ID NO:20) NO:37) NO:25) NO:26)
NO:9) NO:35)
NO :44)
65918 Phos
contact Qm5 none none none Qm5 none
mutants
SBS# 65920
cgGCAGAAGGTGG DRSHLTR LKQHLTR RSAHLSR TSGHLSR QSGNLAR QSGDLTR
GcGGTGGCggcggc (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID
g (SEQ ID NO:20) NO:37) NO:25) NO:26)
NO:9) NO:35)
NO :44)
65920 Phos
contact Qm5 none Qm5 none Qm5 none
mutants
SBS# 57890
tgGTGCTGGAGCT
LRHHLTR RRFTLSK RSDVLSE KHSTRRV RSDVLSE RLYTLHK
GGTGGGTggcggag
(SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID
a (SEQ ID
NO:27) NO:28) NO:29) NO:30) NO:29) NO:30)
NO:6)
[Parent]
SBS# 65894
tgGTGCTGGAGCT LRHHLTR RRFTLSK RSDVLSE KHSTRRV RSDVLSE RLYTLHK
GGTGGGTggcggag (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID
a (SEQ ID NO:27) NO:28) NO:29) NO:30) NO:29) NO:30)
NO: 6)
65894 Phos
contact Qm5 none none none Qm5 none
mutants
SBS# 57947
gcGGCGGCgGCAG
RSAHLSR TSGHLSR QSGNLAR QSGDLTR DRSHLSR DRSHLAR
AAGGTGGGcggtgg
(SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID
c (SEQ ID
NO:25) NO:26) NO:9) NO:35) NO:38) NO:8)
NO:45)
[parent]
SBS# 65968
gcGGCGGCgGCAG RSAHLSR TSGHLSR QSGNLAR QSGDLTR DRSHLSR DRSHLAR
AAGGTGGGcggtgg (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID
c (SEQ ID NO:25) NO:26) NO:9) NO:35) NO:38) NO:8)
NO :45)
65968 Phos
contact Qm5 none Qm5 none Qm5 none
mutants
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SES# 57880
ctGGTGGGtGGCGG DSSHLEL DRSNLTR DRSHLTR DRSHLAR RSAHLSR TSGHLSR
AGACTGAgagcgcg (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID
(SEQ ID NO:5) NO:18) NO:19) NO:20) NO:8) NO:25) NO:26)
[Parent]
SES#65888
DSSHLEL DRSNLTR DRSHLTR DRSHLAR RSAHLSR TSGHLSR
ctGGTGGGtGGCGG
(SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID
AGACTGAgagcgcg
NO:18) NO:19) NO:20) NO:8) NO:25) NO:26)
(SEQ ID NO:5)
65888 Phos
contact Qm5 none Qm5 none Qm5 none
mutants
SES#65887
DSSHLEL DRSNLTR DRSHLTR DRSHLAR RSAHLSR TSGHLSR
ctGGTGGGtGGCGG
(SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID
AGACTGAgagcgcg
NO:18) NO:19) NO:20) NO:8) NO:25) NO:26)
(SEQ ID NO:5)
65887 Phos
contact none none Qm5 none Qm5 none
mutants
SES#52389
ccGTTGCGCCTGAT
ERGTLAR TSANLSR TSGNLTR HRTSLTD RSHSLLR HPSARKR
tGATGCCcagctcc
(SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID
(SEQ ID NO:2)
NO:11) NO:12) NO:13) NO:14) NO:15) NO:16)
[Parent]
SBS#65860
ccGTTGCGCCTGAT ERGTLAR TSANLSR TSGNLTR HRTSLTD RSHSLLR HPSARKR
tGATGCCcagctcc (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID
(SEQ ID NO:2) NO:11) NO:12) NO:13) NO:14) NO:15) NO:16)
65860 Phos
contact none none none none Qm5 none
mutants
Example 2: Tau repression in mouse neurons
[0179] All ZFP repressors were cloned into rAAV2/9 vectors using a CMV
promoter to drive expression. Virus was produced in HEK293T cells, purified
using a
CsC1 density-gradient, and titered by real time qPCR according to methods
known in
the art. The purified virus was used to infect cultured primary mouse cortical
neurons
at 3E5, 1E5, 3E4, and 1E4 VG/cell. After 7 days, total RNA was extracted and
the
expression of MAPT and two reference genes (ATP5b, EIF4a2) was monitored using
real-time RT-qPCR.
[0180] As shown in Figure 1C, all ZFP-TF encoding AAV vectors were
found
to effectively repress mouse MAPT over a broad range of infected doses, with
some
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ZFPs reducing the target by greater than 95% at multiple doses. In contrast,
no
MAPT repression was observed for a rAAV2/9 CMV-GFP virus tested at equivalent
doses, or mock-treated neurons (see Figure 1).
[0181] rAAV2/9 encoding CMV-52389-KRAB was used to infect mouse
cortical neurons at 3E5, 1E5, and 3E4 VG/cell. After 10 days, total protein
was
extracted from one set of replicates; a parallel group of infections was used
for total
RNA extraction. Equal amounts of total cell lysate were probed by western blot
for
total tau protein (anti-tau-5), 52389-KRAB (anti-ZNF10), and GAPDH (anti-
GAPDH) (see Figure 2A). Quantitative fluorescence was measured using the LiCOR
Odyssey system, revealing 98% tau protein reduction at the 3E5 VG/cell dose
compared to mock-treated neurons (see Figure 2). The expression of MAPT, 52389-
KRAB, and two reference genes (ATP5b, EIF4a2) was monitored using real-time RT-
qPCR. MAPT transcript levels were reduced by 98% at the 3E5 VG/cell group
relative to mock-treated neurons (see Figure 2).
[0182] Thus, genetic modulators (e.g., repressors) as described herein,
including those that bind to the target sites as shown in Tables 1 and 2, were
functional repressors when formulated as plasmids, in mRNA form, in Ad vectors
and/or in AAV vectors.
Example 3: Specificity of MAPT repression
[0183] The global specificity of the ZFP-TFs shown in Tables 1 and 2
was
evaluated by microarray analysis in mouse Neuro2A cells. In brief, 300 ng of
ZFP-
TF encoding mRNA was transfected into 150,000 Neuro2A cells in biological
quadruplicate. After 24 hours, total RNA was extracted and processed via the
manufacturer's protocol (Affymetrix Genechip MTA1.0). Robust Multi-array
Average (RMA) was used to normalize raw signals from each probe set. Analysis
was performed using Transcriptome Analysis Console 3.0 (Affymetrix) with the
"Gene Level Differential Expression Analysis" option. ZFP-transfected samples
were
compared to samples that had been treated with an irrelevant ZFP-TF (that does
not
bind to MAPT target site). Change calls are reported for transcripts (probe
sets) with
a >2 fold difference in mean signal relative to control, and a P-value < 0.05
(one-way
ANOVA analysis, unpaired T-test for each probeset).
[0184] As shown in Figure 3, SBS#52322 repressed 5 genes in addition
to
MAPT, and caused an increase in 3 others. SBS#52364 and #52366 repressed 4
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genes in addition to MAPT, whereas SBS#52389 caused repression of only the
MAPT gene and an increase of 1 gene. SBS#57880 reduced 2 genes in addition to
MAPT, and increase 1 gene. SBS#57890 reduced 4 genes in addition to MAPT, and
increased 1 gene. SBS# 52288 repressed 124 genes in addition to MAPT and
increased expression of 25 genes.
[0185] ZFP-TF specificity was also assessed in primary human
fibroblasts,
which are more sensitive to transfection and allow for evaluating specificity
at even
higher effective ZFP-TF levels. Four biological replicates of each treatment
were
used, consisting of 300 ng of ZFP-TF encoding mRNA transfected into 150,000
human fibroblasts. After 24 hours, total RNA was extracted and processed via
the
manufacturer's protocol (Affymetrix Human Primeview). Processing and analysis
were as described for Neuro2A cells. Importantly, human fibroblasts do not
express
MAPT, therefore no change in MAPT levels was detected. Based on the fold-
change
criterion previously outlined, SBS#52322 repressed 58 genes and caused an
increase
in 53 others. SBS#52364 repressed 43 genes and increased 25 genes. SBS#52366
repressed 63 and caused an increase in 63 others. SBS#52389 did not repress
any
genes, and increased 13 others. SBS#57880 reduced 7 genes, and increased 2.
SBS#57890 reduced 1 gene, and increased 2, while SBS#52288 repressed 594 genes
while increasing the expression of 302 genes (see Figure 4).
[0186] ZFP-TF specificity was further assessed in primary mouse cortical
neurons following AAV delivery of the ZFP-TFs. Six biological replicates of
each
treatment were used, consisting of rAAV2/9 encoding the CMV-ZFP-TF infected at
1E5 VG/cell into 160k primary mouse cortical neurons. After 7 days, total RNA
was
extracted and processed via the manufacturer's protocol (Affymetrix Genechip
MTA1.0). Processing and analysis were as described for Neuro2A and fibroblasts
cells.
[0187] Based on the fold-change criterion previously outlined,
SBS#52322
repressed 80 genes in addition to MAPT, and caused an increase in 19 others.
SBS#52364 repressed 29 genes in addition to MAPT, and increased 2 genes.
SBS#52389 repressed 1 gene in addition to MAPT. SBS#57880 reduced 60 genes in
addition to MAPT, and increased 3. SBS#57890 only repressed MAPT, and
increased 1 gene (see Figure 5).
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[0188] Experiments were also carried out to examine the effect on
specificity
that modification(s) (as described herein U.S. Patent Application No.
15/685,580) of
potential phosphate contacts from the zinc finger backbone. Two exemplary ZFP
tau
repressing proteins were compared (57880 and 65888) that have identical helix
domains (see Table 3), but 65888 has mutations in the zinc finger backbone to
remove
potential phosphate contacting amino acid residues.
[0189] As shown in Figure 5B, the number of genes that were either up
and
down regulated by 57800 (75 and 110, respectively) were markedly reduced (3
upregulated and 4 down regulated, including tau) by the 65888 protein,
demonstrating
increased specificity by modifications to the backbone region.
Example 4: Neuronal promoters for CNS-restricted ZFP-TF expression
[0190] Three neuronal promoters were evaluated for their ability to
express
ZFP-TFs in primary mouse neurons: a 469 bp fragment of the human Synapsinl
promoter (SYN1; Kugler etal., (2001)Methods Mol Med 53:139-50), a 375 bp
fragment of the alpha-calcium/calmodulin-dependent protein kinase promoter
(CamKII; Dittgen etal., (2004) Proc Natl Acad Sci USA 101(52):18206-11),), and
a
229 bp fragment of the methyl CpG-binding protein 2 promoter (MeCP2; Adachi et
al., (2005) Hum Mol Genet 14(23):3709-22).). Each fragment was cloned in place
of
the CMV promoter used in Example 2, and rAAV2/9 was manufactured for each
promoter construct driving 52389-KRAB linked by a 2A peptide sequence to
enable
co-expression of both proteins (Nagai et al., (2002) Nat Biotechnol 20(1):87-
90 and
M.D. Ryan etal., (1991) J Gen. Virol. '72 p. 2727).
[0191] The purified virus was used to infect cultured primary mouse
cortical
neurons at 3E5, 1E5, 3E4, and 1E4 VG/cell. After 7 days, total RNA was
extracted
and the expression of MAPT and two reference genes (ATP5b, EIF4a2) was
monitored using real-time RT-qPCR.
[0192] All promoters resulted in ZFP expression, with both SYN1 and
CamKII achieving similar transcript levels to CMV. In contrast, MeCP2 was
expressed at roughly 10-fold lower levels. MAPT was also repressed over the
broad
range of infected doses, with SYN1 showing the strongest response, followed by
CAMKII, then CMV, and finally MeCP2 with the weakest levels of MAPT
repression. At the top dose of 3E5 VG/cell, all promoters were capable of
repressing
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MAPT by >90%, with SYN1 showing the strongest MAPT reduction of 99.4%
(-175x fold lower) compared to mock-treated neurons (see Figure 6A).
[0193] The purified virus was also used to infect cultured primary
hippocampal neurons and samples were analyzed and processed as described for
cortical neurons. All neuronal promoters resulted in ZFP expression and MAPT
repression with the same rank order of activity, similar degree of repression,
and
dose-response profiles observed in primary cortical neurons (see Figure 6B).
[0194] Collectively, these results suggest that any of the neuronal
promoters
would be sufficient to achieve ZFP expression and MAPT repression in the
primary
regions of interest for MAPT-related indication, namely the hippocampus and
cortex.
[0195] Further, the use of a strong neuronal promoter (such as SYN1 or
CAMKII) can be employed to reduce the level of a highly expressed transcript
target,
when adequate delivery of the ZFP-TF is a limiting (for example via
intravenous or
intrathecal delivery), and/or in a case where a low-affinity ZFP-TF might be
selected
to minimize off-target gene regulation. Conversely, a weaker neuronal promoter
(such as MeCP2) can be selected when the target gene of interest is expressed
at
moderate to low levels, when sufficient delivery to the target regions is non-
limiting
(for example direct intracranial delivery), and/or to minimize off-target
activity of a
high-affinity ZFP-TF.
Example 5: Repression of human MAPT in iPS-derived neurons
[0196] Based on a standard SELEX specificity analysis of all ZFPs
described
herein, the ZFPs listed in Tables 2 and 3 were predicted to tolerate
mismatches at the
one (in the case of SBS#57890 and SBS#52366) or two (in the case of SBS#57880)
non-conserved positions in the human MAPT sequence (see Figure 7A, SEQ ID
NO:31 and 32).
[0197] Exemplary ZFP-TFs in Tables 2 and 3 were cloned into an AAV
vector (AAV6, AAV9 AAV2/9, or variants thereof) with the 469 bp SYN1 promoter
fragment described in Example 4. rAAV virus was produced in HEK293T cells,
purified using a CsC1 density-gradient, and titered by qPCR. The purified
virus was
used to infect human iPS-derived neurons at 3E5, 1E5, 3E4, and 1E4 VG/cell
(iCell
Neurons, Cellular Dynamics International Inc). After 18 or 19 days, total RNA
was
extracted and expression of human MAPT, ZFP-KRAB, and three reference genes
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(ATP5b, EIF4a2, GAPDH) was assessed using real-time RT-qPCR. Similar levels of
ZFPs were expressed for each virus across the range of doses tested.
[0198] Exemplary results are shown in Figure 7. At the top dose of 3E5
VG/cell, 57880-KRAB repressed human MAPT by 95%, 52366-KRAB resulted in
87% MAPT reduction, and 57890-KRAB lowered MAPT by 45% compared to mock-
infected cells (see Figure 7B). Similar results were obtained for the other
transcription factors described herein (e.g., ZFP-TFs as shown in Tables 1
through 3).
One explanation for the reduced human MAPT repression activity of SBS#57890 ¨
compared to the behavior observed for mouse MAPT ¨ is its exceptional degree
of
.. genome-wide specificity, consistent with a poor tolerance for mismatches in
its target
site (see Example 3).
[0199] The CMV, SYN1, and CAMKII promoters were evaluated for ZFP-TF
expression and MAPT repression of human MAPT using the ZFP-TF 52366.
rAAV2/9 was manufactured for each promoter construct driving 52366-KRAB and
used to infect human iPS-derived neurons as described. The SYN1 promoter
resulted
in the highest levels of ZFP expression and MAPT reduction (5% of mock-treated
neurons) compared to CAMKII (40% of mock) and CMV (71% of mock) (see Figure
7C).
[0200] The human iPS derived neurons were also subject to microarray
analysis as described above to analyze the amount of off target repression in
a
population of 19,959 coding transcripts. In each case, the ZFP-TFs were
compared to
the 65976 protein based on the fold-change criterion previously outlined, and
the plots
indicate the change in profile for the test ZFP-TF (see Figure 7D). The
results
indicate that several of the human tau ZFP-TFs are highly specific in human
neurons.
Example 6: In vivo MAPT repression driven by AAV-delivered ZFP TFs
[0201] Various ZFP-TFs as described herein were delivered using
various
different AAV vectors to the mouse hippocampus to evaluate repression of MAPT
in
vivo. AAVs encoding Tau repressors were administered either intravenously (IV)
or
intracerebroventricularly (ICV) with 57890.T2A.Venus or intracranially (IC) by
stereotactic injection.
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[0202] In brief, for AAV 2/9, a total dose of 8E9 VGs of rAAV2/9-CMV-
ZFP-TF per hemisphere was administered by stereotactic injection via dual,
bilateral 2
[it injections. For AAV vectors described in U.S. Patent No. 9,585,971, AAV
vectors were administered intracerebroventricularly (ICV) or intravenously
(IV) at
total doses of 1E12. AAV vectors as described in U.S. Provisional Patent
Application
No. 62/503,121 were administered ICV at 1E12 vg or 2E12 vg/animal for IV.
[0203] The animals were sacrificed four (IV and ICV) or five weeks
(IC) post-
injection. For the IV and ICV treated animals, the right hemisphere was
dissected into
the various brain regions for RT-qPCR analysis, and the left hemisphere was
used for
histology. For the IC treated animals, each hippocampus was sectioned into
three
pieces for RT-qPCR analysis (A, Anterior; B, middle; C, posterior
hippocampus).
MAPT and ZFP-TF expression was also analyzed by real time RT-qPCR and
normalized to the geometric mean of three housekeeping genes (ATP5b, EIF4a2
and
GAPDH).
[0204] The data showed that the AAV vectors efficiently transduced neuronal
targets when administered intracerebroventricularly or intravenously and that
the
ZFP-TFs resulted in potent and sustained tau reduction throughout the CNS
(brain and
spinal cord, including in frontal cortex, anterior cortex, posterior cortex,
hippocampus, brain stem, striatum, thalamus, midbrain, cerebellum, lumbar
spinal
cord, thoracic spinal cord and cervical spinal cord). Figures 8A to 8C show
exemplary results using ZFP-TFs SBS#52322, SBS#52389 and SBS#57890 following
administration of AAV vectors described in 62/503,121 ("SGMO" in Figure 8B) as
compared to AAV9 ("9" in Figure 8B). The micrograph depicted in Figure 8A was
taken 4 weeks after delivery of the SGMO vector, with doses of 1 ell vg/mouse
for
ICV delivery and 2e12 vg/mouse for IV delivery. Figure 8C depicts the changes
in
tau mRNA levels following a single administration via ICV or IV dosing from
the
same experiment. The same results were found for all additional ZFP-TFs
tested.
[0205] Figures 8D and 8E show exemplary results using AAV2/9 vector
and
demonstrated that for intracranial delivery to the hippocampus, both 52389-
KRAB
and 52322-KRAB were able to repress MAPT efficiently (see Figure 8D). Coverage
was assessed by the relative ZFP expression levels across the three slices for
each
hemisphere. This analysis revealed that, while many slices had excellent
coverage,
several had little or no ZFP-TF expression, suggesting non-uniform coverage
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throughout the hippocampus for several animals following intracranial
stereotaxic
delivery (see Figure 8E). Similar results were obtained with other ZFP-TFs as
described herein.
[0206] In addition, tau repressors as described herein delivered using
the AAV
vector described in U.S. Patent No. 9,585,971 also significantly repressed tau
in the
CNS (including spinal cord). Exemplary results are shown in Figures 8F-8H. For
all
ZFP-TFs described herein, repression in the CNS was rapid and sustained over
time,
both at the mRNA protein levels. Furthermore, CSF levels of tau correlate with
brain
levels and thus can be used as an indicator of tau repressor function (see,
exemplary
Figure 8H).
[0207] In sum, any tau repressor as described herein, delivered by any
AAV
vector by any route effectively modulates (represses) tau for sustained
periods.
[0208] To further understand the degree of ZFP-TF induced MAPT
repression
in the hippocampus, three analysis methods were used. First, ZFP-TF and MAPT
expression values from all six slices from each animal in a treatment group
were
averaged and compared to the PBS group by ANOVA followed by Sidak's post-test.
Highly significant (P<0.0001) MAPT repression was observed for both 52322
(mean
MAPT reduction 66%) and 52389 (mean MAPT reduction 55%) (see Figure 9A).
[0209] Second, the section from each animal with the highest level of
ZFP-TF
expression was identified and used to calculate the average maximal tau
reduction
under an ideal coverage scenario. Again, highly significant (P<0.0001) MAPT
repression was observed for all ZFP-TFs tested. Exemplary results, shown in
Figure
9B, show reduction by over 70% using 52322 (mean MAPT reduction 73%) and
52389 (mean MAPT reduction 89%).
[0210] Third, the correlation between MAPT and ZFP-TF levels (expressed
here as the absolute value of ZFP copies / ng of RNA input in the RT-qPCR
reaction)
was assessed. A highly significant relationship was observed for both 52322
(P<0.0001, R2 = 0.53) and 52389 (P<0.0001, R2 = 0.82) (see Figure 9C and 9D).
[0211] Tau reduction in the P301L mutant human tau (P301L) transgenic
mouse model of tauopathy (rTg4510, Jackson Labs) is also assessed following
administration of genetic repressors as described herein. In addition, the
clinical and
therapeutic effectiveness of the repressors is evaluated in this and other
mouse models
of AD (e.g., APPswe/PS1d9, Jackson Labs) to determine whether there is a
reduction
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in biomarkers and symptoms of tauopathies, including one or more the
following:
neurotoxicity, gliosis, dystrophic neurites, spine loss, excitotoxicity,
cortical and
hippocampal shrinkage, dendritic tau accumulation, cognitive (e.g., the radial
arm
maze and the Morris water maze, fear conditioning, etc.), and motor deficits.
See,
e.g., Bryan etal., (2009) Chapter 1: Transgenic Mouse Models of Alzheimer's
Disease: Behavioral Testing and Considerations in Methods of Behavior Analysis
in
Neuroscience. 2nd edition, ed. Buccafusco, Boca Raton (FL): CRC Press/Taylor &
Francis. Additionally, chemically induced seizure models, for example, wild-
type
mice treated with excitotoxic compounds such as pentylenetetrazole (PTZ, see
e.g.
Meyers etal., (1975) Epilepsia 16(2):257-67) or kainate (Ferraro etal.,
(1997)Mamm
Genome 8:200- 208, are also assessed at 4-8 weeks following administration of
genetic repressors as described herein, to determine whether tau reduction
confers a
protective effect against seizure, including, fatality related to seizure,
prolonged
latency to seizure, and/or reduction in seizure severity.
[0212] Tau modulators (e.g., repressors) as described herein were delivered
using non-viral or viral (e.g., AAV) vectors as described herein directly into
the brain,
for example by intracranial injection into the hippocampus or cortex,
intracerebroventricular or intravenous delivery. After a period of time post-
administration of the repressors (1-11 months post-repressor administration),
brains
were harvested, sectioned and subjected to immunohistochemistry analysis
and/or
imaging analysis (e.g., 2-photon imaging) to assess neuronal viability,
gliosis,
dystrophic neurites, spine density, cortical and hippocampal thickness, tau
mRNA
expression and tau protein levels.
[0213] Mice receiving the repressors showed an 80-90% or more
repression of
tau (mRNA and protein) in ZFP expressing neurons 8 weeks after administration
of
the ZFP repressors. No significant neuronal loss or elevated gliosis was seen
after
long-term (6 months) tau knock-down. Mouse tau repression also protective
against
neuritic dystrophies. Thus, the tau modulators (e.g., repressors) as described
herein
provide in vivo clinical and therapeutic benefits to subjects with
tauopathies.
Example 7: In vivo MAPT repression and protein knockdown driven by AAV-
delivered ZFP TFs expressed from neuronal promoters
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[0214] Recombinant AAV vectors encoding ZFP-TFs 2A-Venus constructs as
described herein driven by the CMV, SYN1, CAMKII, or MeCP2 promoters
described in Example 5 were delivered to the mouse hippocampus to evaluate
MAPT
mRNA and protein reduction in vivo. In brief, a total dose of 2.4E10 VGs per
hemisphere was administered by stereotactic injection to adult wild-type mice
via
dual, bilateral 1.5 [it injections. The animals were sacrificed six weeks post-
injection
and each hippocampus was finely hom*ogenized with a razor blade, and the
resulting
tissue was distributed into two equal amounts. One half was taken for mRNA
analysis, wherein MAPT, ZFP-TF, GFAP, and IBA1 expression was analyzed by real
time RT-qPCR and normalized to the geometric mean of three housekeeping genes
(ATP5b, EIF4a2 and GAPDH). The other half was taken for quantitative tau
protein
analysis by ELISA, and exemplary results are presented in Figure 10. Relative
to
control-treated animals, tau mRNA was reduced by at least 85% following
administration of the AAV ZFP-TF. Exemplary results following administration
of
rAAV9-CMV-52389V were as follows: 63% reduction in tau expression by the
rAAV9-SYN1-52389V; 74% reduction in tau expression by the rAAV9-CAMKII-
52389V; and 83% reduction by the rAAV9-MeCP2-52389V construct (P<0.0001 for
all promoters). Mean ZFP expression was found to be highest for the vector
encoding
the CAMKII promoter (set to a value of 1), lowest for the MeCP2 promoter (15%
of
the CAMKII levels), and intermediate for the CMV (59% of CAMKII levels) and
SYN1 (90% of CAMKII levels) promoters.
[0215] The presence of microglia and activated astrocytes was also
assessed
using RT-qPCR reagents for IBA1 and GFAP, respectively. The MeCP2 promoter
resulted in no significant changes in IBA1 levels compared to PBS-injected
animals,
SYN1 resulted in 4.7-fold higher IBA1 levels (P<0.0001), CMV resulting in 3.0-
fold
higher levels (P<0.05), and CAMKII resulted in levels 3.2-fold higher
(P<0.001).
Similarly, the MeCP2 promoter resulted in no GFAP elevation compared to PBS-
injected animals, SYN1 resulted in 4.0-fold higher GFAP levels (P<0.0001), CMV
resulting in 3.2-fold higher levels (P<0.01), and CAMKII resulted in levels
2.4-fold
higher (P<0.05).
[0216] Robust reduction in total tau protein was also observed for all
three
neuronal promoter constructs (exemplary results using SBS 52389 shown in
Figure
11). Compared to PBS-injected control levels (269 ng/ml), the rAAV9-SYN1-
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52389V construct resulted in 23% of control levels (61 ng/ml, P<0.0001), the
rAAV9-
CAMKII-52389V construct yielded 18% of control levels (49 ng/ml, P<0.0001),
the
rAAV9-SYN1-52389V construct had 12% of control levels (32 ng/ml, P<0.0001).
Similar or high levels of reduction are obtained with other ZFP-TFs as
described
herein (e.g., 57890, 65894, 57930, 65918, 65920, 57880, 65887, 65888, 57947,
65968, 52322, 52364, 52366, 52288, 52389 and/or 65860).
[0217] Thus, neuronal promoters are capable of driving >80% tau mRNA
and
protein reduction throughout the mouse hippocampus.
Example 8: Histological evidence of tau reduction throughout the hippocampus
and connected brain regions
[0218] Artificial transcription factors as described herein were
delivered to the
mouse hippocampus to evaluate repression of tau in vivo by immunofluorescence
staining. A total dose of 2.4E10 VGs of rAAV-CMV-ZFP-TF (rAAV2/9, rAAV6,
etc.) was administered by stereotactic injection via dual, unilateral 1.5 [IL
injections to
adult wild-type mice. The ZFP-TFs were linked to a fluorescent Venus protein
to
localize TF expression. The animals were sacrificed six weeks post-injection
and the
entire brain was collected and sectioned for histological staining for nuclei
(DAPI),
tau and GFP using standard methods. Fluorescent activated cell sorting (FACS)
analysis was also performed. In brief, to evaluate the tau knock-down
efficiency in
AAV ZFP-dVenus transduced cells, we dissociated hippocampi injected with AAV
CMV ZFP-dVenus using a papain dissociation kit (Worthington Biochem. Corp.)
according to the manufacturer manual and then analyzed the cell suspension
using
flow cytometry (Miltenyi MACSQuant VYB Flow Cytometer). dVenus or GFP
.. expressing cells in the same samples were then separated by FACS using the
green
488 nm laser (BioRadS3 Cell Sorter; 30k-50k GFP positive cells per sample).
After
sorting, cells were spun down by centrifugation for 10 min at 1,000x g and
resuspended in 100 ill RNA (ThermoFisher) for RNA analysis.
[0219] Exemplary results are shown in Figure 12 (animal injected with
rAAV2/9-CMV-ZFP-TF SBS #52389). Figure 12A shows a cross section of the
mouse brain through the anterior hippocampus and identifies four regions boxed
and
numbered 1-4. Injection was into the left hemisphere (the ipsilateral side)
which
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results in higher fluorescence in the left side of the cross section. The
subsequent
panels are close up images of each of these boxes. The boxes are described
below:
[0220] Box 1 (Figure 12B) shows quantitation of fluorescence intensity
across
the retrosplenial cortex where ZFP-TF expression is clearly demarcated between
the
ipsilateral and contralateral hemispheres. In the left panel of Figure 12B,
the
fluorescent signal was higher on the ipsilateral side of the midline ("ipsi")
than on the
contralateral side ("contra"). ZFP expression here likely arises from
hippocampal
axonal projections. The majority of the signal on the right side of the panel
was due
to the DAPI staining of the nuclei. The middle image shows overall
fluorescence with
anti-Tau tagged antibodies where the signal in the ipsilateral (left) side of
the panel
was nearly all due to GFP signal while the signal on the contralateral (right)
side of
the panel was mostly due to the presence of Tau and to underlying DAPI
staining. The
right panel of 12B shows a cross sectional box that was analyzed for
quantification of
the tau signal (depicted in Figure 12C). The figure shows reduced tau-specific
fluorescence from the injected (treated) side ("ipsi" versus "contra").
[0221] Box 2, Figure 12D (ipsilateral) and Box 3, Figure 12E
(contralateral)
are images from the hippocampus showing tau reduction in the ipsilateral
(treated)
side versus the contralateral side, tau knockdown was particularly clear in
the dentate
gyms on the contralateral side versus ipsilateral (indicated by the arrow).
[0222] Box 4 is region of the hypothalamus where there was focal ZFP
expression in the ipsilateral side (top panel shows GFP fluorescence, left
oval) and
bulk tau fluorescence (bottom panel) shows ¨50% reduction in total tau signal
(see
graph between the ipsilateral side and the contralateral side). Similar
results are
obtained using AAV constructs comprising ZFP-TFs as described in Tables 1, 2
and
3.
[0223] Thus, the ZFP-TFs as described herein repress tau expression in
vivo.
Example 9: In vivo safety, tau reduction, and sustained expression of a ZFP-TF
in the mouse hippocampus
[0224] To assess the safety of both short- and long-term hippocampal
expression of a tau targeted artificial transcription factors described
herein, ZFP-TFs
as described herein were delivered in vivo to the mouse hippocampus to monitor
ZFP
expression as well as astrocytes and microglia responses, and gross
hippocampal
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anatomy. In one experiment, a total dose of 2.4E10 VGs of rAAV2/9-CMV-52389-
2A-Venus ("389dV"), rAAV2/9-CMV-ZFP-TF ("389"), rAAV2/9-CMV GFP
("GFP"), or PBS ("PBS") was administered by stereotactic injection via dual,
unilateral 1.5 uL injections to adult wild-type mice. Two cohorts were
sacrificed
either at six weeks or at six months post-injection and the entire brain was
collected
and sectioned for histological staining for nuclei (DAPI), GFP, GFAP (to
detect
activated astrocytes), and IBA1 (to detect microglia) using standard methods
and the
results are shown in Figure 13. Figure 13H comprises images depicting the
fluorescence of two regions of the hippocampus demonstrating that following
the
injections tau was repressed in both the anterior and posterior areas of the
hippocampus.
[0225] For the 6-week time point (Figure 13A), depicting signal for
the
transduced cells in the hippocampus, the signal for the ZFP-TF is nearly the
same as
for the GFP vector. Figure 13B shows the results for the astrocytes and also
shows
nearly the same results across the three treatments, as also seen in the
hippocampal
microglia (Figure 13C).
[0226] For the 6-month time point, the data in Figure 13D demonstrates
very
similar levels of fluorescence for the GFP and ZFP TF vectors as seen in the 6-
week
time point, suggesting that there was not a large change in the amount of
transduced
cells in the hippocampus over time. Figure 13E also shows that the signal
between
the different treatments was very similar and did not change significantly
between the
6-week time point and the 6-month time point (Figure 13B versus 13E- note the
difference in the y axis between the two figures). The microglia at the 6-
month time
point after treatment with the AAV vectors are nearly the same, with a slight
decrease
in the PBS treated samples.
[0227] A measurement of the mean CA1 (one of the hippocampal regions)
thickness shows no significant change after 6 months of ZFP expression (Figure
13G), demonstrating no overt neuronal toxicity from long-term expression of
the ZFP.
[0228] An additional analysis was performed where the ZFP-TFs were
.. administered as above except that only PBS and the 52288, 52364 and 52389
constructs, linked to 2a-Venus were compared. Dosing was as described in
Example
6. After dosing, animals were sacrificed at 4 weeks, and the data is presented
in
Figure 131. The data presented shows that treatment with the 52288 construct
resulted
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in substantially attenuated vector genomes and ZFP expression compared to the
other
constructs, and did not repress tau expression in any significant way. It did
however
result in elevated levels of GFAP and IBA'. The ZFP 52364 had higher vector
genome levels and ZFP expression than the 52288 construct, but led to
similarly high
levels of GFAP and IBA1, and did not significantly repress tau expression. In
contrast, the 52389 construct was able to cause a significant drop in tau
expression
without impacting GFAP or IBA1 expression.
[0229] The lack of tau reduction in vivo and elevated
neuroinflammatory
biomarkers for ZFPs 52288 and 52364 correlate with the ZFP specificity
profiles
assessed in Neuro2A cells, human fibroblasts, and primary neurons (Figures 3,
4, 5),
showing that highly specific ZFPs significantly reduced tau expression and
were
tolerable in vivo.
[0230] Part of the dosing groups receiving PBS or the 52389-2a-Venus
were
extended for 11 months. In addition to analyzing the amount of tau repression,
samples were also analyzed for mRNA expression levels of the amount of ZFP
expressed, and any change in the amount of transcripts encoding GFAP, IBA1,
NeuN,
MAP1A, MAP1B, and MAP2. In brief, each hippocampus was finely hom*ogenized
with a razor blade, and the resulting tissue was distributed into two equal
amounts.
One half was taken for mRNA analysis, wherein MAPT, ZFP, GFAP, IBA1, NeuN,
MAP1A, MAP1B, and MAP2 expression was analyzed by real time RT-qPCR and
normalized to the geometric mean of three housekeeping genes (ATP5b, EIF4a2
and
GAPDH). Exemplary results are presented in Figure 13J which also includes the
6-
week data for comparison. The data demonstrates that in general there was not
a large
change in most data sets analyzed between the 6-week and 11-month time frames.
There was a decrease detected however in the amount of ZFP expression
detected. No
significant compensatory changes were observed for three other Microtubule
Associated Proteins (MAP1A, MAP1B, and MAP2), nor was there any reduction in
NeuN levels, consistent with the preservation of hippocampal volume in ZFP-
treated
hemispheres assessed six months after ZFP delivery. Additional experiments
were
performed using ZFP-TFs as described herein and produced similar results.
[0231] Thus, the results demonstrate that, following a single
administration,
ZFP expression persists over time in vivo and does not result in toxicity to
the CNS of
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the subject, and that ZFP-mediated tau reduction in the adult hippocampus is
sustained and well-tolerated for at least 11 months.
[0232] Another study was performed using C57BL/6 wild type mice (8
week
old females) where the ZFP-TFs were delivered using AAVs described herein via
retro-orbital delivery. 2.5e12 viral genomes (vg) per animal was used and the
animals
were sacrificed ten weeks later. Microarray analysis was performed on the
hippocampal tissue from the treated mice, where the data shown is in
comparison to
an irrelevant ZFP-TF delivered the same way. Figure 17 shows that tau was
specifically repressed by the 57890V and 52389V proteins as compared to
treatment
with a GFP encoding AAV. These data demonstrate that the specificity of these
proteins observed in vitro is also found in vivo.
Example 10: Tau reduction in mouse model of AD
[0233] Studies were performed in a mouse model of AD in addition to
those
done above in wild type mice. APP/PS1 mice (reviewed in Li etal., ibid) were
treated with the ZFP-TFs as described herein to analyze efficacy in this
model.
[0234] In brief, in one experiment, 4 APP/PS1 mice and 2 wild type
controls
were treated at 4.5 months of age. Single injections were performed
introducing 3 [IL
of AAV composition into the opposite cortices (CTX) of the brain. The left CTX
of
each mouse received 3 [IL of AAV9 comprising the ZFP-TF linked to GFP driven
by
the Synapsinl promoter (either an irrelevant ZFP-TF control ("172"; AAV9 synl-
172v)) or the 52389 ZFP-TF ("389"; AAV9 syn1-389v). The right CTX of each
mouse received 3 [IL of AAV9 comprising a RFP expression construct driven by
the
Synapsinl promoter ("AAV9 synl-tRFP").
[0235] Eleven (11) weeks following treatment, the 7-month old mice were
perfused with 4% PFA/PBS. The whole brains were then removed and post-fixed
for
2 days with 4%PFA/PBS at 4 C. The tissue was then cryoprotected in a 30%
sucrose/PBS solution. 50 p.m coronal sections were analyzed for
immunofluorescent
labeling for tRFP, GFP, and amyloid beta ("AP"). 3 brain sections were
analyzed per
mouse and 3-5 images of each section were taken. Dystrophic neurites were
counted
in the AP¨rich plaques such that between 121 and 256 plaques were analyzed per
animal. 708 plaques were counted for the AAV9 syn1-389v treated CTX, and 287
were counted for the AAV9 synl-tRFP CTX. Exemplary data are presented in
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Figures 14A-D, and showed a statistically significant difference in the number
of
dystrophic neurites per numbers of AP plaques in the cortices treated with the
52389
ZFP-TF as compared to the tRFP treated cortices (Figures 15 and 16). In
contrast,
there was no difference in dystrophies per plaque in the animals treated with
the
irrelevant ZFP-TF. In addition, the number and volume of plaques was unchanged
in
both treated and untreated animals. Further experiments performed using other
AAV
vectors, modes of administration and/or ZFP-TFs as described herein produced
similar results.
[0236] Thus, the tau genetic repressors described herein provide
clinical and
therapeutic benefit for tauopathies such as AD, as shown by studies in known
mouse
models of AD.
Example 11: Tau reduction in vivo in a primate brain
[0237] Primate tau-specific ZFP-TFs are tested in cynomolgus monkeys
(M
fascicularis) to observe repression of tau expression in a primate (non-human
primate
(NHP) model). Cynomolgus monkeys are housed in stainless steel cages equipped
with a stainless mesh floor and an automatic watering valve. The study
complies with
all applicable sections of the Final Rules of the Animal Welfare Act
regulations (Code
of Federal Regulations, Title 9).
[0238] Initial experiments are conducted to determine if the hippocampus of
the monkeys can be adequately targeted. Control article (Formulation Buffer,
PBS
and 0.001% Pluronic F-68, pH 7.1) and test article are thawed and dispensed on
Day 1
of the studies where test and control articles are administered via MRI-guided
delivery of AAV9-GFP or AAV9-ZFP-TFs to the hippocampus.
[0239] In the first experiment, AAV9 comprising a hSYN1 driven GFP gene
are delivered at 3.84 ell vg/hemisphere to the left hemisphere and 7.68 ell
vg/hemisphere to the right hemisphere. One animal receives a single dose of
test
article in a volume of 40 [IL in the left and a single dose of 80 [IL in the
right. A
second animal receives two doses of 20 [IL in the left, and two doses of 40
[IL in the
right hemisphere. For both test articles, the dose concentration is 9.6e12
vg/mL.
After 14 days, the animals are sacrificed and the brains are hemisected and
divided
into approximately 17 slices (3mm each). Slices comprising the
hippocampal/entorhinal cortex regions are used to analyze the intrinsic GFP
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fluorescence and GFP immunohistochemistry. Other slices are used to collect
tissue
punches for mRNA analysis via qRT-PCR, where levels of tau and housekeeping
genes are analyzed. Additionally, some punches are collected and retained for
exploratory tau protein analysis. The results show that the hSYN1 promoter is
able to
drive GFP expression which is detectable in the hippocampal region following
delivery.
[0240] A second experiment is performed to evaluate the effect of the
route of
administration on GFP expression and location in the brain. In this study, the
GFP
transgene is carried by an SB3 AAV particle (see U.S. Provisional application
No.
62/503,121), and the dose concentration of the test article is 2.5e13 vg/mL.
The dose
is administered by intracerebroventricular, intrathecal or intravenous routes,
where the
dose volume (mL/animal) is approximately 1, 1.5 and 10-16, respectively. 14
days
following dosing, the animals are sacrificed and the brains are hemisected as
described above. In addition to analyzing the levels of tau and housekeeping
genes by
qRT-PCR, genes associated with inflammation (GFAP and Ibal) are also analyzed.
Exploratory studies are also performed to analyze the levels of tau protein in
the brain
tissue samples and in the cerebrospinal fluid. These studies show that the
dosing
routes are all well tolerated and are able to deliver the GFP transgene to the
hippocampal region.
[0241] Subsequently, a study is performed to analyze the repression of tau
expression by artificial transcription factors as described herein in the
cynomolgus
brain. Three or more ZFP-TFs as described herein (Tables 1 through 3) are
tested,
where the three ZFPs have been characterized as proteins that have high
specificity (0
off targets) and high (> 90% repression) in vitro efficacy (ZFP-TF 1);
moderate
.. specificity (5-10 off targets) and high in vitro efficacy (ZFP-TF2); and
high
specificity and moderate (50-60%) in vitro efficacy (ZFP-TF3). The animals are
sacrificed at 1, 3 and 6-month time points and analyzed as described above.
Significant repression of tau throughout the brain and CSF is observed with no
significant neuronal loss.
[0242] The studies demonstrate that the tau ZFP-TF reagents repress tau
expression (including at therapeutic levels) in a primate brain.
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[0243] All patents, patent applications and publications mentioned
herein are
hereby incorporated by reference for all purposes in their entirety.
[0244] Although disclosure has been provided in some detail by way of
illustration and example for the purposes of clarity of understanding, it will
be
apparent to those skilled in the art that various changes and modifications
can be
practiced without departing from the spirit or scope of the disclosure.
Accordingly,
the foregoing descriptions and examples should not be construed as limiting.
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