U.S. patent application number 15/470405 was filed with the patent office on 2017-09-28 for alteration of neuronal gene expression by synthetic pirnas and by alteration of pirna function.
The applicant listed for this patent is IBIS BIOSCIENCES, INC.. Invention is credited to Danny M. Chou, Lendell L. Cummins, David J. Ecker, Mark W. Eshoo, Todd P. Michael, Stanley T. Motley.
Application Number | 20170275622 15/470405 |
Document ID | / |
Family ID | 51530001 |
Filed Date | 2017-09-28 |
United States Patent
Application |
20170275622 |
Kind Code |
A1 |
Ecker; David J. ; et
al. |
September 28, 2017 |
ALTERATION OF NEURONAL GENE EXPRESSION BY SYNTHETIC piRNAs AND BY
ALTERATION OF piRNA FUNCTION
Abstract
Provided herein are compositions and methods for the alteration
of neuronal methylation by synthetic piRNAs or by alteration of
piRNA function. Such alterations find use in the regulation and
control of neural gene expression and concomitant neural functions.
Further provided herein are systems and methods for the
identification of target sites for regulation by piRNAs.
Inventors: |
Ecker; David J.; (Encinitas,
CA) ; Michael; Todd P.; (Carlsbad, CA) ;
Cummins; Lendell L.; (San Diego, CA) ; Eshoo; Mark
W.; (San Diego, CA) ; Motley; Stanley T.;
(Carlsbad, CA) ; Chou; Danny M.; (Carlsbad,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
IBIS BIOSCIENCES, INC. |
Carlsbad |
CA |
US |
|
|
Family ID: |
51530001 |
Appl. No.: |
15/470405 |
Filed: |
March 27, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14212412 |
Mar 14, 2014 |
9605260 |
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15470405 |
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61784353 |
Mar 14, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 1/6883 20130101;
C12N 2320/11 20130101; C12N 2310/10 20130101; C12Q 2600/154
20130101; C12N 15/113 20130101 |
International
Class: |
C12N 15/113 20060101
C12N015/113; C12Q 1/68 20060101 C12Q001/68 |
Goverment Interests
[0002] This invention was made with government support under
Contract No. FA8650-13-C-7340 awarded by the Department of the Air
Force. The government has certain rights in the invention.
Claims
1. A method for altering neural gene expression, comprising:
administering a synthetic piRNA to a subject under conditions such
that methylation of a regulatory sequence regulating said gene is
altered thereby altering said expression of said neural gene.
2. The method of claim 1, wherein said altered methylation of said
regulatory sequence of said gene is detected by methylome
sequencing.
3. The method of claim 1, wherein said methylome sequencing
comprises sodium bisulfate conversion.
4. The method of claim 1, wherein said methylome sequencing
comprises methylome sequencing of promoter and gene body
regions.
5. The method of claim 1, further comprising strand-specific mRNA
and/or smRNA sequencing.
6. The method of claim 1, wherein said gene expression regulates a
neurological function.
7. The method of claim 6, wherein said neurological function is
memory.
8. The method of claim 6, wherein said neurological function is
learning.
9. The method of claim 6, wherein said neurological function is a
pathology.
10. The method of claim 9, wherein said pathology is a traumatic
brain injury, a psychiatric disease, a cognitive disease, a
neurodegenerative disease, or a post-traumatic stress disorder
(PTSD).
11. The method of claim 1, wherein said synthetic piRNA silences
said neural target gene expression.
12. The method of claim 1, wherein said synthetic piRNA molecule
comprises a chemical modification that improves nuclease stability,
decreases likelihood of triggering an innate immune response,
lowers the incidence of off-target effects, or improves
pharmacodynamics relative to a non-modified piRNA.
13. The method of claim 1, wherein said synthetic piRNA comprises a
nucleotide with at least one chemical modification selected from
the group consisting of a phosphorothioate, a boranophosphate, a
4'-thio-ribose, a locked nucleic acid, a 2'-O-(2'-methoxyethyl), a
2'-O-methyl, a 2'-fluoro, a 2'-deoxy-2'-fluoro-b-D-arabinonucleic
acid, a Morpholino nucleic acid analog, and a Peptide nucleic acid
analog.
14. The method of claim 1, wherein said synthetic piRNA is attached
to a nanoparticle configured to cross the blood-brain barrier.
Description
[0001] The present application is a divisional of U.S. application
Ser. No. 14/212,412, filed Mar. 14, 2014, which claims priority to
U.S. Provisional Application Ser. No. 61/784,353, filed Mar. 14,
2013, which are herein incorporated by reference in their
entirety.
FIELD
[0003] Provided herein are compositions and methods for the
alteration of neuronal methylation by synthetic piRNAs or by
alteration of piRNA function. Such alterations find use in the
regulation and control of neural gene expression and concomitant
neural functions. Further provided herein are systems and methods
for the identification of target sites for regulation by
piRNAs.
BACKGROUND
[0004] Neurological functions and pathologies and resulting
properties and phenotypes (e.g., behavior, memory, disease, etc.)
are fundamentally important aspects of animal (e.g., human)
biology, health, and well-being. Yet the underlying molecular and
cellular biology is poorly understood. In view of this, there is a
dearth of pharmaceutical or research tools for altering these
properties and phenotypes at the molecular level and in a specific
manner.
SUMMARY
[0005] Provided herein are compositions and methods for the
alteration of neuronal methylation by synthetic piRNAs or by
alteration of piRNA function. Such alterations find use in the
regulation and control of neural gene expression and concomitant
neural functions. Further provided herein are systems and methods
for the identification of target sites for regulation by
piRNAs.
[0006] For example, in some embodiments, provided herein are
methods for identifying neural piRNA targets (sequences that are
regulated, e.g., have altered methylation, by endogenous or
synthetic piRNAs), comprising identifying differentially methylated
gene-expression regulatory sequences in neural tissue between a
control subject and a test subject. Any difference can be
identified, including, but not limited to differences associated
with a memory task (where the test subject and control subject
differ in the performance of a memory task), behavior task,
neurological disease or condition, drug administration, therapy
administration (e.g., meditation, etc.).
[0007] Further provided are synthetic piRNA molecules comprising a
chemical modification that improves one or more or all of nuclease
stability, decreased likelihood of triggering an innate immune
response, lowering incidence of off-target effects, and improved
pharmacodynamics relative to a non-modified piRNA. In some
embodiments, the piRNA has a nucleotide with at least one chemical
modification selected from: phosphorothioate, boranophosphate,
4'-thio-ribose, locked nucleic acid, 2'-O-(2'-methoxyethyl),
2'-O-methyl, 2'-fluoro, 2'-deoxy-2'-fluoro-b-D-arabinonucleic acid,
Morpholino nucleic acid analog, and Peptide nucleic acid analog. In
some embodiments, the piRNA is attached to a nanoparticle
configured to cross the blood-brain barrier.
[0008] Also provided herein are molecules useful in the regulation
of endogenous or non-endogenous piRNA regulated neural pathways.
For example, in some embodiments, provided herein are antisense
oligonucleotides having a sequence complementary to an endogenous
piRNA found in neural tissue. In some embodiments, synthetic piRNAs
are provided that alter methylation of an endogenous neural nucleic
acid not known to be regulated by an endogenous piRNA. Thus, in
some embodiments, provided herein are methods for altering neural
gene expression comprising: administering a synthetic piRNA to a
subject under conditions such that methylation of a regulatory
sequence regulating the gene is altered.
[0009] Further provided herein are neural cells or tissue
comprising a synthetic piRNA, a synthetic inhibitor of an
endogenous piRNA (e.g., an antisense oligonucleotide specific for
the piRNA).
DESCRIPTION OF DRAWINGS
[0010] FIG. 1 shows a graphical representation of control of the
CREB gene by a piRNA. The piRNA binds to the PIWI proteins and a
DNA methyl transferase and mediates methylation of a CpG motif that
silences further expression of the CREB gene (15).
[0011] FIG. 2 shows exemplary mass spectral analysis of CREB
nucleic acid molecules having different methylation patterns.
[0012] FIG. 3 shows exemplary mass spectral analysis of CREB
nucleic acid molecules having different methylation patterns.
DEFINITIONS
[0013] The terms "sample" and "specimen" are used in their broadest
sense and encompass samples or specimens obtained from any source.
As used herein, the term "sample" is used to refer to biological
samples obtained from animals (including humans), and encompasses
fluids, solids, tissues, and gases. In some embodiments of this
invention, biological samples include neural tissue or cells,
cerebrospinal fluid (CSF), serous fluid, urine, saliva, blood, and
blood products such as plasma, serum and the like. However, these
examples are not to be construed as limiting the types of samples
that find use with the present invention.
[0014] As used herein, the terms "host," "subject" and "patient"
refer to any animal, including but not limited to, human and
non-human animals (e.g., dogs, cats, cows, horses, sheep, poultry,
fish, crustaceans, etc.) that is studied, analyzed, tested,
diagnosed or treated. As used herein, the terms "host," "subject"
and "patient" are used interchangeably, unless indicated
otherwise.
[0015] As used herein, the term "effective amount" refers to the
amount of a composition (e.g., a synthetic piRNA) sufficient to
effect beneficial or desired results. An effective amount can be
administered in one or more administrations, applications or
dosages and is not intended to be limited to a particular
formulation or administration route.
[0016] As used herein, the terms "administration" and
"administering" refer to the act of giving a drug, prodrug, or
other agent, or therapeutic treatment (e.g., compositions of the
present invention) to a subject (e.g., a subject or in vivo, in
vitro, or ex vivo cells, tissues, and organs). Exemplary routes of
administration to the human body can be through space under the
arachnoid membrane of the brain or spinal cord (intrathecal), the
eyes (ophthalmic), mouth (oral), skin (topical or transdermal),
nose (nasal), lungs (inhalant), oral mucosa (buccal), ear, rectal,
vaginal, by injection (e.g., intravenously, subcutaneously,
intratumorally, intraperitoneally, etc.) and the like.
[0017] As used herein, the terms "co-administration" and
"co-administering" refer to the administration of at least two
agent(s) (e.g., multiple synthetic piRNAs or a piRNA or anti-piRNA
molecule and another therapeutic) or therapies to a subject. In
some embodiments, the co-administration of two or more agents or
therapies is concurrent. In other embodiments, a first
agent/therapy is administered prior to a second agent/therapy.
Those of skill in the art understand that the formulations and/or
routes of administration of the various agents or therapies used
may vary. The appropriate dosage for co-administration can be
readily determined by one skilled in the art. In some embodiments,
when agents or therapies are co-administered, the respective agents
or therapies are administered at lower dosages than appropriate for
their administration alone. Thus, co-administration is especially
desirable in embodiments where the co-administration of the agents
or therapies lowers the requisite dosage of a potentially harmful
(e.g., toxic) agent(s), and/or when co-administration of two or
more agents results in sensitization of a subject to beneficial
effects of one of the agents via co-administration of the other
agent.
[0018] As used herein, the term "treatment" or grammatical
equivalents encompasses the improvement and/or reversal of the
symptoms of disease (e.g., neurodegenerative disease) or condition.
A compound which causes an improvement in any parameter associated
with disease when used in the screening methods of the instant
invention may thereby be identified as a therapeutic compound. The
term "treatment" refers to both therapeutic treatment and
prophylactic or preventative measures. For example, those who may
benefit from treatment with compositions and methods of the present
invention include those already with a disease and/or disorder
(e.g., neurodegenerative disease) as well as those in which a
disease and/or disorder is to be prevented (e.g., using a
prophylactic treatment).
[0019] As used herein, the term "at risk for disease" refers to a
subject (e.g., a human) that is predisposed to experiencing a
particular disease. This predisposition may be genetic (e.g., a
particular genetic tendency to experience the disease, such as
heritable disorders), or due to other factors (e.g., age, weight,
environmental conditions, exposures to detrimental compounds
present in the environment, etc.). Thus, it is not intended that
the present invention be limited to any particular risk, nor is it
intended that the present invention be limited to any particular
disease.
[0020] As used herein, the term "suffering from disease" refers to
a subject (e.g., a human) that is experiencing a particular
disease. It is not intended that the present invention be limited
to any particular signs or symptoms, nor disease. Thus, it is
intended that the present invention encompass subjects that are
experiencing any range of disease (e.g., from sub-clinical
manifestation to full-blown disease) wherein the subject exhibits
at least some of the indicia (e.g., signs and symptoms) associated
with the particular disease.
[0021] As used herein, the terms "disease" and "pathological
condition" are used interchangeably to describe a state, signs,
and/or symptoms that are associated with any impairment of the
normal state of a living animal or of any of its organs or tissues
that interrupts or modifies the performance of normal functions,
and may be a response to environmental factors (such as
malnutrition, industrial hazards, or climate), to specific
infective agents (such as worms, bacteria, or viruses), to inherent
defect of the organism (such as various genetic anomalies, or to
combinations of these and other factors).
[0022] The term "compound" refers to any chemical entity,
pharmaceutical, drug, and the like that can be used to treat or
prevent a disease, illness, sickness, or disorder of bodily
function. Compounds comprise both known and potential therapeutic
compounds. A compound can be determined to be therapeutic by
screening using screening methods. A "known therapeutic compound"
refers to a therapeutic compound that has been shown (e.g., through
animal trials or prior experience with administration to humans) to
be effective in such treatment. In other words, a known therapeutic
compound is not limited to a compound efficacious in the treatment
of disease (e.g., neurodegenerative disease).
[0023] As used herein, the term "pharmaceutical composition" refers
to the combination of an active agent (e.g., a piRNA) with a
carrier, inert or active, making the composition especially
suitable for diagnostic or therapeutic use in vitro, in vivo or ex
vivo.
[0024] As used herein, the term "nucleic acid molecule" refers to
any nucleic acid containing molecule, including but not limited to,
DNA or RNA (e.g., piRNA). The term encompasses sequences that
include any of the known base analogs of DNA and RNA including, but
not limited to, 4-acetylcytosine, 8-hydroxy-N6-methyladenosine,
aziridinylcytosine, pseudoisocytosine,
5-(carboxyhydroxylmethyl)uracil, 5-fluorouracil, 5-bromouracil,
5-carboxymethylaminomethyl-2-thiouracil,
5-carboxymethylaminomethyluracil, dihydrouracil, inosine,
N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil,
1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyl
adenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine,
N6-methyladenine, 7-methylguanine, 5-methylaminomethyluracil,
5-methoxy-aminomethyl-2-thiouracil, beta-D-mannosylqueosine,
5'-methoxycarbonylmethyluracil, 5-methoxyuracil,
2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid
methylester, uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil,
queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil,
4-thiouracil, 5-methyluracil, N-uracil-5-oxyacetic acid
methylester, uracil-5-oxyacetic acid, pseudouracil, queosine,
2-thiocytosine, and 2,6-diaminopurine.
[0025] The term "gene" refers to a nucleic acid (e.g., DNA)
sequence that comprises coding sequences necessary for the
production of a polypeptide, precursor, or RNA (e.g., rRNA, tRNA).
The polypeptide can be encoded by a full length coding sequence or
by any portion of the coding sequence so long as the desired
activity or functional properties (e.g., enzymatic activity, ligand
binding, signal transduction, immunogenicity, etc.) of the
full-length or fragment are retained. The term also encompasses the
coding region of a structural gene and the sequences located
adjacent to the coding region on both the 5' and 3' ends for a
distance of about 1 kb or more on either end such that the gene
corresponds to the length of the full-length mRNA. Sequences
located 5' of the coding region and present on the mRNA are
referred to as 5' non-translated sequences. Sequences located 3' or
downstream of the coding region and present on the mRNA are
referred to as 3' non-translated sequences. The term "gene"
encompasses both cDNA and genomic forms of a gene. A genomic form
or clone of a gene contains the coding region interrupted with
non-coding sequences termed "introns" or "intervening regions" or
"intervening sequences." Introns are segments of a gene that are
transcribed into nuclear RNA (hnRNA); introns may contain
regulatory elements such as enhancers. Introns are removed or
"spliced out" from the nuclear or primary transcript; introns
therefore are absent in the messenger RNA (mRNA) transcript. The
mRNA functions during translation to specify the sequence or order
of amino acids in a nascent polypeptide.
[0026] As used herein, the terms "gene expression" and "expression"
refer to the process of converting genetic information encoded in a
gene into RNA (e.g., mRNA, rRNA, tRNA, or snRNA) through
"transcription" of the gene (i.e., via the enzymatic action of an
RNA polymerase), and for protein encoding genes, into protein
through "translation" of mRNA. Gene expression can be regulated at
many stages in the process. "Up-regulation" or "activation" refer
to regulation that increases and/or enhances the production of gene
expression products (e.g., RNA or protein), while "down-regulation"
or "repression" refer to regulation that decrease production.
Molecules (e.g., transcription factors) that are involved in
up-regulation or down-regulation are often called "activators" and
"repressors," respectively.
[0027] The term "isolated" when used in relation to a nucleic acid,
as in "an isolated oligonucleotide" or "isolated polynucleotide"
refers to a nucleic acid sequence that is identified and separated
from at least one component or contaminant with which it is
ordinarily associated in its natural source. Isolated nucleic acid
is present in a form or setting that is different from that in
which it is found in nature. In contrast, non-isolated nucleic
acids are nucleic acids such as DNA and RNA found in the state they
exist in nature. For example, a given DNA sequence (e.g., a gene)
is found on the host cell chromosome in proximity to neighboring
genes; RNA sequences, such as a specific mRNA sequence encoding a
specific protein, are found in the cell as a mixture with numerous
other mRNAs that encode a multitude of proteins. However, isolated
nucleic acid encoding a given protein includes, by way of example,
such nucleic acid in cells ordinarily expressing the given protein
where the nucleic acid is in a chromosomal location different from
that of natural cells, or is otherwise flanked by a different
nucleic acid sequence than that found in nature. The isolated
nucleic acid, oligonucleotide, or polynucleotide may be present in
single-stranded or double-stranded form.
[0028] The term "synthetic" when used in reference to nucleic acid
molecules (e.g., piRNA) refers to non-natural molecules made
directly (e.g., in a laboratory) or indirectly (e.g., from
expression in a cell of a construct made in a laboratory) by
mankind.
DETAILED DESCRIPTION
[0029] Provided herein are compositions and methods for the
alteration of neuronal methylation by synthetic piRNAs or by
alteration of piRNA function. Such alterations find use in the
regulation and control of neural gene expression and concomitant
neural functions. Further provided herein are systems and methods
for the identification of target sites for regulation by
piRNAs.
[0030] Epigenetic molecular mechanisms, specifically hi stone
post-translational modifications and cytosine methylation of DNA,
have recently been discovered to be critically important regulators
of learning and memory (1). Provided herein are compositions and
methods related to identifying and understanding that association
of methylation with learning and memory, as well as other
neurological functions, properties, and health status. Further
provided herein are drug treatments to augment learning and enhance
memory formation and stabilization. Such enhancement and
stabilization has many uses, including, but not limited to
accelerated training of individuals (e.g., athletes, professionals,
and military personnel) and treatment of diseases and disorders of
the brain and central nervous system (e.g., traumatic brain injury,
psychiatric, cognitive diseases, neurodegenerative disease,
post-traumatic stress disorder (PTSD), etc.).
[0031] Conventional approaches to target identification and
validation have evolved steadily over decades and have benefited by
the availability of newer observational tools such as microarrays
and interventional tools such as antisense and RNA interference.
However, recent breakthroughs is epigenomic science have provided a
far superior approach based on whole methylome sequencing (2). It
is now possible to construct a relevant animal model (e.g., mouse)
for a neural process, such as consolidation of memory, identify and
separate the important cell types (e.g. pyramidal neurons of the
hippocampus), and sequence the complete methylome of these cells
from untrained and memory trained subjects and identify methylation
changes associated with every gene in the target cell type. This
powerful approach avoids the "looking under the lamppost" bias of
previous target identification strategies. In addition to
sequencing the methylome, the expressed RNA from these cells can
also be sequenced to provide a broad picture of the impact of
methylation changes on gene expression. Identification of the
methylation changes in the DNA and expressed RNA provide direct
evidence of what genes are needed for the process being studied and
therefore what genes to turn off to block the process by a
pharmacological intervention. Provided herein are synthetic piRNA
molecules that alter methylation in a specific and directed manner,
providing systematic control over gene expression. Also provided
herein are molecules that regulate endogenous piRNA function.
[0032] Oligonucleotide drugs work by a variety of mechanisms, the
simplest of which is simply hybridizing to the target mRNA and
blocking its natural ability to direct protein synthesis by steric
hindrance (3, 4). However, it was realized early on that the
potency of oligonucleotide drugs can be substantially enhanced by
co-opting a natural pathway such as activating endogenous enzymes
such as RNAse H, resulting destruction of the target RNA through
enzymatic cleavage (5, 6). As the understanding of the various
roles of RNA in the cell grew, opportunities became available to
exploit these newly discovered pathways. The discovery of the siRNA
pathway in the 1990's led to opportunity to create double stranded
RNA mimetics of natural siRNA's to silence genes (7-9). However, a
common feature of all therapeutic uses of oligonucleotides is that
the biological effects brought about by the drug are transient and
require a sustained presence of the drug to maintain the desired
pharmacological effect. This is a problem for treating the brain,
where delivery of the drug to the target cells is much more
challenging than other target tissues. Successful treatment of the
brain requires stable and potent oligonucleotides capable of
surviving the journey to the brain but more importantly, the
oligonucleotides should produce a sustained biological effect in
the brain that persists after the oligonucleotide drug is gone.
[0033] Technology provided herein achieves this result by co-opting
the piRNA pathway to directly impact the silencing or activating
the target genes in the brain through directed changes in DNA
methylation. These changes in DNA methylation can be long acting
and even permanent for the life of the cell if not actively
reversed.
[0034] piRNAs are a distinct set of small non-coding RNA in the
typical size range of 26-32 or 33 nucleotides (ranges from 19-33
have been reported), typically with a U on the 5'-end and a
2'-OMethyl modified 3'-end. The first role discovered for the piRNA
pathway is the protection of the integrity of germ line cells from
parasitic invasion of transposable element DNA (10). Transposable
elements are endogenous genomic parasites that threaten the
integrity of the host genome by jumping to new locations in the
host DNA and possibly landing in regions that might disrupt the
function of normal genes. The cellular countermeasure to protect
the genome from disruption is, by necessity, powerful and
long-lasting, and is accomplished through silencing of the
transposable elements by both directing cleavage of the
transposable element RNA and by directing DNA methylation to
permanently silence the transposable element. piRNA accomplishes
this binding in an antisense fashion to the target gene mRNA and,
through interactions with a specific set of proteins (the Piwi
protein complex), trigger both the destruction of the mRNA and
methylation of the DNA that controls silencing of the target gene
(11, 12). This latter effect is the key to producing a sustained
biological response.
[0035] The piRNA pathway has been recently been studied most for
its role in protection of the germ-line DNA (10, 13). It is now
known that the piRNA pathway is broadly conserved in evolution and
has a broad role in various tissues including the mammalian central
nervous system in the hippocampal neurons, extending into the
dendritic compartment of the cell (14). Most intriguing is a very
recent report from the Kandel laboratory that neuronal piRNA's play
a role in epigenetic control of memory-related synaptic plasticity
in Aplysia. They discovered the presence of abundantly expressed
piRNA in the brain and that piRNA directs serotonin-dependent
methylation and silencing of the CREB2 gene, a major inhibitory
constraint of memory, leading to long term synaptic facilitation
(15).
[0036] This observation is consistent with a growing body of recent
evidence from the laboratory of Sweatt and colleagues who have
shown that DNA methylation is involved in multiple aspects of
memory formation and maintenance (1, 16-21). Epigenetic changes in
cellular differentiation are generally permanent, but not in adult
neurons, where the plastic nature of synaptic neurons requires
long-term but reversible changes in gene expression.
[0037] In some embodiments, provided herein are target
identification systems and methods for identifying fundamental
mechanisms underlying memory formation at the whole genome level
and for identifying targets for therapeutic intervention. Further
provided are therapeutic and research agents for altering
methylation, and thus gene expression, at these target sequences so
as to regulate neurological functions (e.g., memory, learning,
etc.) and pathologies.
[0038] The piRNA pathway provides a molecular targeting mechanism
to link to the epigenetic changes associated with memory. Multiple
mechanisms of intervention are provided including: 1) antisense
oligonucleotide targeting of specific endogenous piRNA's and
interfering with their function to mediate gene silencing,
producing long term gene expression that would otherwise be
silenced, and 2) piRNA mimetic drugs (e.g., synthetic piRNAs) that
specifically silence target genes. It is important to note that the
latter mechanism does not specifically require that the gene (to be
silenced) be naturally regulated by a piRNA, but can be any gene.
All that is required is that the target cell contains an active
piRNA pathway for any purpose that is co-opted for therapeutic
intervention. The existence of this pathway in neurons (14, 15)
allows this strategy.
[0039] In some embodiments, provided herein are synthetic piRNA
molecules. In some embodiments, the piRNA molecules comprise
chemical modification to improve nuclease stability, decrease the
likelihood of triggering an innate immune response, lower the
incidence of off-target effects, and/or improve pharmacodynamics
relative to non-modified molecules so as to increase potency and
specificity. In some embodiments, the molecules are loaded onto
nanoparticles, providing a stabilizing effect (e.g., protecting
against nuclease degradation). These effects are particularly
important for nucleic acids intended to treat the brain, where the
delivery challenges limit the amount of active nucleic acid drug
that will reach the target cells. Therefore, nucleic acid drugs
used to treat the brain should be based on chemistry with high
potency and a long duration of action. Similarly, the
pharmacological targets of the molecules should have high leverage
to create a sustained biological response. Exemplary chemical
modifications of nucleotides (e.g., modifications of the sugars) in
the synthetic piRNA molecules that find use in some embodiments of
the technology include the following: phosphorothioate,
boranophosphate, 4'-thio-ribose, locked nucleic acid,
2'-O-(2'-methoxyethyl), 2'-O-methyl, 2'-fluoro,
2'-deoxy-2'-fluoro-b-D-arabinonucleic acid, Morpholino nucleic acid
analog, and Peptide nucleic acid analog. Additional modification
used with antisense oligonucleotides may be employed (see e.g., US
Pat. Publ. Nos. 2012/0202874 and 2012/0149755, herein incorporated
by reference in their entireties).
[0040] In some embodiments, antisense oligonulceotides that
interact with and interfere with endogenous piRNA sequences are
used.
[0041] Delivery of synthetic piRNA molecules or antisense
oligonucleotides may be accomplished by any desired method. In some
embodiments, molecules are delivered intrathecally. In some
embodiments, a Medtronic infusion system employing an implantable,
battery-powered drug-infusion pump is used to deliver molecules to
the striatum (Dickinson et al., Neuro. Oncol. 12:928-940 (2010);
Sah and Aronin, J. Clin. Invest. 121: 500-507 (2011)). In some
embodiments, intranasal delivery is used. In some embodiments,
nucleic acids are delivered by nanoparticles. For example,
particles comprising an iron-oxide core coated with chitsan may be
used (see e.g., Veiseh et al., Adv. Drug Deliv. Rev., 8:582
(2011)). Chitosan is a transcytosing molecule that is able to cross
the blood brain barrier. In some embodiments, the particles are
associated with a call-penetrating peptide to facilitate delivery
of the nucleic into cells. In some embodiments, endogenous
nanoparticles (e.g., high-density lipoproteins) are used to deliver
molecules across the blood brain barrier.
Example 1
Target Identification:
[0042] This example describes methods for identifying targets for
piRNA regulation, for example, associated with loci where target
sequences are not currently known.
Epigenetic Mouse Model Experiments.
[0043] The approach encompasses two key experiments, using
laboratory animals in molecular, genetic, epigenetic, and
behavioral studies. For both assays, one uses genetically
engineered mice that have a specific neuronal subpopulation
(pyramidal neurons) labeled in the hippocampus, and couple the use
of these mice with FACS in order to isolate a uniquely defined
sample of cells for epigenetic characterization.
[0044] In some embodiments, one can selectively manipulate DNA
methylation in the mouse CNS in vivo, using IntraCerebroVentricular
(ICV) infusion of a DNA MethylTransferase inhibitor (DNMTi).
High-throughput DNA sequencing and subsequent epigenomic
bio-informatic analysis identifies target sites associated with
particular phenotypic changes providing targets for manipulation by
piRNA regulation.
[0045] A second series of experiments utilizes spatial,
hippocampus-dependent learning and memory in the behaving animal as
a stimulus, in order to trigger learning-related epigenetic (DNA
methylation) changes in the hippocampus. Using hippocampal
pyramidal neurons isolated from control and trained animals,
subsequent use of methylomic analysis defines memory associated DNA
methylation changes for the entire cellular epigenenome with
specificity at the single-nucleotide level.
[0046] These assays identify target genes whose methylation is
changed and whose transcription is blocked or increased by DNMT
inhibition. These assays thus correlate a function memory change
(memory blockade) with a specific set of epigenetic alterations
(DNA methylation) and a specific set of transcriptional changes
(mRNA and small non-coding RNA readout).
[0047] One can further identify at single-nucleotide resolution the
complete set of genes whose epigenetic cytosine methylation changes
in response to hippocampus dependent spatial learning, and
correlate this to the list of genes whose transcription is
regulated with memory formation. These assays can define the
specific cellular locus for the changes in the methylome by
utilizing gene engineering-based fluorescent tagging to
specifically isolate hippocampal pyramidal neurons, a neuronal
sub-population known to be involved in spatial learning and
memory.
[0048] The Jackson Lab has commercially available a mouse line
(Stock Number 003782) that selectively labels CNS neurons using
Yellow Fluorescent Protein (YFP) (2). This model is well suited for
the above assays. These mice are beneficial because the genome
databases are well developed for this species, making the
methylomics efficient. Further, this engineered mouse line has
specifically labeled neurons that are advantageous, because one can
use FACS to isolate specific neurons (hippocampal pyramidal
neurons) that are known to be involved in and necessary for spatial
learning and memory.
[0049] This approach is illustrated below for the assessment of
fear response. The mouse model is used for behavioral modeling of
learning, short-term memory, and long-term memory--specifically
contextual fear conditioning. After training animals in this
learning and memory paradigm, bioinformatics and high-throughput
nucleotide sequencing approaches are used to comprehensively
identify memory-associated changes in the methylome.
[0050] Interventive experiment to manipulate the epigenome are also
conducted, using a class of agents that are known to affect memory
capacity: DNMT inhibitors. Animals (control cannulated versus
drug-infused) are treated with this agent and epigenomic and
transcriptomic changes are assessed using the methylomics
approaches described above.
[0051] Mice are trained and evaluated using a behavioral test and
associated control paradigms that assess baseline behaviors,
sensory responses, and hippocampus-dependent memory formation. The
Conditioned Fear Paradigm utilizes automated conditioned fear
chambers. The automated test chambers use a video detection system
and are placed into sound-attenuated chambers. The conditioned fear
test is routinely used to study fear/emotional-based learning and
memory in rodents, and has quickly become one of the most widely
used assays for learning and memory performance in mutant mice. For
these experiments, an aversive stimulus (in this case, a mild foot
shock) is paired either once or 3 times with an auditory
conditioned stimulus (CS, white noise) within a novel environment.
When tested 24 h after training, mice exhibit marked fear, measured
by freezing behavior, in response to re-presentation of either the
context (contextual fear conditioning) or the auditory CS delivered
in a different context (cued fear conditioning). One also evaluates
contextual fear conditioning by itself without presenting the
auditory cue during training in the novel context. Cued fear
conditioning is thought to be dependent upon the amygdala, whereas
contextual fear conditioning also likely involves the hippocampus.
To assess baseline behavior, one monitors animals during the
training phase. One assesses both baseline freezing behavior prior
to presentation of the foot shock on the training day (minutes
1-3), and the freezing of the animal in response to foot shock.
Freezing on the training day in response to application of the foot
shock is also used as a crude estimate of shock sensitivity.
[0052] For the fear conditioning experiments mice are transported
to the laboratory at least 30 min prior to fear conditioning. Fear
conditioned animals are allowed to explore the training chamber for
2 min, after which they receive a series of three electric shocks
(1 s, 0.5 mA) at 2 min intervals. As controls, context exposed
(context only) or latent inhibition plus fear conditioned (latent
inhibition) animals are also placed in the novel training chambers.
Context only animals are placed in the novel training chamber for 7
min without receiving the footshock.
[0053] Latent Inhibition animals are pre-exposed to the context for
2 h before the same 7 min training protocol is administered as
described for the fear conditioned animals. In all shocked groups
the animals are allowed to explore the novel context (training
chamber) for an additional 1 min after the receiving the final
footshock prior to being returned to their home cage. Footshock
alone control animals are taken to the training room, placed in the
training chamber and immediately shocked and removed from the
chamber. Freezing behavior is recorded using Video Freeze software
(Med Associates, St. Albans, Vt.). Another group of age-matched
animals that are handled by the experimenter but do not receive any
experimental manipulations are used as naive controls in these
experiments as well.
[0054] Cannula implantation and intra-CNS Infusion of DNMTi--In
vivo CNS-selective inhibition of DNMTs uses cannula-based direct
infusion of agents into the cerebral ventricles. For DNMT
inhibition, the cytosine analogs zebularine and
5-aza-2-deoxycitidine (in separate experiments) are used, which
selectively inhibit all known DNMTs (i.e., DNMT1, 3A, and 3B). For
stereotaxic surgery, mice are anesthetized with ketamine and
xylazine and secured in a Kopf stereotaxic apparatus. Bilateral
stainless steel guide cannulae (26G; Plastics One, Roankoke, Va.)
are aimed at the ventricles. Clearance through the guide cannulae
is maintained with 33G obdurators (Plastics One) cut to project 1
mm (Area CA1) or 0.2 mm (ACC) beyond the tip of the guide. Animals
are habituated to dummy cannula removal and given 5 days of
recovery and handling before the start of behavioral conditioning,
etc. To ensure accurate cannula placement, brains are collected
from those animals given both fear conditioning training and a
retention test. Sections are collected and stained with cresyl
violet to verify the location of the infusion needle tips.
[0055] Statistical analyses are conducted using the PC software
program Prism. Two-sample comparisons are made using the paired
Student's t test, and multiple comparisons are made using a one-way
analysis of variance (ANOVA), Tukey test, or the Fisher PLSD test.
All behavioral experiments are assessed using analysis of variance
(ANOVA) followed by post-hoc analysis with the Tukey-Kramer test
where appropriate. If data sets do not meet the criteria required
for parametric statistical analysis, then a Kruskal-Wallis ANOVA is
performed, followed by a post-hoc Dunn's multiple comparison
test.
Methylome and RNA Sequencing
[0056] MethylC-Seq library generation: 1-5 .mu.g of genomic DNA is
extracted from frozen tissue using the DNeasy Mini Kit (Qiagen) and
spiked with 25 ng unmethylated c1857 Sam7 Lambda DNA (Promega). The
DNA is fragmented with a Covaris S2 (Covaris) to 100-150 bp,
followed by end repair and addition of a 3' A base. Cytosine
methylated adapters provided by Illumina (Illumina) are ligated to
the sonicated DNA at 16.degree. C. for 16 hours with T4 DNA ligase
(New England Biolabs). Adapter-ligated DNA are isolated by two
rounds of purification with AMPure XP beads (Beckman Coulter
Genomics). Adapter-ligated DNA (S450 ng) are subjected to sodium
bisulfite conversion using the MethylCode kit (Life Technologies)
as per manufacturer's instructions. The bisulfite-converted,
adapter-ligated DNA molecules are enriched by 4 cycles of PCR. The
reaction products are purified using AMPure XP beads (two rounds).
Up to three separate PCR reactions are performed on subsets of the
adapter-ligated, bisulfite-converted DNA, yielding up to three
independent libraries from the same biological sample (Please see
(23) for detailed protocol). One obtains the final sequence
coverage by sequencing all libraries for a sample separately, thus
reducing the incidence of "clonal" reads which share the same
alignment position and likely originate from the same template
molecule in each PCR. The sodium bisulfrte non-conversion rate for
each sample is empirically determined by calculation of the
frequency of cytosines sequenced at cytosine reference positions in
the Lambda genome. Sequencing is performed using the Illumina
HiSeq2000 Sequencing System as per the manufacturer's
instructions.
[0057] Strand-specific mRNA-seq libraries: Samples are processed as
described (23). Briefly, total RNA is isolated from tissue or FACS
isolated cells by treatment with RNA later and using the mirVana
miRNA isolation kit and treated with DNasel (Qiagen) for 30 min at
room temperature. Following ethanol precipitation, biotinylated LNA
oligonucleotide rRNA probes complementary to the 5S, 5.8S, 12S, 18S
and 28S ribosomal RNAs are used to deplete rRNA from 20 .mu.g of
total RNA in two sequential RiboMinus reactions (Life Technologies)
as per manufacturer's instructions.
[0058] Unique 5' and 3' RNA oligonucleotides are then sequentially
ligated to the ends of fragments of RNA devoid of rRNA. Sequencing
is performed using the Illumina HiSeq2000 Sequencing System as per
the manufacturer's instructions.
[0059] smRNA-Seq library generation: RNA fractions enriched for
small RNAs are isolated from tissue or FACS isolated cells by
treatment with RNAlater (Life Technologies) and using the mirVana
miRNA isolation kit (Life Technologies) and treated with DNasel
(Qiagen) for 30 min at room temperature. Following ethanol
precipitation, small RNAs are separated by electrophoresis on a 15%
TBE-urea gel and RNA molecules between approximately 10 and 50 nt
are then excised and eluted from the gel fragments. Following
ethanol precipitation, smRNA-Seq libraries are produced using the
Small RNA Sample Prep v1.5 kit (Iliumina) as per manufacturer's
instructions. Sequencing is performed using the Iliumina HiSeq2000
Sequencing System as per the manufacturer's instructions.
Data Analysis
[0060] MethylC-seq data is mapped and processed as described in
Lister et al (23). Briefly, reads are first be trimmed of any
adapter sequences at the 3' end, and subsequently mapped to the
NCBI m37 reference genome with Bowtie (24), using the following
parameters: -e 90-I 20-n 0-k 10-nomaground-solexa1.3-quals. Mapped
reads are filtered as follows: any read with more than 3 mismatches
is trimmed from the 3' end to contain 3 mismatches, and any read
pair which contained a cytosine mapped to a reference sequence
thymine is removed. Reads are then collapsed to remove clonal reads
potentially produced in the PCR amplification from the same
template molecule, based on common start position of read 1.
Methylcytosines are identified from the mapped and processed read
data as described in (23), including correction of any DNA
methylation incorrectly categorized as non-CG due to SNPs in the
sample versus reference genomes.
[0061] Profiling DNA methylation in promoters and gene bodies.
Promoters are defined as within 2 kb upstream regions starting from
the transcription start site (TSS) of the Ensembl transcript IDs
(NCBI BUILD 37.1). Gene bodies are determined for each Ensembl
transcript 10 as the region spanning from the TSS to the end of
transcription site. Each promoter and gene body is divided in
twenty equally sized bins and the density of CG, and absolute (mCG)
and relative (mCG/CG) methylation is determined for each bin.
Absolute methylation is computed as the average methylation level
(methylated I (methylated+unmethylated) read counts) divided by the
bin size in bp. Relative methylation is determined as the ratio
between the absolute methylation and the CG density (CG/bp) in the
same bin. Similar analyses are performed for any methylation
identified in the non-CG context.
[0062] Mapping of mRNA-Seq data: RNA-Seq read sequences produced by
the Illumina analysis pipeline is aligned with the CLCbio to the
NCBI Build 37.1 C57BU6 mouse reference sequence. Reads that align
to multiple positions are discarded. Reads per kilobase of
transcript per million reads (RPKM) values are calculated with the
Cufflinks software (25) using mouse RefSeq gene models.
[0063] Mapping of smRNA.seq reads. smRNA sequence reads in FastQ
format are produced by the Illumina analysis pipeline. smRNA-Seq
reads that contain at least 5 bases of the 3' adapter sequence are
selected and this adapter sequence removed, retaining the trimmed
reads that are from 16 to 37 nt in length. These processed reads in
FastQ format are then aligned to the mouse reference genome (NCBI
BUILD 37.1) with the Bowtie alignment algorithm using the following
parameters: -solexa-quals-e 1-120-n 0-a -m 1000-best--nomaground.
Consequently, any read that aligns with no mismatches and to no
more than 1000 locations in the NCBI BUILD 37.1 reference genome
sequence is retained for downstream analysis.
[0064] Post-processing data analysis: One performs dimensionality
reduction of the MethylC-Seq and RNA-Seq datasets by keeping only
those genes showing at least 2-fold methylation or expression
changes across the different conditions. The remaining expression
values are then log-transformed for Independent Component Analysis
(26). ICA is an unsupervised machine-learning algorithm that is
used to identify the contribution on methylation states and gene
expression of genes that were most strongly affected. The
expression value of any gene or methylation state is viewed as a
result of several independent sources (components) that contribute
to its expression, treatment and disease state being examples. ICA
allows separation of these components without prior knowledge. The
genes that rank highly on this list are subjected to manual
inspection and interpretation. The ICA sources is mapped into
functional categories using DAVID (27) to identify significantly
enriched gene ontology categories. The categories with most
significant P-values provide insights into the biological processes
involved in discriminating between different treatments and
conditions. Parallel ICA is used to link the ICA analyses of the
MethylC-Seq and the RNA-Seq data sets.
[0065] In some embodiments, mass spectrometry is used to identify
and analyze target methylation sites. FIG. 2 shows data with mass
spectra for nucleic acid sequences bracketing a CpG island in a
promoter region of the CREB2 gene within the Aplysia genome. The
detected nucleic acids were generated synthetically as a means to
illustrate the capability to detect levels of methylation
associated with neuron plasticity. Panel (A) shows both strands of
the genomic sequence with no methylation, Panel (B) show the same
region with approximately equal amount of unmethylated and
methylated cytosines in the CpG island, and Panel (C) illustrates
100% methylation of the CpG island within the promotor sequence.
The relative percentage of methylation and nonmethylation for the
CpG island can be determined by comparison of relative spectral
abundances.
TABLE-US-00001 Avg. mass Label Name Sequence (Da) .alpha. top
GCCAAAAAATTGACTAGCG 18810.18 strand TCTGATTCCACCGCGTTTT
GACACTAATTATTGAGTGA AGAG .beta. bottom CTCTTCACTCAATAATTAG 18752.13
strand TGTCAAAACGCGGTGGAAT CAGACGCTAGTCAATTTTT TGGC .chi.
methylated GCCAAAAAATTGACTAGCG 18838.23 top TCTGATTCCACC5mGC5mG
strand TTTTGACACTAATTATTGA GTGAAGAG .delta. methylated
CTCTTCACTCAATAATTAG 18780.18 bottom TGTCAAAAC5mGC5mGGAA strand
TCAGACGCTAGTCAATTTT TTGGC
[0066] FIG. 3 shows mass spectra for nucleic acid sequences
bracketing a CpG island in a promoter region of the CREB2 gene
within the Aplysia genome. The detected nucleic acids were
generated synthetically as a means to illustrate the capability to
detect levels of methylation associated with neuron plasticity.
Panel (A) shows both strands of the genomic sequence with no
methylation, Panel (B) show the same region with approximately
equal amount of unmethylated and methylated cytosines in the CpG
island--the methylated species are represent by nucleic acids
identical to amplicons resulting from a bisulfite treatment and PCR
amplification process (note that the amplicon products for the
bottom strand (g, h) are near mass degenerate, yielding overlapping
peaks with essentially linear addition of abundance), and Panel (C)
illustrates 100% methylation of the CpG island within the promotor
sequence. The relative percentage of methylation and nonmethylation
for the CpG island can be determined by comparison of relative
spectral abundances.
TABLE-US-00002 Avg. mass Label Name Sequence (Da) .alpha. top
strand GCCAAAAAATTGACTAGC 18810.18 GTCTGATTCCACCGCGTT
TTGACACTAATTATTGAG TGAAGAG .beta. bottom strand CTCTTCACTCAATAATTA
18752.13 GTGTCAAAACGCGGTGGA ATCAGACGCTAGTCAATT TTTTGGC .epsilon.
top bisulfite GCCAAAAAATTGACTAGC 18840.20 amplicon 1
GTCTGATTCCACTGTGTT TTGACACTAATTATTGAG TGAAGAG .phi. top bisulfite
CTCTTCACTCAATAATTA 18720.13 amplicon 2 GTGTCAAAACACAGTGGA
ATCAGACGCTAGTCAATT TTTTGGC .gamma. bottom CTCTTCACTCAATAATTA
18782.15 bisulfite GTGTCAAAATGTGGTGGA amplicon 1 ATCAGACGCTAGTCAATT
TTTTGGC .eta. bottom GCCAAAAAATTGACTAGC 18778.18 bisulfite
GTCTGATTCCACCACATT amplicon 2 TTGACACTAATTATTGAG TGAAGAG
[0067] Using the approaches described above, target methylation
sites for regulation by piRNA are identified for any desired neural
function.
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Sequence CWU 1
1
13161DNAAplysia 1gccaaaaaat tgactagcgt ctgattccac cgcgttttga
cactaattat tgagtgaaga 60g 61261DNAAplysia 2ctcttcactc aataattagt
gtcaaaacgc ggtggaatca gacgctagtc aattttttgg 60c 61361DNAAplysia
3gccaaaaaat tgactagcgt ctgattccac tgtgttttga cactaattat tgagtgaaga
60g 61461DNAAplysia 4ctcttcactc aataattagt gtcaaaacac agtggaatca
gacgctagtc aattttttgg 60c 61561DNAAplysia 5ctcttcactc aataattagt
gtcaaaatgt ggtggaatca gacgctagtc aattttttgg 60c 61661DNAAplysia
6gccaaaaaat tgactagcgt ctgattccac cacattttga cactaattat tgagtgaaga
60g 61727RNAArtificial Sequencesynthetic 7aaagucagcc cucgacacaa
ggguuug 27827RNAArtificial Sequencesynthetic 8aaagucagcc cucgacacaa
ggguuug 27927RNAArtificial Sequencesynthetic 9aaagucagcc cucgacacaa
ggguuug 271027RNAArtificial Sequencesynthetic 10aaagucagcc
cucgacacaa ggguuug 271127RNAArtificial Sequencesynthetic
11aaagucagcc cucgacacaa ggguuug
271261DNAAplysiamisc_feature(31)..(31)methylatedmisc_feature(33)..(33)met-
hylated 12gccaaaaaat tgactagcgt ctgattccac cgcgttttga cactaattat
tgagtgaaga 60g
611361DNAAplysiamisc_feature(28)..(28)methylatedmisc_feature(30)..(30)met-
hylated 13ctcttcactc aataattagt gtcaaaacgc ggtggaatca gacgctagtc
aattttttgg 60c 61
* * * * *