U.S. patent application number 12/751358 was filed with the patent office on 2010-12-09 for compositions and methods comprising aspartyl-trna synthetases having non-canonical biological activities.
Invention is credited to Ryan A. Adams, Eva R. Armour, Kenny D'Arigo, Leslie A. Greene, Fei Hong, Eve Merriman, Kristi Piehl, Ji Zhao.
Application Number | 20100310576 12/751358 |
Document ID | / |
Family ID | 42983072 |
Filed Date | 2010-12-09 |
United States Patent
Application |
20100310576 |
Kind Code |
A1 |
Adams; Ryan A. ; et
al. |
December 9, 2010 |
COMPOSITIONS AND METHODS COMPRISING ASPARTYL-tRNA SYNTHETASES
HAVING NON-CANONICAL BIOLOGICAL ACTIVITIES
Abstract
Isolated aspartyl-tRNA synthetase polypeptides and
polynucleotides having non-canonical biological activities are
provided, as well as compositions and methods related thereto.
Inventors: |
Adams; Ryan A.; (San Diego,
CA) ; Hong; Fei; (San Diego, CA) ; Zhao;
Ji; (San Diego, CA) ; Piehl; Kristi; (San
Diego, CA) ; Armour; Eva R.; (San Diego, CA) ;
D'Arigo; Kenny; (San Diego, CA) ; Greene; Leslie
A.; (San Diego, CA) ; Merriman; Eve; (San
Diego, CA) |
Correspondence
Address: |
SEED INTELLECTUAL PROPERTY LAW GROUP PLLC
701 FIFTH AVE, SUITE 5400
SEATTLE
WA
98104
US
|
Family ID: |
42983072 |
Appl. No.: |
12/751358 |
Filed: |
March 31, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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61165194 |
Mar 31, 2009 |
|
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Current U.S.
Class: |
424/158.1 ;
424/94.1; 424/94.3; 435/183; 435/320.1; 435/325; 435/375; 435/7.1;
435/7.4; 514/1.1; 514/13.3; 514/16.4; 514/17.7; 514/19.2; 514/2.3;
514/44A; 514/44R; 530/300; 530/389.1; 536/23.2; 536/24.31;
536/24.33; 536/24.5 |
Current CPC
Class: |
Y02A 50/423 20180101;
A61P 29/00 20180101; G01N 2500/00 20130101; Y02A 50/58 20180101;
Y02A 50/385 20180101; C07K 2319/30 20130101; G01N 33/573 20130101;
Y02A 50/389 20180101; A61P 31/00 20180101; C12Y 601/01012 20130101;
C12N 9/93 20130101; A61P 3/00 20180101; A61K 38/00 20130101; C07K
2319/33 20130101; Y02A 50/30 20180101; A61P 35/00 20180101; A61P
37/02 20180101; A61P 25/00 20180101; Y02A 50/411 20180101; Y02A
50/53 20180101; C12Q 1/25 20130101; A61P 37/06 20180101; C07K 16/00
20130101; Y02A 50/463 20180101; A61P 9/00 20180101 |
Class at
Publication: |
424/158.1 ;
435/183; 536/23.2; 435/320.1; 435/325; 536/24.31; 536/24.33;
536/24.5; 530/389.1; 530/300; 435/7.4; 435/7.1; 424/94.1; 424/94.3;
514/44.R; 514/44.A; 514/1.1; 514/19.2; 514/17.7; 514/16.4;
514/13.3; 514/2.3; 435/375 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C12N 9/00 20060101 C12N009/00; C07H 21/00 20060101
C07H021/00; C12N 15/63 20060101 C12N015/63; C12N 5/10 20060101
C12N005/10; C07K 16/18 20060101 C07K016/18; C07K 2/00 20060101
C07K002/00; G01N 33/573 20060101 G01N033/573; G01N 33/566 20060101
G01N033/566; A61K 38/43 20060101 A61K038/43; A61K 31/7088 20060101
A61K031/7088; A61K 38/02 20060101 A61K038/02; A61P 35/00 20060101
A61P035/00; A61P 25/00 20060101 A61P025/00; A61P 9/00 20060101
A61P009/00; A61P 31/00 20060101 A61P031/00; A61P 37/02 20060101
A61P037/02; A61P 29/00 20060101 A61P029/00; A61P 3/00 20060101
A61P003/00; C12N 5/07 20100101 C12N005/07 |
Claims
1. An isolated aspartyl-tRNA synthetase (AspRS) polypeptide having
a non-canonical biological activity, or an active variant
thereof.
2. The isolated aspartyl-tRNA synthetase polypeptide of claim 1
where the non-canonical biological activity is selected from the
group consisting of modulation of cell proliferation, modulation of
apoptosis, modulation of inflammation, modulation of cell
differentiation, modulation of angiogenesis, modulation of cell
binding, modulation of Akt-mediated cell signaling, modulation of
cellular metabolism, modulation of cytokine production or activity,
and modulation of toll-like receptor signaling.
3. The isolated aspartyl-tRNA synthetase polypeptide of claim 1
where the polypeptide is a fragment of the full length human
aspartyl-tRNA synthetase sequence set forth in SEQ ID NO: 1.
4. The isolated aspartyl-tRNA synthetase polypeptide of claim 1
where the active variant thereof is a polypeptide having at least
80% or 90% identity along its length to a human aspartyl-tRNA
synthetase sequence set forth in SEQ ID NO: 1.
5. The isolated aspartyl-tRNA synthetase polypeptide of claim 1
where the polypeptide consists essentially of amino acid residues
1-31, 1-154, 1-171, or 1-174 of SEQ ID NO: 1, or an active fragment
or variant thereof.
6. A fusion polypeptide comprising a polypeptide of claim 1 and a
heterologous fusion partner.
7. A dimeric or multimeric complex comprising at least one isolated
aspartyl-tRNA synthetase polypeptide of claim 1.
8. An isolated polynucleotide encoding a polypeptide of claim 1, or
a complement thereof.
9. An expression vector comprising an isolated polynucleotide of
claim 8.
10. A host cell comprising an expression vector of claim 9.
11. An oligonucleotide that specifically hybridizes to a
polynucleotide of claim 8.
12. The oligonucleotide of claim 11, selected from a primer, a
probe, and an antisense oligonucleotide.
13. A binding agent that exhibits binding specificity for an
isolated AspRS polypeptide of claim 1, a cellular binding partner
of the AspRS polypeptide, or both.
14. The binding agent of claim 13, selected from an antibody, an
antigen-binding fragment thereof, a peptide, peptide mimetic, a
small molecule, and an aptamer.
15. The binding agent of claim 13, wherein the binding agent
antagonizes a non-canonical activity of the AspRS polypeptide.
16. The binding agent of claim 13, wherein the binding agent
agonizes a non-canonical activity of the AspRS polypeptide.
17. A method of determining presence or levels of an aspartyl-tRNA
synthetase (AspRS) polypeptide in a sample, comprising: contacting
the sample with one or binding agents that specifically bind to an
AspRS polypeptide according to claim 1, detecting the presence or
absence of the binding agent, and thereby determining the presence
or levels of the AspRS polypeptide: or introducing the sample into
a detector that is capable of specifically identifying an AspRS
polypeptide according to claim 1, and thereby determining the
presence or levels of the AspRS polypeptide.
18. (canceled)
19. The method of claim 17, wherein the molecular detector is a
mass spectrometer (MS).
20. The method of claim 17, comprising comparing the presence or
levels of the AspRS protein fragment to a control sample or a
predetermined value.
21. The method of claim 20, comprising characterizing the state of
the sample to distinguish it from the control.
22. The method of claim 21, wherein the sample and control comprise
a cell or tissue, and the method comprises distinguishing between
cells or tissues of different species, cells of different tissues
or organs, cells at different cellular developmental states, cells
at different cellular differentiation states, or healthy and
diseased cells.
23. A method of identifying a compound that specifically binds to
an aspartyl-tRNA synthetase (AspRS) polypeptide according to claim
1, or one or more of its cellular binding partners, comprising a)
combining the AspRS polypeptide or its cellular binding partner or
both with at least one test compound under suitable conditions, and
b) detecting binding of the AspRS polypeptide or its cellular
binding partner or both to the test compound, thereby identifying a
compound that specifically binds to the AspRS polypeptide or its
cellular binding partner or both.
24. The method of claim 23, wherein the test compound is a
polypeptide or peptide, an antibody or antigen-binding fragment
thereof, a peptide mimetic, or a small molecule.
25. The method of claim 23, wherein the test compound agonizes a
non-canonical biological activity of the AspRS polypeptide or its
cellular binding partner.
26. The method of claim 23, wherein the test compound antagonizes a
non-canonical biological activity of the AspRS polypeptide or its
cellular binding partner.
27. A compound identified by the method of claim 23.
28. A composition comprising a physiologically acceptable carrier
and at least one component selected from the group consisting of:
(i) an isolated aspartyl-tRNA synthetase (AspRS) polypeptide having
a non-canonical biological activity, or an active, variant thereof;
(ii) a fusion protein comprising an AspRS polypeptide of (i); (iii)
a dimeric or multimeric complex comprising an AspRS polypeptide of
(i); (iv) an isolated polynucleotide that encodes an AspRS
polypeptide of (i); (v) an expression vector comprising a
polynucleotide of (iv); (vi) an oligonucleotide that specifically
hybridizes to a polynucleotide of (iv); (vii) a binding agent that
exhibits binding specificity for an AspRS polypeptide of (i); and
(viii) a compound according to claim 27.
29. A method for modulating a cellular activity comprising
contacting a cell or tissue with a composition of claim 28.
30. The method of claim 29, wherein the cellular activity is
selected from the group consisting of cell migration, cell
proliferation, apoptosis, inflammation, cell differentiation,
angiogenesis, modulation of cell binding, Akt-mediated cell
signaling, cellular metabolism, cytokine production, and toll-like
receptor signaling.
31. The method of claim 30, wherein the cellular activity is
cytokine production.
32. The method of claim 31, wherein the cytokine is any one or more
of IL1-.beta., IL-6, IL-8, IL-10, IL-12p40, MIP1-.alpha.,
MIP-1.beta., GRO-.alpha., MCP-1, or IL-1ra.
33. The method of claim 30, wherein the cellular activity is
toll-like receptor (TLR) signaling.
34. The method of claim 33, wherein the TLR is TLR2, TLR4, or
both.
35. The method of claim 34, wherein the method is a method of
stimulating an innate immune response.
36. The method of claim 29, wherein the cell is in a subject.
37. A method for treating a condition comprising administering to a
subject in need thereof a composition of claim 29, wherein the
condition is selected from the group consisting of inflammatory
diseases, autoimmune diseases, neoplastic diseases, metabolic
diseases, neurological diseases, infections, cardiovascular
diseases, and diseases associated with abnormal angiogenesis.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit under 35 U.S.C.
.sctn.119(e) of U.S. Provisional Patent Application No. 61/165,194,
filed Mar. 31, 2009, which is incorporated by reference in its
entirety.
STATEMENT REGARDING SEQUENCE LISTING
[0002] The Sequence Listing associated with this application is
provided in text format in lieu of a paper copy, and is hereby
incorporated by reference into the specification. The name of the
text file containing the Sequence Listing is
120161.sub.--412_SEQUENCE_LISTING.txt. The text file is 13 KB, was
created on Mar. 31, 2010, and is being submitted electronically via
EFS-Web.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] The present invention relates generally to forms of
aspartyl-tRNA synthetase (AspRS) polypeptides, compositions
comprising such polypeptides, and methods of using same.
[0005] 2. Description of the Related Art
[0006] Aminoacyl-tRNA synthetases, which catalyze the
aminoacylation of tRNA molecules, are essential for decoding
genetic information during the process of translation. In higher
eukaryotes, aminoacyl-tRNA synthetases associate with other
polypeptides to form supramolecular multienzyme complexes. Each of
the eukaryotic tRNA synthetases consists of a core enzyme, which is
closely related to the prokaryotic counterpart of the tRNA
synthetase, and an additional domain that is appended to the
amino-terminal or carboxyl-terminal end of the core enzyme. Human
tyrosyl-tRNA synthetase (TyrRS), for example, has a
carboxyl-terminal domain that is not part of prokaryotic and lower
eukaryotic TyrRS molecules.
[0007] Several aminoacyl-tRNA synthetases have been demonstrated to
have non-canonical functions distinct from their involvement in
translation. For example, Mini-tyrosyl tRNA synthetase
(mini-TyrRS), the N-terminal domain of TyrRS which corresponds to
amino acid residues 1-364 and is cleaved by polymorphonuclear cell
elastase and plasmin, is a member of the aminoacyl tRNA synthetase
"AARS" multifunction cytokine-like proteins and peptides. In vitro,
Mini-TyrRS has been shown to stimulate neutrophil activation and
chemotaxis, endothelial cell proliferation and migration, and is
pro-angiogenic in chick chorioallantoic membrane (CAM) and mouse
matrigel assays. Mini-TyrRS has an ELR motif that, like
CXC-chemokines such as IL-8, confers its chemokine and angiogenic
activities. Like other ELR-containing cytokines, mutation of this
motif inhibits mini-TyrRS binding and stimulation of leukocytes and
angiogenesis.
[0008] In addition, truncated forms or TrpRS have been demonstrated
to have angiogenic properties. In normal human cells, there are two
forms of TrpRS that can be detected: a major form consisting of the
full-length molecule (amino acid residues 1-471) and a minor
truncated form. The minor form is generated by the deletion of an
amino-terminal domain through alternative splicing of the pre-mRNA.
The amino-terminus of miniTrpRS has been determined to be the
methionine residue at position 48 of the full-length TrpRS
molecule. Alternatively, truncated TrpRS can be generated by
proteolysis. For example, bovine TrpRS is highly expressed in the
pancreas and is secreted into the pancreatic juice, thus resulting
in the production of a truncated TrpRS molecule. Additional studies
indicate that mini-TrpRS inhibits VEGF-induced cell proliferation
and migration (Wakasugi et al., Proc. Natl. Acad. Sci. 99: 173-177
(2002)). In particular, a chick CAM assay shows that mini TrpRS
blocks angiogenic activity of VEGF. In contrast, the full-length
TrpRS does not inhibit angiogenesis. Thus, removal of the first 48
amino acid residues exposes the anti-angiogenic activity of TrpRS.
Therefore, as with TyrRS, certain forms of TrpRS possess activities
other than the aminoacylation of tRNA.
[0009] Given these observations of non-canonical and
therapeutically relevant activities associated alternative forms of
TyrRS and TrpRS, there is a need to identify biologically relevant
forms and/or activities of other aminoacyl-tRNA synthetase proteins
in order to exploit the full therapeutic potential of this family
of proteins. Accordingly, the present invention addresses these
needs and offers other related advantages.
SUMMARY OF THE INVENTION
[0010] The present invention stems from the discovery that certain
aspartyl-tRNA synthetase (AspRS) polypeptides possess non-canonical
biological activities of therapeutic relevance. Therefore,
according to one aspect, the present invention provides isolated
AspRS polypeptides having at least one non-canonical biological
activity, as well active fragments and variants thereof which
substantially retain said non-canonical activity. "Non-canonical"
activity," as used herein, refers generally to an activity
possessed by a AspRS polypeptide of the invention that is other
than aminoacylation and, more specifically, other than the addition
of aspartic acid onto a tRNA.sup.Asp molecule. As detailed herein,
in certain embodiments, a non-canonical biological activity
exhibited by a AspRS polypeptide of the invention may include, but
is not limited to, modulation of cell proliferation, modulation of
apoptosis, modulation of inflammation, modulation of cell
differentiation, modulation of angiogenesis, modulation of cell
binding, modulation of Akt-mediated cell signaling, modulation of
cellular metabolism, modulation of cytokine production or activity,
and modulation of toll-like receptor signaling, and the like.
[0011] In certain embodiments, the AspRS polypeptide of the
invention is a contiguous fragment of a full length mammalian AspRS
protein. In a more specific embodiment, the AspRS polypeptide is a
contiguous fragment of the human AspRS protein sequence set forth
in SEQ ID NO: 1. Illustratively, the fragments may be of
essentially any length, provided they are not full length and
further provided they retain at least one non-canonical biological
activity of interest. In certain illustrative embodiments, a AspRS
polypeptide of the invention will range in size from about 20-50,
20-100, 20-200, 20-300, 20-400, or 20-500 amino acids in length. In
other embodiments, the AspRS polypeptide of the invention will
range in size from about 50-100, 50-200, 50-300, 50-400, or 50-500
amino acids in length. In other embodiments, the AspRS polypeptide
of the invention will range in size from about 100-200, 100-300,
100-400, or 100-500 amino acids in length. In still other
illustrative embodiments, the AspRS polypeptide of the invention
will range in size from about 200-300, 200-400, or 200-500 amino
acids in length.
[0012] In further embodiments of the invention, an AspRS
polypeptide comprises an active variant (i.e., retains at least one
non-canonical biological activity of interest) of a fragment of an
AspRS protein sequence, such as the human AspRS protein sequence
set forth in SEQ ID NO: 1. In a more specific embodiment, the
active variant is a polypeptide having at least 70%, 80%, 90%, 95%
or 99% identity along its length to a human aspartyl-tRNA
synthetase sequence set forth in SEQ ID NO: 1.
[0013] Other embodiments of the invention provide AspRS splice
variants and point mutants, whether naturally or non-naturally
occurring, that possess one or more non-canonical activities. In
certain embodiments, the AspRS comprises an amphiphilic helix
domain.
[0014] In a more specific embodiment of the invention, an AspRS
polypeptide comprises a fragment of the human AspRS sequence of SEQ
ID NO: 1, consisting essentially of amino acid residues 1-154,
1-171, 1-174, 1-31, 399-425, 413-476 or 397-425, or an active
fragment or variant thereof that substantially retains at least one
non-canonical biological activity of interest.
[0015] In other specific embodiments, the AspRS polypeptide is not
a polypeptide as set forth in NCBI Accession No. NP001340.
[0016] According to another aspect of the invention, there are
provided fusion proteins comprising at least one AspRS polypeptide
as described herein and a heterologous fusion partner.
[0017] According to another aspect of the invention, there are
provided isolated polynucleotides encoding the polypeptides and
fusion proteins as described herein, as well as expression vectors
comprising such polynucleotides, and host cell comprising such
expression vectors. Also included are oligonucleotides that
specifically hybridize to an AspRS polynucleotide. In certain
embodiments, the oligonucleotide is a primer, a probe, or an
antisense oligonucleotide. Other embodiments relate to RNAi agents
that target an AspRS polynucleotide.
[0018] According to another aspect of the invention, there are
provided binding agents (e.g., antibodies and antigen-binding
fragments thereof) that have binding specificity for an AspRS
polypeptide of the invention, or one of its cellular binding
partners. In certain embodiments, the binding agent is an antibody,
an antigen-binding fragment thereof, a peptide, a peptide mimetic,
a small molecule, or an aptamer. In some embodiments, the binding
agent antagonizes a non-canonical activity of the AspRS
polypeptide. In other embodiments, the binding agent agonizes a
non-canonical activity of the AspRS polypeptide.
[0019] According to yet another aspect of the invention, there are
provided compositions, e.g., pharmaceutical compositions,
comprising physiologically acceptable carriers and at least one of
the isolated polypeptides, fusion proteins, binding agents such as
antibodies, isolated polynucleotides, expression vectors, host
cells, etc., of the invention, as described herein.
[0020] Certain embodiments relate to methods of determining
presence or levels of an AspRS polypeptide in a sample, comprising
contacting the sample with one or binding agents that specifically
bind to an AspRS polypeptide as described herein, detecting the
presence or absence of the binding agent, and thereby determining
the presence or levels of the AspRS polypeptide. Certain
embodiments include methods of determining presence or levels of an
AspRS polypeptide in a sample, comprising introducing the sample
into a molecular detector that is capable of specifically
identifying an AspRS polypeptide as described herein, and thereby
determining the presence or levels of the AspRS polypeptide. In
specific embodiments, the molecular detector is a mass spectrometer
(MS). Certain embodiments include comparing the presence or levels
of the AspRS protein fragment to a control sample or a
predetermined value. Some embodiments include characterizing the
state of the sample to distinguish it from the control. In specific
embodiments, the sample and control comprise a cell or tissue, and
the method comprises distinguishing between cells or tissues of
different species, cells of different tissues or organs, cells at
different cellular developmental states, cells at different
cellular differentiation states, or healthy and diseased cells.
[0021] Also included are methods of identifying a compound that
specifically binds to an AspRS polypeptide, or one or more of its
cellular binding partners, comprising a) combining the AspRS
polypeptide or its cellular binding partner or both with at least
one test compound under suitable conditions, and b) detecting
binding of the AspRS polypeptide or its cellular binding partner or
both to the test compound, thereby identifying a compound that
specifically binds to the AspRS polypeptide or its cellular binding
partner or both. In certain embodiments, the test compound is a
polypeptide or peptide, an antibody or antigen-binding fragment
thereof, a peptide mimetic, or a small molecule. In some
embodiments, the test compound agonizes a non-canonical biological
activity of the AspRS polypeptide or its cellular binding partner.
In other embodiments, the test compound antagonizes a non-canonical
biological activity of the AspRS polypeptide or its cellular
binding partner. Also included are compounds identified by any of
the methods provided herein.
[0022] Also provided by the present invention, in other aspects,
are methods for modulating a cellular activity by contacting a cell
or tissue with a composition of the invention, as described herein,
wherein the cellular activity to be modulated is selected from the
group consisting of cell migration, cell proliferation, apoptosis,
inflammation, cell differentiation, angiogenesis, modulation of
cell binding, Akt-mediated cell signaling, cellular metabolism,
cytokine production, and toll-like receptor signaling, and the
like. In certain embodiments, the cellular activity is cytokine
production. In specific embodiments, the cytokine is any one or
more of IL1-.beta., IL-6, IL-8, IL-10, IL-12p40, MIP1-.alpha.,
MIP-1.beta., GRO-.alpha., MCP-1, or IL-1ra. In some embodiments,
the cellular activity is toll-like receptor (TLR) signaling. In
particular embodiments, the TLR is TLR2, TLR4, or both. Certain
embodiments include methods of stimulating an innate immune
response. In some embodiments, the cell is in a subject.
[0023] In other aspects, the present invention provides methods for
treating a disease, disorder or other condition in a subject in
need thereof by administering a composition according to the
present invention. By way of illustration, such diseases, disorders
or conditions may include, but are not limited to, inflammatory
diseases, autoimmune diseases, neoplastic diseases (e.g., cancers),
metabolic diseases, neurological diseases, infections,
cardiovascular diseases, and diseases associated with abnormal
angiogenesis.
BRIEF DESCRIPTION OF SEQUENCE IDENTIFIERS
[0024] SEQ ID NO: 1 is the full length amino acid sequence of human
aspartyl-tRNA synthetase (AspRS).
[0025] SEQ ID NO: 2 is a nucleic acid sequence encoding the AspRS
polypeptide of SEQ ID NO: 1.
[0026] SEQ ID NO:3 is the amino acid sequence of a 32 amino acid
human AspRS peptide.
[0027] SEQ ID NO:4 is the amino acid sequence of a 32 amino acid
rat AspRS peptide.
[0028] SEQ ID NO:5 is a consensus sequence of the positively
charged residues of the AspRS amphiphilic helix.
[0029] SEQ ID NO:6 is the amino acid sequence of a portion of an
anopheles mosquito AspRS N-terminal helix.
[0030] SEQ ID NO:7 is the amino acid sequence of a portion of a
deer tick AspRS N-terminal helix.
[0031] SEQ ID NO:8 is the amino acid sequence of a portion of an
owl limpet AspRS N-terminal helix.
[0032] SEQ ID NO:9 is the amino acid sequence of a portion of a
leach AspRS N-terminal helix.
[0033] SEQ ID NO:10 is the amino acid sequence of a portion of a
Xenopus AspRS N-terminal helix.
[0034] SEQ ID NO:11 is the amino acid sequence of a portion of a
Japanese puffer fish AspRS N-terminal helix.
[0035] SEQ ID NO:12 is the amino acid sequence of a portion of a
green spotted puffer AspRS N-terminal helix.
[0036] SEQ ID NO:13 is the amino acid sequence of a portion of the
stickleback AspRS N-terminal helix.
[0037] SEQ ID NO:14 is the amino acid sequence of a portion of a
chicken AspRS N-terminal helix.
[0038] SEQ ID NO:15 is the amino acid sequence of a portion of a
bovine AspRS N-terminal helix.
[0039] SEQ ID NO:16 is the amino acid sequence of a portion of a
rat AspRS N-terminal helix.
[0040] SEQ ID NO:17 is the amino acid sequence of a portion of a
mouse AspRS N-terminal helix.
[0041] SEQ ID NO:18 is the amino acid sequence of a portion of a
rock hyrax AspRS N-terminal helix.
[0042] SEQ ID NO:19 is the amino acid sequence of a portion of an
opossum AspRS N-terminal helix.
[0043] SEQ ID NO:20 is the amino acid sequence of a portion of a
tarsier AspRS N-terminal helix.
[0044] SEQ ID NO:21 is the amino acid sequence of a portion of an
orangutan AspRS N-terminal helix.
[0045] SEQ ID NO:22 is the amino acid sequence of a portion of a
chimpanzee AspRS N-terminal helix.
[0046] SEQ ID NO:23 is the amino acid sequence of a portion of a
human AspRS N-terminal helix.
BRIEF DESCRIPTION OF THE DRAWINGS
[0047] FIGS. 1A-1D show (A) the domain structure and (B) amino acid
sequence of AspRS (SEQ ID NO:1), and (C and D) illustrate the
SDS-PAGE separation of fragments of AspRS generated by controlled
proteolysis of the full-length AspRS protein with human neutrophil
elastase. FIG. 1C is an SDS-PAGE gel, 4-12% MOPS, showing
full-length AspRS and digestion with PMN elastase. FIG. 1D is an
SDS-PAGE gel, 12% MES, showing full-length AspRS and digestion with
PMN elastase.
[0048] FIGS. 2A-2B demonstrate the activation of Akt in endothelial
cells (bAEC) treated with AspRS (also referred to as DRS) fragments
of the invention. FIG. 2A shows phosphorylation of Akt induced by
treatment with pool of elastase generated AspRS fragments, and FIG.
2B shows a time course of Akt phosphorylation by cut pools of
AspRS.
[0049] FIG. 3 shows the increased secretion of TNF-.alpha. by
peripheral blood mononuclear cells (PBMCs) treated with an AspRS
fragment of the invention, D1, in comparison to TNF-.alpha.
secretion by PBMCs treated with either full-length AspRS (DRS) or
the positive control, endothelial-monocyte-activating polypeptide
II (EMAP). PBMCs were treated for 24 hours with D1, DRS, or EMAP II
protein and assayed for TNF-.alpha. secretion.
[0050] FIG. 4 shows illustrative cytokines that are secreted
following treatment of PBMCs with AspRS fragment D1. PBMCs were
treated for 24 hours, assayed for secretion of 27 different
cytokines, and found to increase secretion of IL1-.beta., IL-6,
IL-8, IL-10, IL-12p40, MIP1-.alpha., MIP-1.beta., GRO-.alpha.,
MCP-1, and IL-1ra.
[0051] FIG. 5 shows that the AspRS fragment D1 activates monocytes
in a cell type specific manner. PBMCs were treated for 24 hours
with PBS as a negative control, PHA as a positive control, and
AspRS fragment D1, and assayed to detect cell-surface markers of
activation on monocytes and lymphocytes.
[0052] FIG. 6 shows that the AspRS fragment D1 induces secretion of
TNF-.alpha. from monocyte (e.g., THP-1) and macrophage (e.g., RAW
267.7) cell lines.
[0053] FIG. 7 shows that the AspRS fragment D1 induces chemotaxis
of a macrophage cell line. FIG. 7A shows the experimental set-up to
assay cell migration using a boudin chamber, and FIG. 1B shows that
RAW 264.7 macrophage cells migrate in a dose-dependent manner
towards the D1 fragment.
[0054] FIG. 8 shows that the TNF-.alpha. secretion mediated by the
AspRS fragment D1 in THP-1 monocytes is inhibited by an inhibitor
of MEK (UO126), a key component in the MAP kinase signaling
pathway, but not by an inhibitor of PI3 kinase signaling
(LY294022). LPS is used as a positive control and its activity is
blocked by both inhibitors.
[0055] FIG. 9 shows that the AspRS fragment D1 inhibits
VEGF-induced angiogenesis. Matrigel solutions containing PBS,
sutent, or D1 fragment in combination with VEGF were implanted into
mice and analyzed for new blood vessel infiltration into the
matrigel plug.
[0056] FIG. 10 shows the results of an experiment which suggests
that the N-terminal region of the AspRS fragment D1 is responsible
for its cytokine activity. The presence of a 6.times.his affinity
tag on the N-terminus of D1, as compared to on the C-terminus of
D1, reduces the TNF-.alpha. secretion activity of the fragment.
[0057] FIG. 11 shows that the AspRS fragment D1 contains a
mammalian-specific 32 amino acid sequence at its N-terminus SEQ ID
NO:3 is the human AspRS 32 amino acid peptide and SEQ ID NO:4 is
the rat AspRS 32 amino acid peptide. A 32 amino acid peptide found
only at the N-terminus of mammalian AspRS, and not found in yeast
AspRS, is dispensable for canonical tRNA synthetase activity and
predicted to contain a putative helix (see Jacobo-Molina and Yang
(1989); and Escalante and Yang, JBC (1992)).
[0058] FIG. 12 shows the identification, evolution and
crystallization of human AspRS fragment D1. FIG. 12A shows the
steps by which RAW264.7 mouse macrophages were subjected to
SDS-PAGE analysis; protein bands were cut out and analyzed by LC
MS/MS and an N-terminal fragment of AspRS was identified as D1.
FIG. 12B shows that the appended N-terminus of AspRS is an evolved
domain. FIG. 12C shows the crystal structure of full length dimeric
human AspRS solved to a resolution of 1.9 .ANG.; the N-terminal
tRNA anticodon-binding domain, the aminoacylation domain, and the
30 amino acid linker connecting the D1 fragment and the
aminoacylation domain are indicated.
[0059] FIG. 13 shows that D1 induces pro-inflammatory and
anti-inflammatory cytokine secretion in vivo and in vitro. FIG. 13A
shows in vivo TNF-.alpha. and IL-10 serum levels from mice injected
intravenously with 10 mg/kg D1. Mice show an increase in
TNF-.alpha. after 2 hours that is quickly cleared by 6 hours while
IL-10 levels continue to increase. FIG. 13B shows in vitro
TNF-.alpha. & IL-10 release from PBMCs after 4 & 24 hours
respectively. Cells show an increase with D1 (250 nM) treatment but
not with full-length AspRS (250 nM); LPS (10 EU) also shows a
strong TNF-.alpha. response. The flow cytometry analysis in FIG.
13C reveals D1 binding to 83% of primary monocytes and 14% of the
total lymphocyte population. Within primary lymphocytes, D1 binds
to 76% of CD19+ B-cells.
[0060] FIG. 14 shows that D1 activates NF-kB via toll-like
receptors 2 and 4 (TLR2 and TLR4). FIG. 14A shows that D1 activates
NF-KB in RAW264.7 mouse macrophages; RAW-Blue cells encoding an
NF-KB-inducible secreted embryonic alkaline phosphatase reporter
gene showed a dose dependent activation of NF-KB with D1 as
compared to the lack of activation by AspRS. As shown in FIG. 14B,
D1 (1 .mu.M) activates both TLR2 and TLR4 over-expressing HEK293
cells whereas AspRS (1 .mu.M) did not show activity; stably
transfected HEK293 cells expressing either TLR2 or TLR4 with an
NF-KB-inducible reporter demonstrated that D1 can induce NF-KB
activation. In FIG. 14C, flow cytometry shows that D1 binds HEK
cells over-expressing TLR2 or TLR4, but does not bind to control
cells.
[0061] FIG. 15 shows a characterization of D1 activity, relating in
part to the N-terminal amphiphilic helix. The helical wheel in FIG.
15A depicts the N-terminus of human AspRS, and reveals an
amphiphilic helix. The alignment in FIG. 15B (SEQ ID NOS:6-23, from
top to bottom) illustrates two D1 mutants that were designed in
view of the N-terminal helix; a triple alanine (AAA) mutation to
neutralize the negatively charged residues, and a partial charge
reversal mutant (SKK) that represents the yeast sequence. FIG. 15C
shows an increase in in vitro TNF-.alpha. & IL-10 release from
PBMCs after 24 treatment with D1 (50 nM) but not with full-length
AspRS (50 nM) or with the .DELTA.22 mutant (50 nM); the charge
mutants (AAA and SKK) also show decreased activity. FIG. 15D
illustrates how D1 can be released from macrophage cells and binds
to monocytes, T-cells and B-cells via TLR2 and TLR4 receptors to
elicit an early proinflammatory response of TNF-.alpha. release,
followed by an anti-inflammatory response of IL-10 release.
[0062] FIG. 16 shows that D1 activity is not due to endotoxin
contamination. In FIG. 16A, mammalian expressed D1 induces cytokine
secretion in peripheral blood mononuclear cells. D1 was expressed
with a conventional secretion sequence in HEK293 cells. Conditioned
media containing secreted D1 was collected, concentrated and
incubated with PBMC; D1 containing media induced TNF-.alpha.
release which was not observed in mock transfected media. As shown
in FIG. 16B, D1 activity is independent of endotoxin contamination;
D1 cytokine release was unaltered in the presence of polymyxin B,
an inactivator of endotoxin, whereas lipopolysaccharide (LPS) was
completely inhibited. FIG. 16C shows that D1 digestion by
proteinase K abolishes PBMC cytokine stimulating ability. D1 was
digested completely by overnight treatment with proteinase K, and
digested D1 was added to PBMC and TNF-.alpha. secretion was
measured by ELISA.
DETAILED DESCRIPTION OF THE INVENTION
[0063] The practice of the present invention will employ, unless
indicated specifically to the contrary, conventional methods of
molecular biology and recombinant DNA techniques within the skill
of the art, many of which are described below for the purpose of
illustration. Such techniques are explained fully in the
literature. See, e.g., Sambrook, et al., Molecular Cloning: A
Laboratory Manual (2nd Edition, 1989); Maniatis et al., Molecular
Cloning: A Laboratory Manual (1982); DNA Cloning: A Practical
Approach, vol. I & II (D. Glover, ed.); Oligonucleotide
Synthesis (N. Gait, ed., 1984); Nucleic Acid Hybridization (B.
Hames & S. Higgins, eds., 1985); Transcription and Translation
(B. Hames & S. Higgins, eds., 1984); Animal Cell Culture (R.
Freshney, ed., 1986); A Practical Guide to Molecular Cloning (B.
Perbal, ed., 1984). All publications, patents and patent
applications cited herein are hereby incorporated by reference in
their entirety.
DEFINITIONS
[0064] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by those
of ordinary skill in the art to which the invention belongs.
Although any methods and materials similar or equivalent to those
described herein can be used in the practice or testing of the
present invention, preferred methods and materials are described.
For the purposes of the present invention, the following terms are
defined below.
[0065] As used in this specification and the appended claims, the
singular forms "a," "an" and "the" include plural references unless
the content clearly dictates otherwise. By way of example, "an
element" means one element or more than one element.
[0066] By "about" is meant a quantity, level, value, number,
frequency, percentage, dimension, size, amount, weight or length
that varies by as much as 30, 25, 20, 25, 10, 9, 8, 7, 6, 5, 4, 3,
2 or 1% to a reference quantity, level, value, number, frequency,
percentage, dimension, size, amount, weight or length.
[0067] An "agonist" refers to a molecule that intensifies or mimics
the non-canonical biological activity of an AspRS. Agonists may
include proteins, nucleic acids, carbohydrates, small molecules, or
any other compound or composition that modulates the activity of an
AspRS either by directly interacting with the AspRS or its binding
partner, or by acting on components of the biological pathway in
which the AspRS participates. Included are partial and full
agonists.
[0068] The term "antagonist" refers to a molecule that inhibits or
attenuates the non-canonical biological activity of an AspRS.
Antagonists may include proteins such as antibodies, nucleic acids,
carbohydrates, small molecules, or any other compound or
composition that modulates the activity of an AspRS or its binding
partner, either by directly interacting with the AspRS or its
binding partner or by acting on components of the biological
pathway in which the AspRS participates. Included are partial and
full antagonists.
[0069] By "coding sequence" is meant any nucleic acid sequence that
contributes to the code for the polypeptide product of a gene. By
contrast, the term "non-coding sequence" refers to any nucleic acid
sequence that does not contribute to the code for the polypeptide
product of a gene.
[0070] Throughout this specification, unless the context requires
otherwise, the words "comprise," "comprises," and "comprising" will
be understood to imply the inclusion of a stated step or element or
group of steps or elements but not the exclusion of any other step
or element or group of steps or elements.
[0071] By "consisting of" is meant including, and limited to,
whatever follows the phrase "consisting of." Thus, the phrase
"consisting of" indicates that the listed elements are required or
mandatory, and that no other elements may be present. By
"consisting essentially of" is meant including any elements listed
after the phrase, and limited to other elements that do not
interfere with or contribute to the activity or action specified in
the disclosure for the listed elements. Thus, the phrase
"consisting essentially of" indicates that the listed elements are
required or mandatory, but that other elements are optional and may
or may not be present depending upon whether or not they materially
affect the activity or action of the listed elements.
[0072] As used herein, the terms "function" and "functional" and
the like refer to a biological, enzymatic, or therapeutic
function.
[0073] By "gene" is meant a unit of inheritance that occupies a
specific locus on a chromosome and consists of transcriptional
and/or translational regulatory sequences and/or a coding region
and/or non-translated sequences (i.e., introns, 5' and 3'
untranslated sequences).
[0074] "Homology" refers to the percentage number of amino acids
that are identical or constitute conservative substitutions.
Homology may be determined using sequence comparison programs such
as GAP (Deveraux et al., 1984, Nucleic Acids Research 12, 387-395),
which is incorporated herein by reference. In this way sequences of
a similar or substantially different length to those cited herein
could be compared by insertion of gaps into the alignment, such
gaps being determined, for example, by the comparison algorithm
used by GAP.
[0075] The term "host cell" includes an individual cell or cell
culture that can be or has been a recipient of any recombinant
vector(s) or isolated polynucleotide of the invention. Host cells
include progeny of a single host cell, and the progeny may not
necessarily be completely identical (in morphology or in total DNA
complement) to the original parent cell due to natural, accidental,
or deliberate mutation and/or change. A host cell includes cells
transfected or infected in vivo or in vitro with a recombinant
vector or a polynucleotide of the invention. A host cell which
comprises a recombinant vector of the invention is a recombinant
host cell.
[0076] By "isolated" is meant material that is substantially or
essentially free from components that normally accompany it in its
native state. For example, an "isolated polynucleotide," as used
herein, includes a polynucleotide that has been purified from the
sequences that flank it in its naturally-occurring state, e.g., a
DNA fragment which has been removed from the sequences that are
normally adjacent to the fragment. Alternatively, an "isolated
peptide" or an "isolated polypeptide" and the like, as used herein,
includes the in vitro isolation and/or purification of a peptide or
polypeptide molecule from its natural cellular environment, and
from association with other components of the cell; i.e., it is not
significantly associated with in vivo substances.
[0077] The term "mRNA" or sometimes refer by "mRNA transcripts" as
used herein, include, but not limited to pre-mRNA transcript(s),
transcript processing intermediates, mature mRNA(s) ready for
translation and transcripts of the gene or genes, or nucleic acids
derived from the mRNA transcript(s). Transcript processing may
include splicing, editing and degradation. As used herein, a
nucleic acid derived from an mRNA transcript refers to a nucleic
acid for whose synthesis the mRNA transcript or a subsequence
thereof has ultimately served as a template. A cDNA reverse
transcribed from an mRNA, an RNA transcribed from that cDNA, a DNA
amplified from the cDNA, an RNA transcribed from the amplified DNA,
etc., are all derived from the mRNA transcript and detection of
such derived products is indicative of the presence and/or
abundance of the original transcript in a sample. Thus, mRNA
derived samples include, but are not limited to, mRNA transcripts
of the gene or genes, cDNA reverse transcribed from the mRNA, cRNA
transcribed from the cDNA, DNA amplified from the genes, RNA
transcribed from amplified DNA, and the like.
[0078] "Non-canonical" activity as used herein, refers generally to
an activity possessed by an AspRS polypeptide of the invention that
is other than aminoacylation and, more specifically, other than the
addition of its cognate amino acid onto its cognate tRNA molecule.
Non-limiting examples of non-canonical activities include
RNA-binding, amino acid-binding, modulation of cell proliferation,
modulation of cell migration, modulation of cell differentiation
(e.g., hematopoiesis), modulation of apoptosis or other forms of
cell death, modulation of cell signaling, modulation of
angiogenesis, modulation of cell binding, modulation of cellular
metabolism, modulation of cytokine production or activity,
modulation of cytokine receptor activity, modulation of
inflammation, and the like.
[0079] The term "modulating" includes "increasing" or
"stimulating," as well as "decreasing" or "reducing," typically in
a statistically significant or a physiologically significant amount
as compared to a control. An "increased" or "enhanced" amount is
typically a "statistically significant" amount, and may include an
increase that is 1.1, 1.2, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30
or more times (e.g., 500, 1000 times) (including all integers and
decimal points in between and above 1, e.g., 1.5, 1.6, 1.7. 1.8,
etc.) the amount produced by no composition (the absence of an
agent or compound) or a control composition. A "decreased" or
reduced amount is typically a "statistically significant" amount,
and may include a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%,
12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%,
45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%
decrease in the amount produced by no composition (the absence of
an agent or compound) or a control composition, including all
integers in between. Other examples of "statistically significant"
amounts are described herein.
[0080] By "obtained from" is meant that a sample such as, for
example, a polynucleotide extract or polypeptide extract is
isolated from, or derived from, a particular source of the subject.
For example, the extract can be obtained from a tissue or a
biological fluid isolated directly from the subject. "Derived" or
"obtained from" can also refer to the source of a polypeptide or
polynucleotide sequence. For instance, an AspRS sequence of the
present invention may be "derived" from the sequence information of
an AspRS proteolytic fragment or AspRS splice variant, or a portion
thereof, whether naturally-occurring or artificially generated, and
may thus comprise, consist essentially of, or consist of that
sequence.
[0081] The recitations "sequence identity" or, for example,
comprising a "sequence 50% identical to," as used herein, refer to
the extent that sequences are identical on a
nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis
over a window of comparison. Thus, a "percentage of sequence
identity" may be calculated by comparing two optimally aligned
sequences over the window of comparison, determining the number of
positions at which the identical nucleic acid base (e.g., A, T, C,
G, I) or the identical amino acid residue (e.g., Ala, Pro, Ser,
Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu,
Asn, Gln, Cys and Met) occurs in both sequences to yield the number
of matched positions, dividing the number of matched positions by
the total number of positions in the window of comparison (i.e.,
the window size), and multiplying the result by 100 to yield the
percentage of sequence identity.
[0082] A "splice junction" as used herein includes the region in a
mature mRNA transcript or the encoded polypeptide where the 3' end
of a first exon joins with the 5' end of a second exon. The size of
the region may vary, and may include 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55,
60, 65, 70, 75, 80, 85, 90, 95, 100 or more (including all integers
in between) nucleotide or amino acid residues on either side of the
exact residues where the 3' end of one exon joins with the 5' end
of another exon. An "exon" refers to a nucleic acid sequence that
is represented in the mature form of an RNA molecule after either
portions of a precursor RNA (introns) have been removed by
cis-splicing or two or more precursor RNA molecules have been
ligated by trans-splicing. The mature RNA molecule can be a
messenger RNA or a functional form of a non-coding RNA such as rRNA
or tRNA. Depending on the context, an exon can refer to the
sequence in the DNA or its RNA transcript. An "intron" refers to a
non-coding nucleic acid region within a gene, which is not
translated into a protein. Non-coding intronic sections are
transcribed to precursor mRNA (pre-mRNA) and some other RNAs (such
as long noncoding RNAs), and subsequently removed by splicing
during the processing to mature RNA.
[0083] A "splice variant" refers to a mature mRNA and its encoded
protein that are produced by alternative splicing, a process by
which the exons of the RNA (a primary gene transcript or pre-mRNA)
are reconnected in multiple ways during RNA splicing. The resulting
different mRNAs may be translated into different protein isoforms,
allowing a single gene to code for multiple proteins.
[0084] A "subject," as used herein, includes any animal that
exhibits a symptom, or is at risk for exhibiting a symptom, which
can be treated or diagnosed with an AspRS polynucleotide or
polypeptide of the invention. Suitable subjects (patients) include
laboratory animals (such as mouse, rat, rabbit, or guinea pig),
farm animals, and domestic animals or pets (such as a cat or dog).
Non-human primates and, preferably, human patients, are
included.
[0085] "Treatment" or "treating," as used herein, includes any
desirable effect on the symptoms or pathology of a disease or
condition that can be effected by the non-canonical activities of
an AspRS polynucleotide or polypeptide, as described herein, and
may include even minimal changes or improvements in one or more
measurable markers of the disease or condition being treated. Also
included are treatments that relate to non-AspRS therapies, in
which an AspRS sequence described herein provides a clinical marker
of treatment. "Treatment" or "treating" does not necessarily
indicate complete eradication or cure of the disease or condition,
or associated symptoms thereof. The subject receiving this
treatment is any subject in need thereof. Exemplary markers of
clinical improvement will be apparent to persons skilled in the
art.
[0086] By "vector" or "nucleic acid construct" is meant a
polynucleotide molecule, preferably a DNA molecule derived, for
example, from a plasmid, bacteriophage, yeast or virus, into which
a polynucleotide can be inserted or cloned. A vector preferably
contains one or more unique restriction sites and can be capable of
autonomous replication in a defined host cell including a target
cell or tissue or a progenitor cell or tissue thereof, or be
integrable with the genome of the defined host such that the cloned
sequence is reproducible. Accordingly, the vector can be an
autonomously replicating vector, i.e., a vector that exists as an
extra-chromosomal entity, the replication of which is independent
of chromosomal replication, e.g., a linear or closed circular
plasmid, an extra-chromosomal element, a mini-chromosome, or an
artificial chromosome. The vector can contain any means for
assuring self-replication. Alternatively, the vector can be one
which, when introduced into the host cell, is integrated into the
genome and replicated together with the chromosome(s) into which it
has been integrated.
[0087] The terms "wild-type" and "naturally occurring" are used
interchangeably to refer to a gene or gene product that has the
characteristics of that gene or gene product when isolated from a
naturally occurring source. A wild-type gene or gene product (e.g.,
a polypeptide) is that which is most frequently observed in a
population and is thus arbitrarily designed the "normal" or
"wild-type" form of the gene.
Aspartyl-tRNA Synthetase Polypeptides
[0088] The present invention relates generally to isolated AspRS
polypeptides, polynucleotides encoding such polypeptides, binding
agents that bind such polypeptides, analogs, variants and fragments
of such polypeptides, etc., as well as compositions and methods of
using any of the foregoing. Therefore, according to one aspect of
the invention, there are provided AspRS polypeptides having
non-canonical activities of therapeutic relevance, as well as
compositions comprising the same.
[0089] The terms "polypeptide," "peptide," and "protein" are used
interchangeably herein to refer to a polymer of amino acid residues
and to variants and synthetic analogues of the same. Thus, these
terms apply to amino acid polymers in which one or more amino acid
residues are synthetic non-naturally occurring amino acids, such as
a chemical analogue of a corresponding naturally occurring amino
acid, as well as to naturally-occurring amino acid polymers.
[0090] Polypeptides are not limited to a specific length, but, in
the context of the present invention, typically represent a
fragment of a full length protein, and may include
post-translational modifications, for example, glycosylations,
acetylations, phosphorylations and the like, as well as other
modifications known in the art, both naturally occurring and
non-naturally occurring. Polypeptides and proteins of the invention
may be prepared using any of a variety of well known recombinant
and/or synthetic techniques, illustrative examples of which are
further discussed below.
[0091] The recitation "polypeptide variant" refers to polypeptides
that are distinguished from a reference AspRS polypeptide (e.g.,
SEQ ID NO: 1, or any of its fragments such as D1, including
fragments that consist of amino acid residues 1-154, 1-171, 1-174,
1-177, 1-31, 399-425, 413-476 or 397-425 of SEQ ID NO:1) by the
addition, deletion, and/or substitution of at least one amino acid
residue, and which typically retain at least one non-canonical
activity, as described herein. In certain embodiments, a
polypeptide variant is distinguished from a reference polypeptide
by one or more substitutions, which may be conservative or
non-conservative, as described herein and known in the art. In
certain embodiments, the polypeptide variant comprises conservative
substitutions and, in this regard, it is well understood in the art
that some amino acids may be changed to others with broadly similar
properties without changing the nature of the activity of the
polypeptide.
[0092] Polypeptide variants encompassed by the present invention
will typically exhibit at least about 70%, 75%, 80%, 85%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity (determined as
described below), along their lengths, to the corresponding region
of a wild-type mammalian AspRS protein, such as SEQ ID NO: 1, or
any of its fragments such as D1, including fragments that consist
of amino acid residues 1-154, 1-171, 1-174, 1-177, 1-31, 399-425,
413-476 or 397-425 of SEQ ID NO:1. Also included are sequences
differing from the reference AspRS sequences by the addition,
deletion, or substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100,
110, 120, 130, 140, 150 or more amino acids but which retain the
properties of a reference AspRS polypeptide, such as a
non-canonical activity. In certain embodiments, the amino acid
additions or deletions occur at the C-terminal end and/or the
N-terminal end of SEQ ID NO:1 or fragments thereof that consist of
amino acid residues 1-154, 1-171, 1-174, 1-177, 1-31, 399-425,
413-476 or 397-425 of SEQ ID NO:1. In certain embodiments, the
amino acid additions include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50 or more wild-type
residues (i.e., from the corresponding full-length AARS
polypeptide) that are proximal to the C-terminal end and/or the
N-terminal end of these AspRS fragments.
[0093] In other embodiments, variant polypeptides differ from the
corresponding AspRS reference sequences by at least 1% but less
than 20%, 15%, 10% or 5% of the residues. (If this comparison
requires alignment, the sequences should be aligned for maximum
similarity. "Looped" out sequences from deletions or insertions, or
mismatches, are considered differences.) The differences are,
suitably, differences or changes at a non-essential residue or a
conservative substitution.
[0094] Also included are biologically active "fragments" of the
AspRS reference polypeptides. Representative biologically active
fragments generally participate in an interaction, e.g., an
intramolecular or an inter-molecular interaction. An
inter-molecular interaction can be a specific binding interaction
or an enzymatic interaction. An inter-molecular interaction can be
between an AspRS polypeptide and a cellular binding partner, such
as a cellular receptor or other host molecule that participates in
the non-canonical activity of the AspRS polypeptide.
[0095] Typically, biologically active fragments comprise a domain
or motif with at least one activity of an AspRS reference
polypeptide and may include one or more (and in some cases all) of
the various active domains, and include fragments having a
non-canonical activity. In some cases, biologically active
fragments of an AspRS polypeptide have a biological activity that
is unique to the particular, truncated fragment, such that the
full-length AspRS polypeptide may not have that activity. In
certain cases, the biological activity may be revealed by
separating the biologically active AspRS polypeptide fragment from
the other full-length AspRS polypeptide sequences, or by altering
certain residues of the full-length AspRS wild-type polypeptide
sequence to unmask the biologically active domains. For example, in
certain illustrative embodiments, an AspRS polypeptide may comprise
all or a portion of an amphiphilic helix, as illustrated herein
(see, e.g., SEQ ID NO:3), and/or a region of positive charged
residues (see, e.g., SEQ ID NO:5). In certain embodiments, the
amphiphilic helix is a 22 amino acid region, as described
herein.
[0096] A biologically active fragment of an AspRS reference
polypeptide can be a polypeptide fragment which is, for example,
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, 55, 60, 65, 70, 75, 80, 85, 90, 95,
100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240,
250, 260, 280, 300 or more contiguous or non-contiguous amino
acids, including all integers in between, of the amino acid
sequences set forth SEQ ID NO:1. In other illustrative embodiments,
an AspRS fragment of SEQ ID NO:1 may range in size from about
20-30, 20-40, 20-50, 20-60, 20-70, 20-80, 20-90, 20-100, 20-125,
20-150 or 20-175 amino acids in length. In other embodiments, the
fragment will range in size from about 30-40, 30-50, 30-60, 30-70,
30-80, 30-90, 30-100, 30-125, 30-150 or 30-175 amino acids in
length. In other embodiments, the fragment will range in size from
about 40-50, 40-60, 40-70, 40-80, 40-90, 40-100, 40-125, 40-150 or
40-175 amino acids in length. In still other illustrative
embodiments, the fragment will range in size from about 50-60,
50-70, 50-80, 50-90, 50-100, 50-125, 50-150 or 50-175 amino acids
in length.
[0097] In certain embodiments, the AspRS polypeptide is a truncated
AspRS polypeptide. A "truncated" AspRS, as used herein, refers to
an aspartyl-tRNA synthetase protein which is shorter than its
corresponding full length AspRS protein, for example, due to
removal of amino acids from its N- and/or C-terminal ends. The
extent of the truncation, that is, the number of N- and/or
C-terminal amino acid residues removed from a full length AspRS
protein can vary considerably while still providing desired
cellular effects when administered to a cell, tissue or subject, as
described herein. In certain embodiments, at least about 5, 10, 15,
20, 25, 50, 75, 100, 150, 200, 250, 300, 350 amino acids, or more,
including all intermediate lengths, are truncated from the N-
and/or C-terminus of a full length mammalian AspRS protein.
Intermediate lengths are intended to include all integers
therebetween, for example, 6, 7, 8, etc., 51, 52, 53, etc., 201,
202, 203, etc. Suitably, the biologically-active fragment has no
less than about 1%, 10%, 25%, or 50% of a non-canonical
biologically-activity of an AspRS reference polypeptide.
[0098] Also included are proteolytic fragments of an AspRS
polypeptide, which can be characterized, identified, or derived
according to a variety of techniques. For instance, proteolytic
fragments can be identified in vitro, such as by incubating
full-length or other AspRS polypeptides with selected proteases, or
they can be identified endogenously (i.e., in vivo). In certain
embodiments, protein fragments such as endogenous proteolytic
fragments can be generated or identified, for instance, by
recombinantly expressing full-length or other AspRS polypeptides in
a selected microorganism or eukaryotic cell that has been either
modified to contain one or more selected proteases, or that
naturally contains one or more proteases that are capable of acting
on a selected AspRS polypeptide, and isolating and characterizing
the endogenously produced protein fragments therefrom.
[0099] In certain embodiments, protein fragments such as endogenous
(e.g., naturally-occurring) proteolytic fragments can be generated
or identified, for instance, from various cellular fractions (e.g.,
cytosolic, membrane, nuclear) and/or growth medium of various
cell-types, including, for example, macrophages such as RAW
macrophages (e.g., RAW 264.7 macrophages), T-cells, including
primary T-cells and T-cell lines such as Jurkats, and natural
killer (NK) cells, among others. In certain embodiments, protein
fragments such as endogenous proteolytic fragments, however
generated, can be identified by techniques such as
mass-spectrometry, or equivalent techniques. Once an in vitro or
endogenously identified protein fragment has been generated or
identified, it can be mapped or sequenced, and, for example, cloned
into an expression vector for recombinant production, or produced
synthetically.
[0100] A wide variety of proteases can be used to produce,
identify, derive, or characterize the sequence of AspRS proteolytic
fragments. Generally, proteases are usually classified according to
three major criteria: (i) the reaction catalysed, (ii) the chemical
nature of the catalytic site, and (iii) the evolutionary
relationship, as revealed by the structure. General examples of
proteases or proteinases, as classified by mechanism of catalysis,
include aspartic proteases, serine proteases, cysteine proteases,
and metalloproteases.
[0101] Most aspartic proteases belong to the pepsin family. This
family includes digestive enzymes, such as pepsin and chymosin, as
well as lysosomal cathepsins D and processing enzymes such as
renin, and certain fungal proteases (e.g., penicillopepsin,
rhizopuspepsin, endothiapepsin). A second family of aspartic
proteases includes viral proteinases such as the protease from the
AIDS virus (H IV), also called retropepsin.
[0102] Serine proteases include two distinct families. First, the
chymotrypsin family, which includes the mammalian enzymes such as
chymotrypsin, trypsin, elastase, and kallikrein, and second, the
substilisin family, which includes the bacterial enzymes such as
subtilisin. The general 3D structure between these two families is
different, but they have the same active site geometry, and
catalysis proceeds via the same mechanism. The serine proteases
exhibit different substrate specificities, differences which relate
mainly to amino acid substitutions in the various enzyme subsites
(substrate residue interacting sites). Some serine proteases have
an extended interaction site with the substrate whereas others have
a specificity that is restricted to the P1 substrate residue.
[0103] The cysteine protease family includes the plant proteases
such as papain, actinidin, and bromelain, several mammalian
lysosomal cathepsins, the cytosolic calpains (calcium-activated),
as well as several parasitic proteases (e.g., Trypanosoma,
Schistosoma). Papain is the archetype and the best studied member
of the family. Recent elucidation of the X-ray structure of the
Interleukin-1-beta Converting Enzyme has revealed a novel type of
fold for cysteine proteinases.
[0104] The metalloproteases are one of the older classes of
proteases, found in bacteria, fungi, and higher organisms. They
differ widely in their sequences and their 3D structures, but the
great majority of enzymes contain a zinc atom that is catalytically
active. In some cases, zinc may be replaced by another metal such
as cobalt or nickel without loss of proteolytic activity. Bacterial
thermolysin has been well characterized and its crystallographic
structure indicates that zinc is bound by two histidines and one
glutamic acid. Many metalloproteases contain the sequence motif
HEXXH, which provides two histidine ligands for the zinc. The third
ligand is either a glutamic acid (thermolysin, neprilysin, alanyl
aminopeptidase) or a histidine (astacin, serralysin).
[0105] In certain illustrative embodiments, truncated AspRS
polypeptides may be produced using any of a variety of proteolytic
enzymes using techniques known and available in the art.
Illustrative proteases include, for example, achromopeptidase,
aminopeptidase, ancrod, angiotensin converting enzyme, bromelain,
calpain, calpain I, calpain II, carboxypeptidase A,
carboxypeptidase B, carboxypeptidase G, carboxypeptidase P,
carboxypeptidase W, carboxypeptidase Y, caspase 1, caspase 2,
caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8,
caspase 9, caspase 10, caspase 11, caspase 12, caspase 13,
cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin G,
cathepsin H, cathepsin L, chymopapain, chymase, chymotrypsin,
clostripain, collagenase, complement C1r, complement C1s,
complement Factor D, complement factor I, cucumisin, dipeptidyl
peptidase IV, elastase (leukocyte), elastase (pancreatic),
endoproteinase Arg-C, endoproteinase Asp-N, endoproteinase Glu-C,
endoproteinase Lys-C, enterokinase, factor Xa, ficin, furin,
granzyme A, granzyme B, HIV Protease, IGase, kallikrein tissue,
leucine aminopeptidase (general), leucine aminopeptidase (cytosol),
leucine aminopeptidase (microsomal), matrix metalloprotease,
methionine aminopeptidase, neutrase, papain, pepsin, plasmin,
prolidase, pronase E, prostate specific antigen, protease
alkalophilic from Streptomyces griseus, protease from Aspergillus,
protease from Aspergillus saitoi, protease from Aspergillus sojae,
protease (B. licheniformis) (alkaline or alcalase), protease from
Bacillus polymyxa, protease from Bacillus sp., protease from
Rhizopus sp., protease S, proteasomes, proteinase from Aspergillus
oryzae, proteinase 3, proteinase A, proteinase K, protein C,
pyroglutamate aminopeptidase, rennin, streptokinase, subtilisin,
thermolysin, thrombin, tissue plasminogen activator, trypsin,
tryptase and urokinase.
[0106] Certain embodiments relate to isolated AspRS polypeptides,
comprising, consisting essentially of, or consisting of amino acid
sequences that have been derived from endogenous,
naturally-occurring AspRS polypeptide fragments, and pharmaceutical
compositions comprising said fragments, and methods of use thereof.
In certain embodiments, as noted above, the sequences of AspRS
protein fragments such as endogenous proteolytic fragments can be
generated or identified, for instance, from various cellular
fractions (e.g., cytosolic, membrane, nuclear) and/or conditioned
medium from various cell-types, including primary cells and cell
lines. Examples of such cell types include, without limitation,
immune cells such as monocytes, dendritic cells, macrophages (e.g.,
RAW 264.7 macrophages; see Example 5), neutrophils, eosinophils,
basophils, and lymphocytes, such as B-cells and T-cells (e.g., CD4+
helper and CD8+ killer cells), including primary T-cells and T-cell
lines such as Jurkat T-cells, as well as natural killer (NK)
cells.
[0107] In certain embodiments, AspRS protein fragments can be
identified by techniques such as mass-spectrometry, or equivalent
techniques. Merely by way of illustration and not limitation, in
certain embodiments the proteomes from various cell types, tissues,
or body fluids from a variety of physiological states (e.g.,
hyposia, diet, age, disease) or fractions thereof may be separated
by 1D SDS-PAGE and the gel lanes cut into bands at fixed intervals;
after which the bands may be optionally digested with an
appropriate protease, such as trypsin, to release the peptides,
which may then be analyzed by 1D reverse phase LC-MS/MS. The
resulting proteomic data may be integrated into so-called
peptographs, which plot, in the left panel, sequence coverage for a
given protein in the horizontal dimension (N to C terminus, left to
right) versus SDS-PAGE migration in the vertical dimension (high to
low molecular weight, top to bottom). The specific peptide
fragments can then be sequenced or mapped. In certain embodiments,
the AspRS reference fragment may be characterized by its unique
molecular weight, as compared, for example, to the molecular weight
of the corresponding full-length AspRS.
[0108] As noted above, a polypeptide variant may differ from an
AspRS polypeptide of the invention in one or more substitutions,
deletions, additions and/or insertions. Such variants may be
naturally occurring or may be synthetically generated, for example,
by modifying one or more of the above polypeptide sequences of the
invention and evaluating their biological activity as described
herein using any of a number of techniques well known in the
art.
[0109] In other illustrative embodiments, the variant may be a
splice variant, whether naturally or non-naturally occurring,
wherein the polypeptide possesses at least one non-canonical
activity, e.g., as described herein. In other illustrative
embodiments, the variant contains one or more point mutations
relative to the wild type AspRS polypeptide sequence, whether
naturally or non-naturally occurring, wherein the polypeptide
possesses at least one non-canonical activity, e.g., as described
herein.
[0110] In certain embodiments, a variant will contain conservative
substitutions. A "conservative substitution" is one in which an
amino acid is substituted for another amino acid that has similar
properties, such that one skilled in the art of peptide chemistry
would expect the secondary structure and hydropathic nature of the
polypeptide to be substantially unchanged. Modifications may be
made in the structure of the polynucleotides and polypeptides of
the present invention and still obtain a functional molecule that
encodes a variant or derivative polypeptide with desirable
characteristics. When it is desired to alter the amino acid
sequence of a polypeptide to create an equivalent, or even an
improved, variant of an AspRS polypeptide of the invention, one
skilled in the art, for example, can change one or more of the
codons of the encoding DNA sequence according to Table 1.
[0111] For example, certain amino acids may be substituted for
other amino acids in a protein structure without appreciable loss
of interactive binding capacity with structures such as, for
example, antigen-binding regions of antibodies or binding sites on
substrate molecules. Since it is the interactive capacity and
nature of a protein that generally defines that protein's
biological functional activity, certain amino acid sequence
substitutions can be made in a protein sequence, and, of course,
its underlying DNA coding sequence, and nevertheless obtain a
protein with like properties. It is thus contemplated that various
changes may be made in the polypeptide sequences of the disclosed
compositions, or corresponding DNA sequences which encode said
polypeptides without appreciable loss of their desired utility or
activity.
TABLE-US-00001 TABLE 1 Amino Acids Codons Alanine Ala A GCA GCC GCG
GCU Cysteine Cys C UGC UGU Aspartic acid Asp D GAC GAU Glutamic
acid Glu E GAA GAG Phenylalanine Phe F UUC UUU Glycine Gly G GGA
GGC GGG GGU Histidine His H CAC CAU Isoleucine Ile I AUA AUC AUU
Lysine Lys K AAA AAG Leucine Leu L UUA UUG CUA CUC CUG CUU
Methionine Met M AUG Asparagine Asn N AAC AAU Proline Pro P CCA CCC
CCG CCU Glutamine Gln Q CAA CAG Arginine Arg R AGA AGG CGA CGC CGG
CGU Serine Ser S AGC AGU UCA UCC UCG UCU Threonine Thr T ACA ACC
ACG ACU Valine Val V GUA GUC GUG GUU Tryptophan Trp W UGG Tyrosine
Tyr Y UAC UAU
[0112] In making such changes, the hydropathic index of amino acids
may also be considered. The importance of the hydropathic amino
acid index in conferring interactive biologic function on a protein
is generally understood in the art (Kyte and Doolittle, 1982,
incorporated herein by reference). For example, it is known that
the relative hydropathic character of the amino acid contributes to
the secondary structure of the resultant protein, which in turn
defines the interaction of the protein with other molecules, for
example, enzymes, substrates, receptors, DNA, antibodies, antigens,
and the like. Each amino acid has been assigned a hydropathic index
on the basis of its hydrophobicity and charge characteristics (Kyte
and Doolittle, 1982). These values are: isoleucine (+4.5); valine
(+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine (+2.5);
methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine
(-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline
(-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5);
aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine
(-4.5).
[0113] It is known in the art that certain amino acids may be
substituted by other amino acids having a similar hydropathic index
or score and still result in a protein with similar biological
activity, i.e. still obtain a biological functionally equivalent
protein. In making such changes, the substitution of amino acids
whose hydropathic indices are within .+-.2 is preferred, those
within .+-.1 are particularly preferred, and those within .+-.0.5
are even more particularly preferred.
[0114] It is also understood in the art that the substitution of
like amino acids can be made effectively on the basis of
hydrophilicity. As detailed in U.S. Pat. No. 4,554,101, the
following hydrophilicity values have been assigned to amino acid
residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0.+-.1);
glutamate (+3.0.+-.1); serine (+0.3); asparagine (+0.2); glutamine
(+0.2); glycine (0); threonine (-0.4); proline (-0.5.+-.1); alanine
(-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3);
valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3);
phenylalanine (-2.5); tryptophan (-3.4). It is understood that an
amino acid can be substituted for another having a similar
hydrophilicity value and still obtain a biologically equivalent
protein. In such changes, the substitution of amino acids whose
hydrophilicity values are within .+-.2 is preferred, those within
.+-.1 are particularly preferred, and those within .+-.0.5 are even
more particularly preferred.
[0115] As outlined above, amino acid substitutions may be based on
the relative similarity of the amino acid side-chain substituents,
for example, their hydrophobicity, hydrophilicity, charge, size,
and the like. Exemplary substitutions that take various of the
foregoing characteristics into consideration are well known to
those of skill in the art and include: arginine and lysine;
glutamate and aspartate; serine and threonine; glutamine and
asparagine; and valine, leucine and isoleucine.
[0116] In addition, any polynucleotide may be further modified to
increase stability in vivo. Possible modifications include, but are
not limited to, the addition of flanking sequences at the 5' and/or
3' ends; the use of phosphorothioate or 2' O-methyl rather than
phosphodiesterase linkages in the backbone; and/or the inclusion of
nontraditional bases such as inosine, queosine and wybutosine, as
well as acetyl- methyl-, thio- and other modified forms of adenine,
cytidine, guanine, thymine and uridine.
[0117] Amino acid substitutions may further be made on the basis of
similarity in polarity, charge, solubility, hydrophobicity,
hydrophilicity and/or the amphipathic nature of the residues. For
example, negatively charged amino acids include aspartic acid and
glutamic acid; positively charged amino acids include lysine and
arginine; and amino acids with uncharged polar head groups having
similar hydrophilicity values include leucine, isoleucine and
valine; glycine and alanine; asparagine and glutamine; and serine,
threonine, phenylalanine and tyrosine. Other groups of amino acids
that may represent conservative changes include: (1) ala, pro, gly,
glu, asp, gln, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile,
leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his.
A variant may also, or alternatively, contain nonconservative
changes. In a preferred embodiment, variant polypeptides differ
from a native sequence by substitution, deletion or addition of
five amino acids or fewer. Variants may also (or alternatively) be
modified by, for example, the deletion or addition of amino acids
that have minimal influence on secondary structure and hydropathic
nature of the polypeptide.
[0118] Polypeptides may comprise a signal (or leader) sequence at
the N-terminal end of the protein, which co-translationally or
post-translationally directs transfer of the protein. The
polypeptide may also be conjugated to a linker or other sequence
for ease of synthesis, purification or identification of the
polypeptide (e.g., poly-His), or to enhance binding of the
polypeptide to a solid support. For example, a polypeptide may be
conjugated to an immunoglobulin Fc region.
[0119] When comparing polypeptide sequences, two sequences are said
to be "identical" if the sequence of amino acids in the two
sequences is the same when aligned for maximum correspondence, as
described below. Comparisons between two sequences are typically
performed by comparing the sequences over a comparison window to
identify and compare local regions of sequence similarity. A
"comparison window" as used herein, refers to a segment of at least
about 20 contiguous positions, usually 30 to about 75, 40 to about
50, in which a sequence may be compared to a reference sequence of
the same number of contiguous positions after the two sequences are
optimally aligned.
[0120] Optimal alignment of sequences for comparison may be
conducted, for example, using the Megalign program in the Lasergene
suite of bioinformatics software (DNASTAR, Inc., Madison, Wis.),
using default parameters. This program embodies several alignment
schemes described in the following references: Dayhoff, M. O.
(1978) A model of evolutionary change in proteins--Matrices for
detecting distant relationships. In Dayhoff, M. O. (ed.) Atlas of
Protein Sequence and Structure, National Biomedical Research
Foundation, Washington D.C. Vol. 5, Suppl. 3, pp. 345-358; Hein J.
(1990) Unified Approach to Alignment and Phylogenes pp. 626-645
Methods in Enzymology vol. 183, Academic Press, Inc., San Diego,
Calif.; Higgins, D. G. and Sharp, P. M. (1989) CABIOS 5:151-153;
Myers, E. W. and Muller W. (1988) CABIOS 4:11-17; Robinson, E. D.
(1971) Comb. Theor 11:105; Santou, N. Nes, M. (1987) Mol. Biol.
Evol. 4:406-425; Sneath, P. H. A. and Sokal, R. R. (1973) Numerical
Taxonomy--the Principles and Practice of Numerical Taxonomy,
Freeman Press, San Francisco, Calif.; Wilbur, W. J. and Lipman, D.
J. (1983) Proc. Nat'l Acad., Sci. USA 80:726-730.
[0121] Alternatively, optimal alignment of sequences for comparison
may be conducted by the local identity algorithm of Smith and
Waterman (1981) Add. APL. Math 2:482, by the identity alignment
algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by
the search for similarity methods of Pearson and Lipman (1988)
Proc. Nat'l Acad. Sci. USA 85: 2444, by computerized
implementations of these algorithms (GAP, BESTFIT, BLAST, FASTA,
and TFASTA in the Wisconsin Genetics Software Package, Genetics
Computer Group (GCG), 575 Science Dr., Madison, Wis.), or by
inspection.
[0122] Examples of algorithms that are suitable for determining
percent sequence identity and sequence similarity are the BLAST and
BLAST 2.0 algorithms, which are described in Altschul et al. (1977)
Nucl. Acids Res. 25:3389-3402 and Altschul et al. (1990) J. Mol.
Biol. 215:403-410, respectively. BLAST and BLAST 2.0 can be used,
for example with the parameters described herein, to determine
percent sequence identity for the polynucleotides and polypeptides
of the invention. Software for performing BLAST analyses is
publicly available through the National Center for Biotechnology
Information. For amino acid sequences, a scoring matrix can be used
to calculate the cumulative score. Extension of the word hits in
each direction are halted when: the cumulative alignment score
falls off by the quantity X from its maximum achieved value; the
cumulative score goes to zero or below, due to the accumulation of
one or more negative-scoring residue alignments; or the end of
either sequence is reached. The BLAST algorithm parameters W, T and
X determine the sensitivity and speed of the alignment.
[0123] In one illustrative approach, the "percentage of sequence
identity" is determined by comparing two optimally aligned
sequences over a window of comparison of at least 20 positions,
wherein the portion of the polypeptide sequence in the comparison
window may comprise additions or deletions (i.e., gaps) of 20
percent or less, usually 5 to 15 percent, or 10 to 12 percent, as
compared to the reference sequences (which does not comprise
additions or deletions) for optimal alignment of the two sequences.
The percentage is calculated by determining the number of positions
at which the identical amino acid residue occurs in both sequences
to yield the number of matched positions, dividing the number of
matched positions by the total number of positions in the reference
sequence (i.e., the window size) and multiplying the results by 100
to yield the percentage of sequence identity.
[0124] In certain embodiments of the invention, there are provided
fusion polypeptides, and polynucleotides encoding fusion
polypeptides. Fusion polypeptides refer to AspRS polypeptides of
the invention that have been covalently linked, either directly or
indirectly via an amino acid linker, to one or more heterologous
polypeptide sequences (fusion partners). The polypeptides forming
the fusion protein are typically linked C-terminus to N-terminus,
although they can also be linked C-terminus to C-terminus,
N-terminus to N-terminus, or N-terminus to C-terminus. The
polypeptides of the fusion protein can be in any order.
[0125] The fusion partner may be designed and included for
essentially any desired purpose provided they do not adversely
affect the desired activity of the polypeptide. For example, in one
embodiment, a fusion partner comprises a sequence that assists in
expressing the protein (an expression enhancer) at higher yields
than the native recombinant protein. Other fusion partners may be
selected so as to increase the solubility of the protein or to
enable the protein to be targeted to desired intracellular
compartments. Still further fusion partners include affinity tags,
which facilitate purification of the protein.
[0126] More generally, fusion to heterologous sequences, such as an
Fc fragment, may be utilized to remove unwanted characteristics or
to improve the desired characteristics (e.g., pharmacokinetic
properties) of an AspRS polypeptide. For example, fusion to a
heterologous sequence may increase chemical stability, decrease
immunogenicity, improve in vivo targeting, and/or increase
half-life in circulation of an AspRS polypeptide.
[0127] Fusion to heterologous sequences may also be used to create
bi-functional fusion proteins, such as bi-functional proteins that
are not only possess a selected non-canonical activity through the
AspRS polypeptide, but are also capable of modifying (i.e.,
stimulating or inhibiting) other pathways through the heterologous
polypeptide. Examples of such pathways include, but are not limited
to, various immune system-related pathways, such as innate or
adaptive immune activation pathways, or cell-growth regulatory
pathways, such as angiogenesis. In certain aspects, the
heterologous polypeptide may act synergistically with the AspRS
polypeptide to modulate a cellular pathway in a subject. Examples
of heterologous polypeptides that may be utilized to create a
bi-functional fusion protein include, but are not limited to,
thrombopoietin, cytokines (e.g., IL-11), chemokines, and various
hematopoietic growth factors, in addition to biologically active
fragments and/or variants thereof.
[0128] Fusion proteins may generally be prepared using standard
techniques. For example, DNA sequences encoding the polypeptide
components of a desired fusion may be assembled separately, and
ligated into an appropriate expression vector. The 3' end of the
DNA sequence encoding one polypeptide component is ligated, with or
without a peptide linker, to the 5' end of a DNA sequence encoding
the second polypeptide component so that the reading frames of the
sequences are in phase. This permits translation into a single
fusion protein that retains the biological activity of both
component polypeptides.
[0129] A peptide linker sequence may be employed to separate the
first and second polypeptide components by a distance sufficient to
ensure that each polypeptide folds into its secondary and tertiary
structures, if desired. Such a peptide linker sequence is
incorporated into the fusion protein using standard techniques well
known in the art. Certain peptide linker sequences may be chosen
based on the following factors: (1) their ability to adopt a
flexible extended conformation; (2) their inability to adopt a
secondary structure that could interact with functional epitopes on
the first and second polypeptides; and (3) the lack of hydrophobic
or charged residues that might react with the polypeptide
functional epitopes. Preferred peptide linker sequences contain
Gly, Asn and Ser residues. Other near neutral amino acids, such as
Thr and Ala may also be used in the linker sequence. Amino acid
sequences which may be usefully employed as linkers include those
disclosed in Maratea et al., Gene 40:39 46 (1985); Murphy et al.,
Proc. Natl. Acad. Sci. USA 83:8258 8262 (1986); U.S. Pat. No.
4,935,233 and U.S. Pat. No. 4,751,180. The linker sequence may
generally be from 1 to about 50 amino acids in length. Linker
sequences are not required when the first and second polypeptides
have non-essential N-terminal amino acid regions that can be used
to separate the functional domains and prevent steric
interference.
[0130] The ligated DNA sequences are operably linked to suitable
transcriptional or translational regulatory elements. The
regulatory elements responsible for expression of DNA are located
only 5' to the DNA sequence encoding the first polypeptides.
Similarly, stop codons required to end translation and
transcription termination signals are only present 3' to the DNA
sequence encoding the second polypeptide.
[0131] In general, polypeptides and fusion polypeptides (as well as
their encoding polynucleotides) are isolated. An "isolated"
polypeptide or polynucleotide is one that is removed from its
original environment. For example, a naturally-occurring protein is
isolated if it is separated from some or all of the coexisting
materials in the natural system. Preferably, such polypeptides are
at least about 90% pure, more preferably at least about 95% pure
and most preferably at least about 99% pure. A polynucleotide is
considered to be isolated if, for example, it is cloned into a
vector that is not a part of the natural environment.
[0132] In still other embodiments, an AspRS polypeptide of the
invention may be part of a dimer. Dimers may include, for example,
homodimers between two identical AspRS polypeptides, heterodimers
between two different AspRS polypeptides (e.g., a full-length AspRS
polypeptide and a truncated AspRS polypeptide or two different
truncated AspRS polypeptides), and/or heterodimers between an AspRS
polypeptide and a heterologous polypeptide. The monomers and/or
dimmers may be soluble and may be isolated or purified to
homogeneity. Certain heterodimers, such as those between an AspRS
polypeptide and a heterologous polypeptide, may be
bi-functional.
[0133] Also included are monomers of AspRS polypeptides, including
isolated AspRS monomers that do not substantially dimerize with
themselves (homodomerize) or with a second AspRS polypeptide
(heterodimerize), whether due to one or more substitutions,
truncations, deletions, additions, chemical modifications, or a
combination of these alterations. In certain embodiments, monomeric
AspRS polypeptides possess biological activities, including
non-canonical activities, which are not possessed by dimeric or
multimeric AspRS polypeptide complexes.
[0134] In other embodiments, an AspRS polypeptide of the invention
may be part of a multi-unit complex. A multi-unit complex of the
present invention can include, for example, at least 2, 3, 4, or 5
or more monomers. The monomers and/or multi-unit complexes of the
present invention may be soluble and may be isolated or purified to
homogeneity. Monomer units of a multi-unit complex may be
different, homologous, substantially homologous, or identical to
one another. However, a multi-unit complex of the invention
includes at least one monomer comprising an AspRS polypeptide as
described herein or, in other embodiments, at least two or more
AspRS polypeptides as described herein.
[0135] Covalently linked monomers can be linked directly (by bonds)
or indirectly (e.g., via a linker). For directly linking the
polypeptide monomers herein, it may be beneficial to modify the
polypeptides herein to enhance dimerization. For example, one or
more amino acid residues of an AspRS polypeptide may be modified by
the addition or substation by one or more cysteines. Methods for
creating amino acid substitutions, such as cysteine substitutions,
or other modifications to facilitate linking, are well known to
those skilled in the art.
[0136] Certain embodiments of the present invention also
contemplate the use of modified AspRS polypeptides, including
modifications that improve desired characteristics of an AspRS
polypeptide, as described herein. Illustrative modifications of
AspRS polypeptides of the invention include, but are not limited
to, chemical and/or enzymatic derivatizations at one or more
constituent amino acids, including side chain modifications,
backbone modifications, and N- and C-terminal modifications
including acetylation, hydroxylation, methylation, amidation, and
the attachment of carbohydrate or lipid moieties, cofactors, and
the like. Exemplary modifications also include pegylation of an
AspRS polypeptide (see, e.g., Veronese and Harris, Advanced Drug
Delivery Reviews 54: 453-456, 2002, herein incorporated by
reference).
[0137] In certain aspects, chemoselective ligation technology may
be utilized to modify truncated AspRS polypeptides of the
invention, such as by attaching polymers in a site-specific and
controlled manner. Such technology typically relies on the
incorporation of chemoselective anchors into the protein backbone
by either chemical or recombinant means and subsequent modification
with a polymer carrying a complementary linker. As a result, the
assembly process and the covalent structure of the resulting
protein-polymer conjugate may be controlled, enabling the rational
optimization of drug properties, such as efficacy and
pharmacokinetic properties (see, e.g., Kochendoerfer, Current
Opinion in Chemical Biology 9:555-560, 2005).
[0138] The AspRS polypeptides described herein may be prepared by
any suitable procedure known to those of skill in the art, such as
by recombinant techniques. For example, AspRS polypeptides may be
prepared by a procedure including the steps of: (a) preparing a
construct comprising a polynucleotide sequence that encodes an
AspRS polypeptide and that is operably linked to a regulatory
element; (b) introducing the construct into a host cell; (c)
culturing the host cell to express the AspRS polypeptide; and (d)
isolating the AspRS polypeptide from the host cell. Recombinant
AspRS polypeptides can be conveniently prepared using standard
protocols as described for example in Sambrook, et al., (1989,
supra), in particular Sections 16 and 17; Ausubel et al., (1994,
supra), in particular Chapters 10 and 16; and Coligan et al.,
Current Protocols in Protein Science (John Wiley & Sons, Inc.
1995-1997), in particular Chapters 1, 5 and 6.
[0139] In addition to recombinant production methods, polypeptides
of the invention, and fragments thereof, may be produced by direct
peptide synthesis using solid-phase techniques (Merrifield, J. Am.
Chem. Soc. 85:2149-2154 (1963)). Protein synthesis may be performed
using manual techniques or by automation. Automated synthesis may
be achieved, for example, using Applied Biosystems 431A Peptide
Synthesizer (Perkin Elmer). Alternatively, various fragments may be
chemically synthesized separately and combined using chemical
methods to produce the desired molecule.
Polynucleotide Compositions
[0140] The present invention also provides isolated polynucleotides
that encode the AspRS polypeptides of the invention, as well as
compositions comprising such polynucleotides. Also included within
the AspRS polynucleotides of the present invention are primers,
probes, antisense oligonucleotides, and RNA interference agents
that comprise all or a portion of the AspRS reference
polynucleotides, which are complementary to all or a portion of
these reference polynucleotides, or which specifically hybridize to
these reference polynucleotides, as described herein.
[0141] As used herein, the terms "DNA" and "polynucleotide" and
"nucleic acid" refer to a DNA molecule that has been isolated free
of total genomic DNA of a particular species. Therefore, a DNA
segment encoding a polypeptide refers to a DNA segment that
contains one or more coding sequences yet is substantially isolated
away from, or purified free from, total genomic DNA of the species
from which the DNA segment is obtained. Included within the terms
"DNA segment" and "polynucleotide" are DNA segments and smaller
fragments of such segments, and also recombinant vectors,
including, for example, plasmids, cosmids, phagemids, phage,
viruses, and the like.
[0142] As will be understood by those skilled in the art, the
polynucleotide sequences of this invention can include genomic
sequences, extra-genomic and plasmid-encoded sequences and smaller
engineered gene segments that express, or may be adapted to
express, proteins, polypeptides, peptides and the like. Such
segments may be naturally isolated, or modified synthetically by
the hand of man.
[0143] As will be recognized by the skilled artisan,
polynucleotides may be single-stranded (coding or antisense) or
double-stranded, and may be DNA (genomic, cDNA or synthetic) or RNA
molecules. Additional coding or non-coding sequences may, but need
not, be present within a polynucleotide of the present invention,
and a polynucleotide may, but need not, be linked to other
molecules and/or support materials.
[0144] Polynucleotides may comprise a native sequence (i.e., an
endogenous sequence that encodes an AspRS or a portion thereof) or
may comprise a variant, or a biological functional equivalent of
such a sequence. Polynucleotide variants may contain one or more
substitutions, additions, deletions and/or insertions, as further
described below, preferably such that the desired activity of the
encoded polypeptide is not substantially diminished relative to the
unmodified polypeptide. The effect on the activity of the encoded
polypeptide may generally be assessed as described herein.
[0145] In additional embodiments, the present invention provides
isolated polynucleotides comprising various lengths of contiguous
stretches of sequence identical to or complementary to an
aspartyl-tRNA synthetase, wherein the isolated polynucleotides
encode an AspRS as described herein. For example, polynucleotides
are provided by this invention that encode at least about 100, 150,
200, 250, 300, 350, 400, 450 or 500, or more, contiguous amino acid
residues of an AspRS polypeptide of the invention, as well as all
intermediate lengths. It will be readily understood that
"intermediate lengths", in this context, means any length between
the quoted values, such as 101, 102, 103, etc.; 151, 152, 153,
etc.; 201, 202, 203, etc.
[0146] The polynucleotides of the present invention, regardless of
the length of the coding sequence itself, may be combined with
other DNA sequences, such as promoters, polyadenylation signals,
additional restriction enzyme sites, multiple cloning sites, other
coding segments, and the like, such that their overall length may
vary considerably. It is therefore contemplated that a
polynucleotide fragment of almost any length may be employed; with
the total length preferably being limited by the ease of
preparation and use in the intended recombinant DNA protocol.
[0147] Moreover, it will be appreciated by those of ordinary skill
in the art that, as a result of the degeneracy of the genetic code,
there are many nucleotide sequences that encode a polypeptide as
described herein. Some of these polynucleotides bear minimal
homology to the nucleotide sequence of any native gene.
Nonetheless, polynucleotides that vary due to differences in codon
usage are specifically contemplated by the present invention, for
example polynucleotides that are optimized for human and/or primate
codon selection. Further, alleles of the genes comprising the
polynucleotide sequences provided herein are within the scope of
the present invention. Alleles are endogenous genes that are
altered as a result of one or more mutations, such as deletions,
additions and/or substitutions of nucleotides. The resulting mRNA
and protein may, but need not, have an altered structure or
function. Alleles may be identified using standard techniques (such
as hybridization, amplification and/or database sequence
comparison).
[0148] Polynucleotides and fusions thereof may be prepared,
manipulated and/or expressed using any of a variety of well
established techniques known and available in the art. For example,
polynucleotide sequences which encode polypeptides of the
invention, or fusion proteins or functional equivalents thereof,
may be used in recombinant DNA molecules to direct expression of an
AspRS polypeptide in appropriate host cells. Due to the inherent
degeneracy of the genetic code, other DNA sequences that encode
substantially the same or a functionally equivalent amino acid
sequence may be produced and these sequences may be used to clone
and express a given polypeptide.
[0149] As will be understood by those of skill in the art, it may
be advantageous in some instances to produce polypeptide-encoding
nucleotide sequences possessing non-naturally occurring codons. For
example, codons preferred by a particular prokaryotic or eukaryotic
host can be selected to increase the rate of protein expression or
to produce a recombinant RNA transcript having desirable
properties, such as a half-life which is longer than that of a
transcript generated from the naturally occurring sequence.
[0150] Moreover, the polynucleotide sequences of the present
invention can be engineered using methods generally known in the
art in order to alter polypeptide encoding sequences for a variety
of reasons, including but not limited to, alterations which modify
the cloning, processing, expression and/or activity of the gene
product.
[0151] In order to express a desired polypeptide, a nucleotide
sequence encoding the polypeptide, or a functional equivalent, may
be inserted into appropriate expression vector, i.e., a vector
which contains the necessary elements for the transcription and
translation of the inserted coding sequence. Methods which are well
known to those skilled in the art may be used to construct
expression vectors containing sequences encoding a polypeptide of
interest and appropriate transcriptional and translational control
elements. These methods include in vitro recombinant DNA
techniques, synthetic techniques, and in vivo genetic
recombination. Such techniques are described in Sambrook et al.,
Molecular Cloning, A Laboratory Manual (1989), and Ausubel et al.,
Current Protocols in Molecular Biology (1989).
[0152] A variety of expression vector/host systems are known and
may be utilized to contain and express polynucleotide sequences.
These include, but are not limited to, microorganisms such as
bacteria transformed with recombinant bacteriophage, plasmid, or
cosmid DNA expression vectors; yeast transformed with yeast
expression vectors; insect cell systems infected with virus
expression vectors (e.g., baculovirus); plant cell systems
transformed with virus expression vectors (e.g., cauliflower mosaic
virus, CaMV; tobacco mosaic virus, TMV) or with bacterial
expression vectors (e.g., Ti or pBR322 plasmids); or animal cell
systems, such as viral-based expression systems.
[0153] The "control elements" or "regulatory sequences" present in
an expression vector are those non-translated regions of the
vector--enhancers, promoters, 5' and 3' untranslated regions--which
interact with host cellular proteins to carry out transcription and
translation. Such elements may vary in their strength and
specificity. Depending on the vector system and host utilized, any
number of suitable transcription and translation elements,
including constitutive and inducible promoters, may be used. For
example, when cloning in bacterial systems, inducible promoters
such as the hybrid lacZ promoter of the PBLUESCRIPT phagemid
(Stratagene, La Jolla, Calif.) or PSPORT1 plasmid (Gibco BRL,
Gaithersburg, Md.) and the like may be used. In mammalian cell
systems, promoters from mammalian genes or from mammalian viruses
are generally preferred. If it is necessary to generate a cell line
that contains multiple copies of the sequence encoding a
polypeptide, vectors based on SV40 or EBV may be advantageously
used with an appropriate selectable marker.
[0154] Specific initiation signals may also be used to achieve more
efficient translation of sequences encoding a polypeptide of
interest. Such signals include the ATG initiation codon and
adjacent sequences. In cases where sequences encoding the
polypeptide, its initiation codon, and upstream sequences are
inserted into the appropriate expression vector, no additional
transcriptional or translational control signals may be needed.
However, in cases where only coding sequence, or a portion thereof,
is inserted, exogenous translational control signals including the
ATG initiation codon should be provided. Furthermore, the
initiation codon should be in the correct reading frame to ensure
translation of the entire insert. Exogenous translational elements
and initiation codons may be of various origins, both natural and
synthetic. The efficiency of expression may be enhanced by the
inclusion of enhancers which are appropriate for the particular
cell system which is used, such as those described in the
literature (Scharf. et al., Results Probl. Cell Differ. 20:125-162
(1994)).
[0155] In addition, a host cell strain may be chosen for its
ability to modulate the expression of the inserted sequences or to
process the expressed protein in the desired fashion. Such
modifications of the polypeptide include, but are not limited to,
acetylation, carboxylation, glycosylation, phosphorylation,
lipidation, and acylation. Post-translational processing which
cleaves a "prepro" form of the protein may also be used to
facilitate correct insertion, folding and/or function. Different
host cells such as CHO, HeLa, MDCK, HEK293, and W138, which have
specific cellular machinery and characteristic mechanisms for such
post-translational activities, may be chosen to ensure the correct
modification and processing of the foreign protein.
[0156] For long-term, high-yield production of recombinant
proteins, stable expression is generally preferred. For example,
cell lines which stably express a polynucleotide of interest may be
transformed using expression vectors which may contain viral
origins of replication and/or endogenous expression elements and a
selectable marker gene on the same or on a separate vector.
Following the introduction of the vector, cells may be allowed to
grow for 1-2 days in an enriched media before they are switched to
selective media. The purpose of the selectable marker is to confer
resistance to selection, and its presence allows growth and
recovery of cells which successfully express the introduced
sequences. Resistant clones of stably transformed cells may be
proliferated using tissue culture techniques appropriate to the
cell type.
[0157] Any number of selection systems may be used to recover
transformed cell lines. These include, but are not limited to, the
herpes simplex virus thymidine kinase (Wigler et al., Cell
11:223-232 (1977)) and adenine phosphoribosyltransferase (Lowy et
al., Cell 22:817-823 (1990)) genes which can be employed in tk- or
aprt- cells, respectively. Also, antimetabolite, antibiotic or
herbicide resistance can be used as the basis for selection; for
example, dhfr which confers resistance to methotrexate (Wigler et
al., Proc. Natl. Acad. Sci. U.S.A. 77:3567-70 (1980)); npt, which
confers resistance to the aminoglycosides, neomycin and G-418
(Colbere-Garapin et al., J. Mol. Biol. 150:1-14 (1981)); and als or
pat, which confer resistance to chlorsulfuron and phosphinotricin
acetyltransferase, respectively (Murry, supra).
[0158] A variety of protocols for detecting and measuring the
expression of polynucleotide-encoded products, using either
polyclonal or monoclonal antibodies specific for the product are
known in the art. Examples include enzyme-linked immunosorbent
assay (ELISA), radioimmunoassay (RIA), and fluorescence activated
cell sorting (FACS). These and other assays are described, among
other places, in Hampton et al., Serological Methods, a Laboratory
Manual (1990) and Maddox et al., J. Exp. Med. 158:1211-1216
(1983).
[0159] A wide variety of labels and conjugation techniques are
known by those skilled in the art and may be used in various
nucleic acid and amino acid assays. Means for producing labeled
hybridization or PCR probes for detecting sequences related to
polynucleotides include oligolabeling, nick translation,
end-labeling or PCR amplification using a labeled nucleotide.
Alternatively, the sequences, or any portions thereof may be cloned
into a vector for the production of an mRNA probe. Such vectors are
known in the art, are commercially available, and may be used to
synthesize RNA probes in vitro by addition of an appropriate RNA
polymerase such as T7, T3, or SP6 and labeled nucleotides. These
procedures may be conducted using a variety of commercially
available kits. Suitable reporter molecules or labels, which may be
used include radionuclides, enzymes, fluorescent, chemiluminescent,
or chromogenic agents as well as substrates, cofactors, inhibitors,
magnetic particles, and the like.
[0160] Host cells transformed with a polynucleotide sequence of
interest may be cultured under conditions suitable for the
expression and recovery of the protein from cell culture. The
protein produced by a recombinant cell may be secreted or contained
intracellularly depending on the sequence and/or the vector used.
As will be understood by those of skill in the art, expression
vectors containing polynucleotides of the invention may be designed
to contain signal sequences which direct secretion of the encoded
polypeptide through a prokaryotic or eukaryotic cell membrane.
Other recombinant constructions may be used to join sequences
encoding a polypeptide of interest to nucleotide sequence encoding
a polypeptide domain which will facilitate purification of soluble
proteins.
[0161] According to another aspect of the invention,
polynucleotides encoding polypeptides of the invention may be
delivered to a subject in vivo, e.g., using gene therapy
techniques. Gene therapy refers generally to the transfer of
heterologous nucleic acids to the certain cells, target cells, of a
mammal, particularly a human, with a disorder or conditions for
which such therapy is sought. The nucleic acid is introduced into
the selected target cells in a manner such that the heterologous
DNA is expressed and a therapeutic product encoded thereby is
produced.
[0162] Various viral vectors that can be utilized for gene therapy
as taught herein include adenovirus, herpes virus, vaccinia,
adeno-associated virus (AAV), or, preferably, an RNA virus such as
a retrovirus. Preferably, the retroviral vector is a derivative of
a murine or avian retrovirus, or is a lentiviral vector. The
preferred retroviral vector is a lentiviral vector. Examples of
retroviral vectors in which a single foreign gene can be inserted
include, but are not limited to: Moloney murine leukemia virus
(MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary
tumor virus (MuMTV), SIV, BIV, HIV and Rous Sarcoma Virus (RSV). A
number of additional retroviral vectors can incorporate multiple
genes. All of these vectors can transfer or incorporate a gene for
a selectable marker so that transduced cells can be identified and
generated. By inserting a zinc finger derived-DNA binding
polypeptide sequence of interest into the viral vector, along with
another gene that encodes the ligand for a receptor on a specific
target cell, for example, the vector may be made target specific.
Retroviral vectors can be made target specific by inserting, for
example, a polynucleotide encoding a protein (dimer). Illustrative
targeting may be accomplished by using an antibody to target the
retroviral vector. Those of skill in the art will know of, or can
readily ascertain without undue experimentation, specific
polynucleotide sequences which can be inserted into the retroviral
genome to allow target specific delivery of the retroviral vector
containing the zinc finger-nucleotide binding protein
polynucleotide.
[0163] Since recombinant retroviruses are defective, they require
assistance in order to produce infectious vector particles. This
assistance can be provided, for example, by using helper cell lines
that contain plasmids encoding all of the structural genes of the
retrovirus under the control of regulatory sequences within the
LTR. These plasmids are missing a nucleotide sequence which enables
the packaging mechanism to recognize an RNA transcript for
encapsulation. Helper cell lines which have deletions of the
packaging signal include but are not limited to PSI.2, PA317 and
PA12, for example. These cell lines produce empty virions, since no
genome is packaged. If a retroviral vector is introduced into such
cells in which the packaging signal is intact, but the structural
genes are replaced by other genes of interest, the vector can be
packaged and vector virion produced. The vector virions produced by
this method can then be used to infect a tissue cell line, such as
NIH 3T3 cells, to produce large quantities of chimeric retroviral
virions.
[0164] "Non-viral" delivery techniques for gene therapy can also be
used including, for example, DNA-ligand complexes,
adenovirus-ligand-DNA complexes, direct injection of DNA,
CaPO.sub.4 precipitation, gene gun techniques, electroporation,
liposomes, lipofection, and the like. Any of these methods are
widely available to one skilled in the art and would be suitable
for use in the present invention. Other suitable methods are
available to one skilled in the art, and it is to be understood
that the present invention can be accomplished using any of the
available methods of transfection. Lipofection can be accomplished
by encapsulating an isolated DNA molecule within a liposomal
particle and contacting the liposomal particle with the cell
membrane of the target cell. Liposomes are self-assembling,
colloidal particles in which a lipid bilayer, composed of
amphiphilic molecules such as phosphatidyl serine or phosphatidyl
choline, encapsulates a portion of the surrounding media such that
the lipid bilayer surrounds a hydrophilic interior. Unilammellar or
multilammellar liposomes can be constructed such that the interior
contains a desired chemical, drug, or, as in the instant invention,
an isolated DNA molecule.
[0165] Certain embodiments include polynucleotides that hybridize
to a reference AspRS polynucleotide sequence, or to their
complements, under stringency conditions described below. As used
herein, the term "hybridizes under low stringency, medium
stringency, high stringency, or very high stringency conditions"
describes conditions for hybridization and washing. Guidance for
performing hybridization reactions can be found in Ausubel et al.,
(1998, supra), Sections 6.3.1-6.3.6. Aqueous and non-aqueous
methods are described in that reference and either can be used.
[0166] Reference herein to low stringency conditions include and
encompass from at least about 1% v/v to at least about 15% v/v
formamide and from at least about 1 M to at least about 2 M salt
for hybridization at 42.degree. C., and at least about 1 M to at
least about 2 M salt for washing at 42.degree. C. Low stringency
conditions also may include 1% Bovine Serum Albumin (BSA), 1 mM
EDTA, 0.5 M NaHPO.sub.4 (pH 7.2), 7% SDS for hybridization at
65.degree. C., and (i) 2.times.SSC, 0.1% SDS; or (ii) 0.5% BSA, 1
mM EDTA, 40 mM NaHPO.sub.4 (pH 7.2), 5% SDS for washing at room
temperature. One embodiment of low stringency conditions includes
hybridization in 6.times. sodium chloride/sodium citrate (SSC) at
about 45.degree. C., followed by two washes in 0.2.times.SSC, 0.1%
SDS at least at 50.degree. C. (the temperature of the washes can be
increased to 55.degree. C. for low stringency conditions).
[0167] Medium stringency conditions include and encompass from at
least about 16% v/v to at least about 30% v/v formamide and from at
least about 0.5 M to at least about 0.9 M salt for hybridization at
42.degree. C., and at least about 0.1 M to at least about 0.2 M
salt for washing at 55.degree. C. Medium stringency conditions also
may include 1% Bovine Serum Albumin (BSA), 1 mM EDTA, 0.5 M
NaHPO.sub.4 (pH 7.2), 7% SDS for hybridization at 65.degree. C.,
and (i) 2.times.SSC, 0.1% SDS; or (ii) 0.5% BSA, 1 mM EDTA, 40 mM
NaHPO.sub.4 (pH 7.2), 5% SDS for washing at 60-65.degree. C. One
embodiment of medium stringency conditions includes hybridizing in
6.times.SSC at about 45.degree. C., followed by one or more washes
in 0.2.times.SSC, 0.1% SDS at 60.degree. C. High stringency
conditions include and encompass from at least about 31% v/v to at
least about 50% v/v formamide and from about 0.01 M to about 0.15 M
salt for hybridization at 42.degree. C., and about 0.01 M to about
0.02 M salt for washing at 55.degree. C.
[0168] High stringency conditions also may include 1% BSA, 1 mM
EDTA, 0.5 M NaHPO.sub.4 (pH 7.2), 7% SDS for hybridization at
65.degree. C., and (i) 0.2.times.SSC, 0.1% SDS; or (ii) 0.5% BSA, 1
mM EDTA, 40 mM NaHPO.sub.4 (pH 7.2), 1% SDS for washing at a
temperature in excess of 65.degree. C. One embodiment of high
stringency conditions includes hybridizing in 6.times.SSC at about
45.degree. C., followed by one or more washes in 0.2.times.SSC,
0.1% SDS at 65.degree. C. One embodiment of very high stringency
conditions includes hybridizing in 0.5 M sodium phosphate, 7% SDS
at 65.degree. C., followed by one or more washes in 0.2.times.SSC,
1% SDS at 65.degree. C.
[0169] Other stringency conditions are well known in the art and a
skilled artisan will recognize that various factors can be
manipulated to optimize the specificity of the hybridization.
Optimization of the stringency of the final washes can serve to
ensure a high degree of hybridization. For detailed examples, see
Ausubel et al., supra at pages 2.10.1 to 2.10.16 and Sambrook et
al. (1989, supra) at sections 1.101 to 1.104.
[0170] While stringent washes are typically carried out at
temperatures from about 42.degree. C. to 68.degree. C., one skilled
in the art will appreciate that other temperatures may be suitable
for stringent conditions. Maximum hybridization rate typically
occurs at about 20.degree. C. to 25.degree. C. below the T.sub.m
for formation of a DNA-DNA hybrid. It is well known in the art that
the T.sub.m is the melting temperature, or temperature at which two
complementary polynucleotide sequences dissociate. Methods for
estimating T.sub.m are well known in the art (see Ausubel et al.,
supra at page 2.10.8).
[0171] In general, the T.sub.m of a perfectly matched duplex of DNA
may be predicted as an approximation by the formula:
T.sub.m=81.5+16.6 (log.sub.10 M)+0.41 (% G+C)-0.63 (%
formamide)-(600/length) wherein: M is the concentration of
Na.sup.+, preferably in the range of 0.01 molar to 0.4 molar; % G+C
is the sum of guanosine and cytosine bases as a percentage of the
total number of bases, within the range between 30% and 75% G+C; %
formamide is the percent formamide concentration by volume; length
is the number of base pairs in the DNA duplex. The T.sub.m of a
duplex DNA decreases by approximately 1.degree. C. with every
increase of 1% in the number of randomly mismatched base pairs.
Washing is generally carried out at T.sub.m-15.degree. C. for high
stringency, or T.sub.m-30.degree. C. for moderate stringency.
[0172] In one example of a hybridization procedure, a membrane
(e.g., a nitrocellulose membrane or a nylon membrane) containing
immobilized DNA is hybridized overnight at 42.degree. C. in a
hybridization buffer (50% deionized formamide, 5.times.SSC,
5.times.Denhardt's solution (0.1% ficoll, 0.1% polyvinylpyrollidone
and 0.1% bovine serum albumin), 0.1% SDS and 200 mg/mL denatured
salmon sperm DNA) containing a labeled probe. The membrane is then
subjected to two sequential medium stringency washes (i.e.,
2.times.SSC, 0.1% SDS for 15 min at 45.degree. C., followed by
2.times.SSC, 0.1% SDS for 15 min at 50.degree. C.), followed by two
sequential higher stringency washes (i.e., 0.2.times.SSC, 0.1% SDS
for 12 min at 55.degree. C. followed by 0.2.times.SSC and 0.1% SDS
solution for 12 min at 65-68.degree. C.
[0173] Embodiments of the present invention also include
oligonucleotides, whether for detection, amplification, antisense
therapies, or other purpose. For these and related purposes, the
term "oligonucleotide" or "oligo" or "oligomer" is intended to
encompass a singular "oligonucleotide" as well as plural
"oligonucleotides," and refers to any polymer of two or more of
nucleotides, nucleosides, nucleobases or related compounds used as
a reagent in the amplification methods of the present invention, as
well as subsequent detection methods. The oligonucleotide may be
DNA and/or RNA and/or analogs thereof.
[0174] The term oligonucleotide does not necessarily denote any
particular function to the reagent, rather, it is used generically
to cover all such reagents described herein. An oligonucleotide may
serve various different functions, e.g., it may function as a
primer if it is capable of hybridizing to a complementary strand
and can further be extended in the presence of a nucleic acid
polymerase, it may provide a promoter if it contains a sequence
recognized by an RNA polymerase and allows for transcription, and
it may function to prevent hybridization or impede primer extension
if appropriately situated and/or modified. An oligonucleotide may
also function as a probe, or an antisense agent. An oligonucleotide
can be virtually any length, limited only by its specific function,
e.g., in an amplification reaction, in detecting an amplification
product of the amplification reaction, or in an antisense or RNA
interference application. Any of the oligonucleotides described
herein can be used as a primer, a probe, an antisense oligomer, or
an RNA interference agent.
[0175] The term "primer" as used herein refers to a single-stranded
oligonucleotide capable of acting as a point of initiation for
template-directed DNA synthesis under suitable conditions defined,
for example, by buffer and temperature, in the presence of four
different nucleoside triphosphates and an agent for polymerization,
such as a DNA or RNA polymerase or reverse transcriptase. The
length of the primer, in any given case, depends on, for example,
the intended use of the primer, and generally ranges from about 15
to 30 nucleotides, although shorter and longer primers may be used.
Short primer molecules generally require cooler temperatures to
form sufficiently stable hybrid complexes with the template. A
primer need not reflect the exact sequence of the template but must
be sufficiently complementary to hybridize with such template. The
primer site is the area of the template to which a primer
hybridizes. The primer pair is a set of primers including a 5'
upstream primer that hybridizes with the 5' end of the sequence to
be amplified and a 3' downstream primer that hybridizes with the
complement of the 3' end of the sequence to be amplified.
[0176] The term "probe" as used herein refers to a
surface-immobilized molecule that can be recognized by a particular
target. See, e.g., U.S. Pat. No. 6,582,908 for an example of arrays
having all possible combinations of probes with 10, 12, and more
bases. Probes and primers as used herein typically comprise at
least 10-15 contiguous nucleotides of a known sequence. In order to
enhance specificity, longer probes and primers may also be
employed, such as probes and primers that comprise at least 20, 25,
30, 40, 50, 60, 70, 80, 90, 100, or at least 150 nucleotides of an
AspRS reference sequence or its complement. Probes and primers may
be considerably longer than these examples, and it is understood
that any length supported by the knowledge in the art and the
specification, including the tables, figures, and Sequence Listing,
may be used.
[0177] Methods for preparing and using probes and primers are
described in the references, for example Sambrook, J. et al. (1989)
Molecular Cloning: A Laboratory Manual, 2.sup.nd ed., vol. 1-3,
Cold Spring Harbor Press, Plainview N.Y.; Ausubel, F. M. et al.
(1987) Current Protocols in Molecular Biology, Greene Publ. Assoc.
& Wiley-Intersciences, New York N.Y.; Innis, M. et al. (1990)
PCR Protocols. A Guide to Methods and Applications, Academic Press,
San Diego Calif. PCR primer pairs can be derived from a known
sequence, for example, by using computer programs intended for that
purpose such as Primer (Version 0.5, 1991, Whitehead Institute for
Biomedical Research, Cambridge Mass.).
[0178] Oligonucleotides for use as primers or probes may be
selected using software known in the art. For example, OLIGO 4.06
software is useful for the selection of PCR primer pairs of up to
100 nucleotides each, and for the analysis of oligonucleotides and
larger polynucleotides of up to 5,000 nucleotides from an input
polynucleotide sequence of up to 32 kilobases. The Primer3 primer
selection program (available to the public from the Whitehead
Institute/MIT Center for Genome Research, Cambridge Mass.) allows
the user to input a "mispriming library," in which sequences to
avoid as primer binding sites are user-specified. The
oligonucleotides and polynucleotide fragments identified by any of
the above selection methods are useful in hybridization
technologies, for example, as PCR or sequencing primers, microarray
elements, or specific probes to identify fully or partially
complementary polynucleotides in a sample of nucleic acids. Methods
of oligonucleotide selection are not limited to those described
herein.
[0179] The terms "antisense oligomer" or "antisense compound" or
"antisense oligonucleotide" are used interchangeably and refer to a
sequence of cyclic subunits, each bearing a base-pairing moiety,
linked by intersubunit linkages that allow the base-pairing
moieties to hybridize to a target sequence in a nucleic acid
(typically an RNA) by Watson-Crick base pairing, to form a nucleic
acid:oligomer heteroduplex within the target sequence, and
typically thereby prevent translation of that RNA. Also included
are methods of use thereof to modulate expression of a selected
AspRS transcript, such as a splice variant or proteolytic fragment,
and/or its corresponding polyeptide.
[0180] Antisense oligonucleotides may contain between about 8 and
40 subunits, typically about 8-25 subunits, and preferably about 12
to 25 subunits. In certain embodiments, oligonucleotides may have
exact sequence complementarity to the target sequence or near
complementarity, as defined below. In certain embodiments, the
degree of complementarity between the target and antisense
targeting sequence is sufficient to form a stable duplex. The
region of complementarity of the antisense oligomers with the
target RNA sequence may be as short as 8-11 bases, but is
preferably 12-15 bases or more, e.g., 12-20 bases, or 12-25 bases,
including all integers in between these ranges. An antisense
oligomer of about 14-15 bases is generally long enough to have a
unique complementary sequence in targeting the selected AspRS
transcript.
[0181] In certain embodiments, antisense oligomers as long as 40
bases may be suitable, where at least a minimum number of bases,
e.g., 10-12 bases, are complementary to the target sequence. In
general, however, facilitated or active uptake in cells is
optimized at oligomer lengths less than about 30. For certain
oligomers, described further below, an optimum balance of binding
stability and uptake generally occurs at lengths of 18-25 bases.
Included are antisense oligomers (e.g., PNAs, LNAs, 2'-OMe, MOE,
morpholinos) that consist of about 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,
35, 36, 37, 38, 39, or 40 bases, in which at least about 6, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,
27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40
contiguous or non-contiguous bases are complementary to their AspRS
target sequence, or variants thereof.
[0182] In certain embodiments, antisense oligomers may be 100%
complementary to the AspRS nucleic acid target sequence, or it may
include mismatches, e.g., to accommodate variants, as long as a
heteroduplex formed between the oligomer and AspRS nucleic acid
target sequence is sufficiently stable to withstand the action of
cellular nucleases and other modes of degradation which may occur
in vivo. Oligomer backbones which are less susceptible to cleavage
by nucleases are discussed below. Mismatches, if present, are less
destabilizing toward the end regions of the hybrid duplex than in
the middle. The number of mismatches allowed will depend on the
length of the oligomer, the percentage of G:C base pairs in the
duplex, and the position of the mismatch(es) in the duplex,
according to well understood principles of duplex stability.
Although such an antisense oligomer is not necessarily 100%
complementary to the AspRS nucleic acid target sequence, it is
effective to stably and specifically bind to the target sequence,
such that a biological activity of the nucleic acid target, e.g.,
expression of AspRS protein(s), is modulated.
[0183] The stability of the duplex formed between an oligomer and a
target sequence is a function of the binding Tm and the
susceptibility of the duplex to cellular enzymatic cleavage. The Tm
of an antisense oligonucleotide with respect to
complementary-sequence RNA may be measured by conventional methods,
such as those described by Hames et al., Nucleic Acid
Hybridization, IRL Press, 1985, pp. 107-108 or as described in
Miyada C. G. and Wallace R. B., 1987, Oligonucleotide hybridization
techniques, Methods Enzymol. Vol. 154 pp. 94-107. In certain
embodiments, antisense oligomer may have a binding Tm, with respect
to a complementary-sequence RNA, of greater than body temperature
and preferably greater than 50.degree. C. Tm's in the range
60-80.degree. C. or greater are preferred. According to well known
principles, the Tm of an oligomer compound, with respect to a
complementary-based RNA hybrid, can be increased by increasing the
ratio of C:G paired bases in the duplex, and/or by increasing the
length (in base pairs) of the heteroduplex.
[0184] Antisense oligomers can be designed to block or inhibit
translation of mRNA or to inhibit natural pre-mRNA splice
processing, or induce degradation of targeted mRNAs, and may be
said to be "directed to" or "targeted against" a target sequence
with which it hybridizes. In certain embodiments, the target
sequence may include any coding or non-coding sequence of an AspRS
mRNA transcript, and may thus by within an exon or within an
intron. In certain embodiments, the target sequence is relatively
unique or exceptional among AspRS s and is selective for reducing
expression of a selected AspRS proteolytic fragment or splice
variant. In certain embodiments, the target site includes a 3' or
5' splice site of a pre-processed mRNA, or a branch point. The
target sequence for a splice site may include an mRNA sequence
having its 5' end 1 to about 25 to about 50 base pairs downstream
of a splice acceptor junction or upstream of a splice donor
junction in a preprocessed mRNA. An oligomer is more generally said
to be "targeted against" a biologically relevant target, such as
reference AspRS polynucleotide, when it is targeted against the
nucleic acid of the target in the manner described herein.
[0185] A "subunit" of an oligonucleotide refers to one nucleotide
(or nucleotide analog) unit. The term may refer to the nucleotide
unit with or without the attached intersubunit linkage, although,
when referring to a "charged subunit", the charge typically resides
within the intersubunit linkage (e.g., a phosphate or
phosphorothioate linkage or a cationic linkage).
[0186] The cyclic subunits of an oligonucleotide may be based on
ribose or another pentose sugar or, in certain embodiments,
alternate or modified groups. Examples of modified oligonucleotide
backbones include, without limitation, phosphorothioates, chiral
phosphorothioates, phosphorodithioates, phosphotriesters,
aminoalkylphosphotriesters, methyl and other alkyl phosphonates
including 3'-alkylene phosphonates and chiral phosphonates,
phosphinates, phosphoramidates including 3'-amino phosphoramidate
and aminoalkylphosphoramidates, thionophosphoramidates,
thionoalkylphosphonates, thionoalkylphosphotriesters, and
boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs
of these, and those having inverted polarity wherein the adjacent
pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to
5'-2'. Also contemplated are peptide nucleic acids (PNAs), locked
nucleic acids (LNAs), 2'-O-Methyl oligonucleotides (2'-OMe),
2'-methoxyethoxy oligonucleotides (MOE), morpholinos, among other
oligonucleotides known in the art.
[0187] The purine or pyrimidine base pairing moiety is typically
adenine, cytosine, guanine, uracil, thymine or inosine. Also
included are bases such as pyridin-4-one, pyridin-2-one, phenyl,
pseudouracil, 2,4,6-trime115thoxy benzene, 3-methyl uracil,
dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidines (e.g.,
5-methylcytidine), 5-alkyluridines (e.g., ribothymidine),
5-halouridine (e.g., 5-bromouridine) or 6-azapyrimidines or
6-alkylpyrimidines (e.g. 6-methyluridine), propyne, quesosine,
2-thiouridine, 4-thiouridine, wybutosine, wybutoxosine,
4-acetyltidine, 5-(carboxyhydroxymethyl)uridine,
5''-carboxymethylaminomethyl-2-thiouridine,
5-carboxymethylaminomethyluridine, .beta.-D-galactosylqueosine,
1-methyladenosine, 1-methylinosine, 2,2-dimethylguanosine,
3-methylcytidine, 2-methyladenosine, 2-methylguanosine,
N6-methyladenosine, 7-methylguanosine,
5-methoxyaminomethyl-2-thiouridine, 5-methylaminomethyluridine,
5-methylcarbonylmethyluridine, 5-methyloxyuridine,
5-methyl-2-thiouridine, 2-methylthio-N6-isopentenyladenosine,
.beta.-D-mannosylqueosine, uridine-5-oxyacetic acid,
2-thiocytidine, threonine derivatives and others (Burgin et al.,
1996, Biochemistry, 35, 14090; Uhlman & Peyman, supra). By
"modified bases" in this aspect is meant nucleotide bases other
than adenine (A), guanine (G), cytosine (C), thymine (T), and
uracil (U), as illustrated above; such bases can be used at any
position in the antisense molecule. Persons skilled in the art will
appreciate that depending on the uses of the oligomers, Ts and Us
are interchangeable. For instance, with other antisense chemistries
such as 2'-O-methyl antisense oligonucleotides that are more
RNA-like, the T bases may be shown as U.
[0188] An oligonucleotide is typically complementary to a target
sequence, such as a target DNA or RNA. The terms "complementary"
and "complementarity" refer to polynucleotides (i.e., a sequence of
nucleotides) related by the base-pairing rules. For example, the
sequence "A-G-T," is complementary to the sequence "T-C-A."
Complementarity may be "partial," in which only some of the nucleic
acids' bases are matched according to the base pairing rules. Or,
there may be "complete" or "total" complementarity (100%) between
the nucleic acids. The degree of complementarity between nucleic
acid strands has significant effects on the efficiency and strength
of hybridization between nucleic acid strands. While perfect
complementarity is often desired, some embodiments can include one
or more but preferably 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10,
9, 8, 7, 6, 5, 4, 3, 2, or 1 mismatches with respect to the target
sequence. Variations at any location within the oligomer are
included. In certain embodiments, variations in sequence near the
termini of an oligomer are generally preferable to variations in
the interior, and if present are typically within about 10, 9, 8,
7, 6, 5, 4, 3, 2, or 1 nucleotides of the 5' and/or 3'
terminus.
[0189] The term "target sequence" refers to a portion of the target
RNA against which the oligonucleotide is directed, that is, the
sequence to which the oligonucleotide will hybridize by
Watson-Crick base pairing of a complementary sequence. In certain
embodiments, the target sequence may be a contiguous region of an
AspRS mRNA (e.g., a unique splice junction of an AspRS mRNA), or
may be composed of non-contiguous regions of the mRNA.
[0190] The term "targeting sequence" or in certain embodiments
"antisense targeting sequence" refers to the sequence in an
oligonucleotide that is complementary (meaning, in addition,
substantially complementary) to the target sequence in the DNA or
RNA target molecule. The entire sequence, or only a portion, of the
antisense compound may be complementary to the target sequence. For
example, in an oligonucleotide having 20-30 bases, about 6, 7, 8,
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,
26, 27, 28, or 29 may be targeting sequences that are complementary
to the target region. Typically, the targeting sequence is formed
of contiguous bases, but may alternatively be formed of
non-contiguous sequences that when placed together, e.g., from
opposite ends of the oligonucleotide, constitute sequence that
spans the target sequence.
[0191] Target and targeting sequences are described as
"complementary" to one another when hybridization occurs in an
antiparallel configuration. A targeting sequence may have "near" or
"substantial" complementarity to the target sequence and still
function for the purpose of the present invention, that is, it may
still be functionally "complementary."
[0192] An oligonucleotide "specifically hybridizes" to a target
polynucleotide if the oligomer hybridizes to a target (e.g., an
AspRS reference polynucleotide or its complement) under
physiological conditions, with a Tm substantially greater than
45.degree. C., preferably at least 50.degree. C., and typically
60.degree. C.-80.degree. C. or higher. Such hybridization
preferably corresponds to stringent hybridization conditions. At a
given ionic strength and pH, the Tm is the temperature at which 50%
of a target sequence hybridizes to a complementary polynucleotide.
Again, such hybridization may occur with "near" or "substantial"
complementarity of the antisense oligomer to the target sequence,
as well as with exact complementarity.
[0193] The terms specifically binds or specifically hybridizes
refer generally to an oligonucleotide probe or polynucleotide
sequence that not only binds to its intended target gene sequence
in a sample under selected hybridization conditions, but does not
bind significantly to other target sequences in the sample, and
thereby discriminates between its intended target and all other
targets in the target pool. A probe that specifically hybridizes to
its intended target sequence may also detect concentration
differences under the selected hybridization conditions, as
described herein.
[0194] As noted above, certain oligonucleotides provided herein
include peptide nucleic acids (PNAs). Also included are "locked
nucleic acid" subunits (LNAs). The structures of LNAs are known in
the art: for example, Wengel, et al., Chemical Communications
(1998) 455; Tetrahedron (1998) 54, 3607, and Accounts of Chem.
Research (1999) 32, 301); Obika, et al., Tetrahedron Letters (1997)
38, 8735; (1998) 39, 5401, and Bioorganic Medicinal Chemistry
(2008) 16, 9230. Certain oligonucleotides may comprise
morpholino-based subunits bearing base-pairing moieties, joined by
uncharged or substantially uncharged linkages. The terms
"morpholino oligomer" or "PMO" (phosphoramidate- or
phosphorodiamidate morpholino oligomer) refer to an oligonucleotide
analog composed of morpholino subunit structures, where (i) the
structures are linked together by phosphorus-containing linkages,
one to three atoms long, preferably two atoms long, and preferably
uncharged or cationic, joining the morpholino nitrogen of one
subunit to a 5' exocyclic carbon of an adjacent subunit, and (ii)
each morpholino ring bears a purine or pyrimidine or an equivalent
base-pairing moiety effective to bind, by base specific hydrogen
bonding, to a base in a polynucleotide.
[0195] In certain embodiments, oligonucleotides can be prepared by
stepwise solid-phase synthesis, employing methods detailed in the
references cited above, and below with respect to the synthesis of
oligonucleotides having a mixture or uncharged and cationic
backbone linkages. In some cases, it may be desirable to add
additional chemical moieties to the oligonucleotide, e.g., to
enhance pharmacokinetics or to facilitate capture or detection of
the compound. Such a moiety may be covalently attached, typically
to a terminus of the oligomer, according to standard synthetic
methods. For example, addition of a polyethylene glycol moiety or
other hydrophilic polymer, e.g., one having 10-100 monomeric
subunits, may be useful in enhancing solubility. One or more
charged groups, e.g., anionic charged groups such as an organic
acid, may enhance cell uptake.
[0196] A variety of detectable molecules may be used to render an
oligonucleotide detectable, such as a radioisotopes, fluorochromes,
dyes, enzymes, nanoparticles, chemiluminescent markers, biotin, or
other monomer known in the art that can be detected directly (e.g.,
by light emission) or indirectly (e.g., by binding of a
fluorescently-labeled antibody).
[0197] Certain embodiments relate to RNA interference (RNAi) agents
that target one or more mRNA transcripts of an AspRS reference
polynucleotide, including fragments and variants thereof. Also
included are methods of use thereof to modulate the levels of a
selected AspRS transcript, such as an AspRS splice variant or
proteolytic fragment.
[0198] The term "double-stranded" means two separate nucleic acid
strands comprising a region in which at least a portion of the
strands are sufficiently complementary to hydrogen bond and form a
duplex structure. The term "duplex" or "duplex structure" refers to
the region of a double stranded molecule wherein the two separate
strands are substantially complementary, and thus hybridize to each
other. "dsRNA" refers to a ribonucleic acid molecule having a
duplex structure comprising two complementary and anti-parallel
nucleic acid strands (i.e., the sense and antisense strands). Not
all nucleotides of a dsRNA must exhibit Watson-Crick base pairs;
the two RNA strands may be substantially complementary. The RNA
strands may have the same or a different number of nucleotides.
[0199] The strands of a dsRNA are sufficiently complementary to
hybridize to form a duplex structure. In certain embodiments, the
complementary RNA strand may be less than 30 nucleotides, less than
25 nucleotides in length, or even 19 to 24 nucleotides in length.
In certain aspects, the complementary nucleotide sequence may be
20-23 nucleotides in length, or 22 nucleotides in length.
[0200] In certain embodiments, at least one of the RNA strands
comprises a nucleotide overhang of 1 to 4 nucleotides in length. In
other embodiments, one or both of the strands are blunt-ended. In
certain embodiments, the dsRNA may further comprise at least one
chemically modified nucleotide.
[0201] Certain embodiments of the present invention may comprise
microRNAs. Micro-RNAs represent a large group of small RNAs
produced naturally in organisms, some of which regulate the
expression of target genes. Micro-RNAs are formed from an
approximately 70 nucleotide single-stranded hairpin precursor
transcript by Dicer. (V. Ambros et al. Current Biology 13:807,
2003).
[0202] Certain embodiments may also employ short-interfering RNAs
(siRNA). Each strand of an siRNA agent can be equal to or less than
35, 30, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, or 15 nucleotides
in length. The strand is preferably at least 19 nucleotides in
length. For example, each strand can be between 21 and 25
nucleotides in length. Preferred siRNA agents have a duplex region
of 17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs, and one
or more overhangs, preferably one or two 3' overhangs, of 2-3
nucleotides.
[0203] A "single strand RNAi agent" as used herein, is an RNAi
agent which is made up of a single molecule. It may include a
duplexed region, formed by intra-strand pairing, e.g., it may be,
or include, a hairpin or pan-handle structure. A single strand RNAi
agent is at least 14, and more preferably at least 15, 20, 25, 29,
35, 40, or 50 nucleotides in length. It is preferably less than
200, 100, or 60 nucleotides in length.
[0204] Hairpin RNAi modulating agents may have a duplex region
equal to or at least 17, 18, 19, 29, 21, 22, 23, 24, or 25
nucleotide pairs. The duplex region may preferably be equal to or
less than 200, 100, or 50, in length. Certain ranges for the duplex
region are 15-30, 17 to 23, 19 to 23, and 19 to 21 nucleotides
pairs in length. The hairpin may have a single strand overhang or
terminal unpaired region, preferably the 3', and preferably of the
antisense side of the hairpin. In certain embodiments, overhangs
are 2-3 nucleotides in length.
[0205] The present invention further encompasses oligonucleotides
employing ribozymes. Also included are vector delivery systems that
are capable of expressing the AspRS-targeting sequences described
herein. Included are vectors that express siRNA or other
duplex-forming RNA interference molecules. Exemplary delivery
systems may include viral vector systems (i.e., viral-mediated
transduction) including, but not limited to, retroviral (e.g.,
lentiviral) vectors, adenoviral vectors, adeno-associated viral
vectors, and herpes viral vectors, among others known in the
art.
[0206] Oligonucleotides and RNAi agents that target one or more
portions of an AspRS polynucleotide reference sequence or its
complement may be used in any of the therapeutic, diagnostic, or
drug screening methods described herein and apparent to persons
skilled in the art.
Antibody Compositions, Fragments thereof and Other Binding
Agents
[0207] According to another aspect, the present invention further
provides binding agents, such as antibodies, antigen-binding
fragments thereof, soluble receptors, small molecules, aptamers
etc., that exhibit binding specificity for a polypeptide disclosed
herein, or to a portion, variant or derivative thereof, and methods
of using same. Preferably, such binding agents are effective for
modulating one or more of the non-canonical activities mediated by
an AspRS polypeptide of the invention. In certain embodiments, for
example, the binding agent is one that binds to an AspRS
polypeptide of the invention and inhibits its ability to bind to
one or more of its cellular binding partners. Accordingly, such
binding agents may be used to treat or prevent diseases, disorders
or other conditions that are mediated by an AspRS polypeptide of
the invention by antagonizing it activity.
[0208] An antibody, or antigen-binding fragment thereof, is said to
"specifically bind," "immunologically bind," and/or is
"immunologically reactive" to a polypeptide of the invention if it
reacts at a detectable level (within, for example, an ELISA assay)
with the polypeptide, and does not react detectably with unrelated
polypeptides under similar conditions.
[0209] Immunological binding, as used in this context, generally
refers to the non-covalent interactions of the type which occur
between an immunoglobulin molecule and an antigen for which the
immunoglobulin is specific. The strength, or affinity of
immunological binding interactions can be expressed in terms of the
dissociation constant (K.sub.d) of the interaction, wherein a
smaller K.sub.d represents a greater affinity. Immunological
binding properties of selected polypeptides can be quantified using
methods well known in the art. One such method entails measuring
the rates of antigen-binding site/antigen complex formation and
dissociation, wherein those rates depend on the concentrations of
the complex partners, the affinity of the interaction, and on
geometric parameters that equally influence the rate in both
directions. Thus, both the "on rate constant" (K.sub.on) and the
"off rate constant" (K.sub.off) can be determined by calculation of
the concentrations and the actual rates of association and
dissociation. The ratio of K.sub.off/K.sub.on enables cancellation
of all parameters not related to affinity, and is thus equal to the
dissociation constant K.sub.d. See, e.g., Davies et al. (1990)
Annual Rev. Biochem. 59:439-473.
[0210] An "antigen-binding site," or "binding portion" of an
antibody, refers to the part of the immunoglobulin molecule that
participates in antigen binding. The antigen binding site is formed
by amino acid residues of the N-terminal variable ("V") regions of
the heavy ("H") and light ("L") chains. Three highly divergent
stretches within the V regions of the heavy and light chains are
referred to as "hypervariable regions" which are interposed between
more conserved flanking stretches known as "framework regions," or
"FRs". Thus the term "FR" refers to amino acid sequences which are
naturally found between and adjacent to hypervariable regions in
immunoglobulins. In an antibody molecule, the three hypervariable
regions of a light chain and the three hypervariable regions of a
heavy chain are disposed relative to each other in three
dimensional space to form an antigen-binding surface. The
antigen-binding surface is complementary to the three-dimensional
surface of a bound antigen, and the three hypervariable regions of
each of the heavy and light chains are referred to as
"complementarity-determining regions," or "CDRs."
[0211] A binding agent may be, for example, a ribosome, with or
without a peptide component, an RNA molecule or a polypeptide. In a
preferred embodiment, a binding agent is an antibody or an
antigen-binding fragment thereof. Antibodies may be prepared by any
of a variety of techniques known to those of ordinary skill in the
art. See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual,
Cold Spring Harbor Laboratory, 1988. Monoclonal antibodies specific
for a polypeptide of interest may be prepared, for example, using
the technique of Kohler and Milstein, Eur. J. Immunol. 6:511-519,
1976, and improvements thereto. The polypeptides of this invention
may be used in the purification process in, for example, an
affinity chromatography step.
[0212] An "Fv" fragment can be produced by preferential proteolytic
cleavage of an IgM, and on rare occasions IgG or IgA immunoglobulin
molecule. Fv fragments are, however, more commonly derived using
recombinant techniques known in the art. The Fv fragment includes a
non-covalent V.sub.H::V.sub.L heterodimer including an
antigen-binding site which retains much of the antigen recognition
and binding capabilities of the native antibody molecule. Inbar et
al. (1972) Proc. Nat. Acad. Sci. USA 69:2659-2662; Hochman et al.
(1976) Biochem 15:2706-2710; and Ehrlich et al. (1980) Biochem
19:4091-4096.
[0213] A single chain Fv ("sFv") polypeptide is a covalently linked
V.sub.H::V.sub.L heterodimer which is expressed from a gene fusion
including V.sub.H- and V.sub.L-encoding genes linked by a
peptide-encoding linker. Huston et al. (1988) Proc. Nat. Acad. Sci.
USA 85(16):5879-5883. A number of methods have been described to
discern chemical structures for converting the naturally
aggregated--but chemically separated--light and heavy polypeptide
chains from an antibody V region into an sFv molecule which will
fold into a three dimensional structure substantially similar to
the structure of an antigen-binding site. See, e.g., U.S. Pat. Nos.
5,091,513 and 5,132,405, to Huston et al.; and U.S. Pat. No.
4,946,778, to Ladner et al.
[0214] Each of the above-described molecules includes a heavy chain
and a light chain CDR set, respectively interposed between a heavy
chain and a light chain FR set which provide support to the CDRS
and define the spatial relationship of the CDRs relative to each
other. As used herein, the term "CDR set" refers to the three
hypervariable regions of a heavy or light chain V region.
Proceeding from the N-terminus of a heavy or light chain, these
regions are denoted as "CDR1," "CDR2," and "CDR3" respectively. An
antigen-binding site, therefore, includes six CDRs, comprising the
CDR set from each of a heavy and a light chain V region. A
polypeptide comprising a single CDR, (e.g., a CDR1, CDR2 or CDR3)
is referred to herein as a "molecular recognition unit."
Crystallographic analysis of a number of antigen-antibody complexes
has demonstrated that the amino acid residues of CDRs form
extensive contact with bound antigen, wherein the most extensive
antigen contact is with the heavy chain CDR3. Thus, the molecular
recognition units are primarily responsible for the specificity of
an antigen-binding site.
[0215] As used herein, the term "FR set" refers to the four
flanking amino acid sequences which frame the CDRs of a CDR set of
a heavy or light chain V region. Some FR residues may contact bound
antigen; however, FRs are primarily responsible for folding the V
region into the antigen-binding site, particularly the FR residues
directly adjacent to the CDRs. Within FRs, certain amino residues
and certain structural features are very highly conserved. In this
regard, all V region sequences contain an internal disulfide loop
of around 90 amino acid residues. When the V regions fold into a
binding-site, the CDRs are displayed as projecting loop motifs
which form an antigen-binding surface. It is generally recognized
that there are conserved structural regions of FRs which influence
the folded shape of the CDR loops into certain "canonical"
structures--regardless of the precise CDR amino acid sequence.
Further, certain FR residues are known to participate in
non-covalent interdomain contacts which stabilize the interaction
of the antibody heavy and light chains.
[0216] A number of "humanized" antibody molecules comprising an
antigen-binding site derived from a non-human immunoglobulin have
been described, including chimeric antibodies having rodent V
regions and their associated CDRs fused to human constant domains
(Winter et al. (1991) Nature 349:293-299; Lobuglio et al. (1989)
Proc. Nat. Acad. Sci. USA 86:4220-4224; Shaw et al. (1987) J.
Immunol. 138:4534-4538; and Brown et al. (1987) Cancer Res.
47:3577-3583), rodent CDRs grafted into a human supporting FR prior
to fusion with an appropriate human antibody constant domain
(Riechmann et al. (1988) Nature 332:323-327; Verhoeyen et al.
(1988) Science 239:1534-1536; and Jones et al. (1986) Nature
321:522-525), and rodent CDRs supported by recombinantly veneered
rodent FRs (European Patent Publication No. 519,596, published Dec.
23, 1992). These "humanized" molecules are designed to minimize
unwanted immunological response toward rodent antihuman antibody
molecules which limits the duration and effectiveness of
therapeutic applications of those moieties in human recipients.
[0217] As noted above, "peptides" are included as binding agents.
The term peptide typically refers to a polymer of amino acid
residues and to variants and synthetic analogues of the same. In
certain embodiments, the term "peptide" refers to relatively short
polypeptides, including peptides that consist of about 2, 3, 4, 5,
6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35,
40, 45, or 50 amino acids, including all integers and ranges (e.g.,
5-10, 8-12, 10-15) in between, and interact with an AspRS
polypeptide, its cellular binding partner, or both. Peptides can be
composed of naturally-occurring amino acids and/or non-naturally
occurring amino acids, as described herein.
[0218] A binding agent may include a peptide mimetic or other small
molecule. A "small molecule" refers to an organic compound that is
of synthetic or biological origin (biomolecule), but is typically
not a polymer. Organic compounds refer to a large class of chemical
compounds whose molecules contain carbon, typically excluding those
that contain only carbonates, simple oxides of carbon, or cyanides.
A "biomolecule" refers generally to an organic molecule that is
produced by a living organism, including large polymeric molecules
(biopolymers) such as peptides, polysaccharides, and nucleic acids
as well, and small molecules such as primary secondary metabolites,
lipids, phospholipids, glycolipids, sterols, glycerolipids,
vitamins, and hormones. A "polymer" refers generally to a large
molecule or macromolecule composed of repeating structural units,
which are typically connected by covalent chemical bond.
[0219] In certain embodiments, a small molecule has a molecular
weight of less than 1000 Daltons, typically between about 300 and
700 Daltons, and including about 50, 100, 150, 200, 250, 300, 350,
400, 450, 500, 550, 500, 650, 600, 750, 700, 850, 800, 950, or 1000
Daltons.
[0220] Aptamers are also included as binding agents. Examples of
aptamers included nucleic acid aptamers (e.g., DNA aptamers, RNA
aptamers) and peptide aptamers. Nucleic acid aptamers refer
generally to nucleic acid species that have been engineered through
repeated rounds of in vitro selection or equivalent method, such as
SELEX (systematic evolution of ligands by exponential enrichment),
to bind to various molecular targets such as small molecules,
proteins, nucleic acids, and even cells, tissues and organisms.
Hence, included are nucleic acid aptamers that bind to the AspRS
polypeptides described herein and/or their cellular binding
partners.
[0221] Peptide aptamers typically include a variable peptide loop
attached at both ends to a protein scaffold, a double structural
constraint that typically increases the binding affinity of the
peptide aptamer to levels comparable to that of an antibody's
(e.g., in the nanomolar range). In certain embodiments, the
variable loop length may be composed of about 10-20 amino acids
(including all integers in between), and the scaffold may include
any protein that has good solubility and compacity properties.
Certain exemplary embodiments may utilize the bacterial protein
Thioredoxin-A as a scaffold protein, the variable loop being
inserted within the reducing active site (-Cys-Gly-Pro-Cys- loop in
the wild protein), with the two cysteines lateral chains being able
to form a disulfide bridge. Hence, included are peptide aptamers
that bind to the AspRS polypeptides described herein and/or their
cellular binding partners. Peptide aptamer selection can be
performed using different systems known in the art, including the
yeast two-hybrid system.
[0222] As noted above, the AspRS polypeptides and binding agents of
the present invention can be used in any of the diagnostic, drug
discovery, or therapeutic methods described herein.
[0223] In another embodiment of the invention, binding agents such
as monoclonal antibodies of the present invention may be coupled to
one or more agents of interest. For example, a therapeutic agent
may be coupled (e.g., covalently bonded) to a suitable monoclonal
antibody either directly or indirectly (e.g., via a linker group).
A direct reaction between an agent and an antibody is possible when
each possesses a substituent capable of reacting with the other.
For example, a nucleophilic group, such as an amino or sulfhydryl
group, on one may be capable of reacting with a carbonyl-containing
group, such as an anhydride or an acid halide, or with an alkyl
group containing a good leaving group (e.g., a halide) on the
other.
[0224] Alternatively, it may be desirable to couple a therapeutic
agent and an antibody via a linker group. A linker group can
function as a spacer to distance an antibody from an agent in order
to avoid interference with binding capabilities. A linker group can
also serve to increase the chemical reactivity of a substituent on
an agent or an antibody, and thus increase the coupling efficiency.
An increase in chemical reactivity may also facilitate the use of
agents, or functional groups on agents, which otherwise would not
be possible.
[0225] It will be evident to those skilled in the art that a
variety of bifunctional or polyfunctional reagents, both homo- and
hetero-functional (such as those described in the catalog of the
Pierce Chemical Co., Rockford, Ill.), may be employed as the linker
group. Coupling may be effected, for example, through amino groups,
carboxyl groups, sulfhydryl groups or oxidized carbohydrate
residues. There are numerous references describing such
methodology, e.g., U.S. Pat. No. 4,671,958, to Rodwell et al.
[0226] Where a therapeutic agent is more potent when free from the
antibody portion of the immunoconjugates of the present invention,
it may be desirable to use a linker group which is cleavable during
or upon internalization into a cell. A number of different
cleavable linker groups have been described. The mechanisms for the
intracellular release of an agent from these linker groups include
cleavage by reduction of a disulfide bond (e.g., U.S. Pat. No.
4,489,710, to Spitler), by irradiation of a photolabile bond (e.g.,
U.S. Pat. No. 4,625,014, to Senter et al.), by hydrolysis of
derivatized amino acid side chains (e.g., U.S. Pat. No. 4,638,045,
to Kohn et al.), by serum complement-mediated hydrolysis (e.g.,
U.S. Pat. No. 4,671,958, to Rodwell et al.), and acid-catalyzed
hydrolysis (e.g., U.S. Pat. No. 4,569,789, to Blattler et al.).
[0227] It may be desirable to couple more than one agent to an
antibody. In one embodiment, multiple molecules of an agent are
coupled to one antibody molecule. In another embodiment, more than
one type of agent may be coupled to one antibody. Regardless of the
particular embodiment, immunoconjugates with more than one agent
may be prepared in a variety of ways. For example, more than one
agent may be coupled directly to an antibody molecule, or linkers
that provide multiple sites for attachment can be used.
Formulation and Administration
[0228] The compositions of the invention (e.g., polypeptides,
polynucleotides, antibodies, etc.) are generally formulated in
pharmaceutically-acceptable or physiologically-acceptable solutions
for administration to a cell, tissue or animal, either alone, or in
combination with one or more other modalities of therapy. It will
also be understood that, if desired, the compositions of the
invention may be administered in combination with other agents as
well, such as, e.g., other proteins or polypeptides or various
pharmaceutically-active agents. There is virtually no limit to
other components that may also be included in the compositions,
provided that the additional agents do not adversely affect the
desired effects desired to be achieved with an AspRS polypeptide of
the invention.
[0229] In the pharmaceutical compositions of the invention,
formulation of pharmaceutically-acceptable excipients and carrier
solutions is well-known to those of skill in the art, as is the
development of suitable dosing and treatment regimens for using the
particular compositions described herein in a variety of treatment
regimens, including e.g., oral, parenteral, intravenous,
intranasal, intracranial and intramuscular administration and
formulation.
[0230] In certain applications, the pharmaceutical compositions
disclosed herein may be delivered via oral administration to a
subject. As such, these compositions may be formulated with an
inert diluent or with an assimilable edible carrier, or they may be
enclosed in hard- or soft-shell gelatin capsule, or they may be
compressed into tablets, or they may be incorporated directly with
the food of the diet.
[0231] In certain circumstances it will be desirable to deliver the
pharmaceutical compositions disclosed herein parenterally,
intravenously, intramuscularly, or even intraperitoneally as
described, for example, in U.S. Pat. No. 5,543,158; U.S. Pat. No.
5,641,515 and U.S. Pat. No. 5,399,363 (each specifically
incorporated herein by reference in its entirety). Solutions of the
active compounds as free base or pharmacologically acceptable salts
may be prepared in water suitably mixed with a surfactant, such as
hydroxypropylcellulose. Dispersions may also be prepared in
glycerol, liquid polyethylene glycols, and mixtures thereof and in
oils. Under ordinary conditions of storage and use, these
preparations contain a preservative to prevent the growth of
microorganisms.
[0232] The pharmaceutical forms suitable for injectable use include
sterile aqueous solutions or dispersions and sterile powders for
the extemporaneous preparation of sterile injectable solutions or
dispersions (U.S. Pat. No. 5,466,468, specifically incorporated
herein by reference in its entirety). In all cases the form should
be sterile and should be fluid to the extent that easy
syringability exists. It should be stable under the conditions of
manufacture and storage and should be preserved against the
contaminating action of microorganisms, such as bacteria and fungi.
The carrier can be a solvent or dispersion medium containing, for
example, water, ethanol, polyol (e.g., glycerol, propylene glycol,
and liquid polyethylene glycol, and the like), suitable mixtures
thereof, and/or vegetable oils. Proper fluidity may be maintained,
for example, by the use of a coating, such as lecithin, by the
maintenance of the required particle size in the case of dispersion
and by the use of surfactants. The prevention of the action of
microorganisms can be facilitated by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
sorbic acid, thimerosal, and the like. In many cases, it will be
preferable to include isotonic agents, for example, sugars or
sodium chloride. Prolonged absorption of the injectable
compositions can be brought about by the use in the compositions of
agents delaying absorption, for example, aluminum monostearate and
gelatin.
[0233] For parenteral administration in an aqueous solution, for
example, the solution should be suitably buffered if necessary and
the liquid diluent first rendered isotonic with sufficient saline
or glucose. These particular aqueous solutions are especially
suitable for intravenous, intramuscular, subcutaneous and
intraperitoneal administration. In this connection, a sterile
aqueous medium that can be employed will be known to those of skill
in the art in light of the present disclosure. For example, one
dosage may be dissolved in 1 ml of isotonic NaCl solution and
either added to 1000 ml of hypodermoclysis fluid or injected at the
proposed site of infusion (see, e.g., Remington's Pharmaceutical
Sciences, 15th Edition, pp. 1035-1038 and 1570-1580). Some
variation in dosage will necessarily occur depending on the
condition of the subject being treated. The person responsible for
administration will, in any event, determine the appropriate dose
for the individual subject. Moreover, for human administration,
preparations should meet sterility, pyrogenicity, and the general
safety and purity standards as required by FDA Office of Biologics
standards.
[0234] Sterile injectable solutions can be prepared by
incorporating the active compounds in the required amount in the
appropriate solvent with the various other ingredients enumerated
above, as required, followed by filtered sterilization. Generally,
dispersions are prepared by incorporating the various sterilized
active ingredients into a sterile vehicle which contains the basic
dispersion medium and the required other ingredients from those
enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, the preferred methods
of preparation are vacuum-drying and freeze-drying techniques which
yield a powder of the active ingredient plus any additional desired
ingredient from a previously sterile-filtered solution thereof.
[0235] The compositions disclosed herein may be formulated in a
neutral or salt form. Pharmaceutically-acceptable salts, include
the acid addition salts (formed with the free amino groups of the
protein) and which are formed with inorganic acids such as, for
example, hydrochloric or phosphoric acids, or such organic acids as
acetic, oxalic, tartaric, mandelic, and the like. Salts formed with
the free carboxyl groups can also be derived from inorganic bases
such as, for example, sodium, potassium, ammonium, calcium, or
ferric hydroxides, and such organic bases as isopropylamine,
trimethylamine, histidine, procaine and the like. Upon formulation,
solutions will be administered in a manner compatible with the
dosage formulation and in such amount as is therapeutically
effective. The formulations are easily administered in a variety of
dosage forms such as injectable solutions, drug-release capsules,
and the like.
[0236] As used herein, "carrier" includes any and all solvents,
dispersion media, vehicles, coatings, diluents, antibacterial and
antifungal agents, isotonic and absorption delaying agents,
buffers, carrier solutions, suspensions, colloids, and the like.
The use of such media and agents for pharmaceutical active
substances is well known in the art. Except insofar as any
conventional media or agent is incompatible with the active
ingredient, its use in the therapeutic compositions is
contemplated. Supplementary active ingredients can also be
incorporated into the compositions.
[0237] The phrase "pharmaceutically-acceptable" refers to molecular
entities and compositions that do not produce an allergic or
similar untoward reaction when administered to a human. The
preparation of an aqueous composition that contains a protein as an
active ingredient is well understood in the art. Typically, such
compositions are prepared as injectables, either as liquid
solutions or suspensions; solid forms suitable for solution in, or
suspension in, liquid prior to injection can also be prepared. The
preparation can also be emulsified.
[0238] In certain embodiments, the pharmaceutical compositions may
be delivered by intranasal sprays, inhalation, and/or other aerosol
delivery vehicles. Methods for delivering genes, polynucleotides,
and peptide compositions directly to the lungs via nasal aerosol
sprays has been described e.g., in U.S. Pat. No. 5,756,353 and U.S.
Pat. No. 5,804,212 (each specifically incorporated herein by
reference in its entirety). Likewise, the delivery of drugs using
intranasal microparticle resins (Takenaga et al., 1998) and
lysophosphatidyl-glycerol compounds (U.S. Pat. No. 5,725,871,
specifically incorporated herein by reference in its entirety) are
also well-known in the pharmaceutical arts. Likewise, transmucosal
drug delivery in the form of a polytetrafluoroetheylene support
matrix is described in U.S. Pat. No. 5,780,045 (specifically
incorporated herein by reference in its entirety).
[0239] In certain embodiments, the delivery may occur by use of
liposomes, nanocapsules, microparticles, microspheres, lipid
particles, vesicles, and the like, for the introduction of the
compositions of the present invention into suitable host cells. In
particular, the compositions of the present invention may be
formulated for delivery either encapsulated in a lipid particle, a
liposome, a vesicle, a nanosphere, a nanoparticle or the like. The
formulation and use of such delivery vehicles can be carried out
using known and conventional techniques.
Kits Comprising Compositions of the Invention
[0240] The invention, in other aspects, provides kits comprising
one or more containers filled with one or more of the polypeptides,
polynucleotides, antibodies, multiunit complexes, compositions
thereof, etc., of the invention, as described herein. The kits can
include written instructions on how to use such compositions (e.g.,
to modulate cellular signaling, angiogenesis, cancer, inflammatory
conditions, etc.).
[0241] The kits herein may also include a one or more additional
therapeutic agents or other components suitable or desired for the
indication being treated. An additional therapeutic agent may be
contained in a second container, if desired. Examples of additional
therapeutic agents include, but are not limited to antineoplastic
agents, anti-inflammatory agents, antibacterial agents, antiviral
agents, angiogenic agents, etc.
[0242] The kits herein can also include one or more syringes or
other components necessary or desired to facilitate an intended
mode of delivery (e.g., stents, implantable depots, etc.).
Methods of Use
[0243] Embodiments of the present invention also include methods of
using the AspRS compositions or "agents" described herein for
diagnostic, drug discovery, and/or therapeutic purposes. The term
AspRS "agents" refers generally to the AspRS polynucleotides, AspRS
polypeptides, binding agents, and other compounds described herein.
For diagnostic purposes, the AspRS agents can be used in a variety
of non-limiting ways, such as to distinguish between different cell
types or different cellular states, or to identify subjects having
a relevant disease or condition. For drug discovery purposes, the
AspRS agents can be used to identify one or more cellular "binding
partners" of an AspRS polypeptide, characterize one or more
"non-canonical" activities of an AspRS polypeptide, identify agents
that selectively or non-selectively agonize or antagonize the
interaction of an AspRS polypeptide with its binding partner(s),
and/or identify agents that selectively or non-selectively agonize
or antagonize one or more "non-canonical" activities of an AspRS
polypeptide. For therapeutic purposes, the AspRS agents or
compositions provided herein can be used to treat a variety of
diseases or conditions, detailed below.
[0244] A. Diagnostics
[0245] As noted above, AspRS agents described herein can be used in
diagnostic assays. These embodiments include the detection of the
AspRS polynucleotide sequence(s) or corresponding polypeptide
sequence(s) or portions thereof of one or more newly identified
AspRS protein fragments. In certain embodiments, the presence or
levels of one or more newly identified AspRS sequences associates
or correlates with one or more cellular types or cellular states.
Hence, as noted above, the presence or levels of an AspRS sequence
can be used to distinguish between different cellular types or
different cellular states. The presence or levels of AspRS
sequences can be detected according to polynucleotide and/or
polypeptide-based diagnostic techniques.
[0246] Certain of the methods provided herein rely on the
differential expression of an AspRS sequence to characterize the
condition or state of a cell, tissue, or subject, and to
distinguish it from another cell, tissue, or subject. Non-limiting
examples include methods of detecting the presence or levels of an
AspRS sequence in a biological sample to distinguish between cells
or tissues of different species, cells of different tissues or
organs, cellular developmental states such as neonatal and adult,
cellular differentiation states, conditions such as healthy,
diseased and treated, intracellular and extracellular fractions, in
addition to primary cell cultures and other cell cultures, such as
immortalized cell cultures.
[0247] Differential expression refers generally to a statistically
significant difference in one or more gene expression levels of an
AspRS polynucleotide or polypeptide sequence compared to the
expression levels of the same sequence in an appropriate control.
The statistically significant difference may relate to either an
increase or a decrease in expression levels, as measured by RNA
levels, protein levels, protein function, or any other relevant
measure of gene expression such as those described herein.
[0248] A result is typically referred to as statistically
significant if it is unlikely to have occurred by chance. The
significance level of a test or result relates traditionally to a
frequentist statistical hypothesis testing concept. In simple
cases, statistical significance may be defined as the probability
of making a decision to reject the null hypothesis when the null
hypothesis is actually true (a decision known as a Type I error, or
"false positive determination"). This decision is often made using
the p-value: if the p-value is less than the significance level,
then the null hypothesis is rejected. The smaller the p-value, the
more significant the result. Bayes factors may also be utilized to
determine statistical significance (see, e.g., Goodman S., Ann
Intern Med 130:1005-13, 1999).
[0249] In more complicated, but practically important cases, the
significance level of a test or result may reflect an analysis in
which the probability of making a decision to reject the null
hypothesis when the null hypothesis is actually true is no more
than the stated probability. This type of analysis allows for those
applications in which the probability of deciding to reject may be
much smaller than the significance level for some sets of
assumptions encompassed within the null hypothesis.
[0250] In certain exemplary embodiments, statistically significant
differential expression may include situations wherein the
expression level of a given AspRS sequence provides at least about
a 1.2.times., 1.3.times., 1.4.times., 1.5.times., 1.6.times.,
1.7.times., 1.8.times., 1.9.times.. 2.0.times., 2.2.times.,
2.4.times., 2.6.times., 2.8.times., 3.0.times., 4.0.times.,
5.0.times., 6.0.times., 7.0.times., 8.0.times., 9.0.times.,
10.0.times., 15.0.times., 20.0.times., 50.0.times., 100.0.times.,
or greater difference in expression (i.e., differential expression
that may be higher or lower expression) in a suspected biological
sample as compared to an appropriate control, including all
integers and decimal points in between (e.g., 1.24.times.,
1.25.times., 2.1.times., 2.5.times., 60.0.times., 75.0.times.,
etc.). In certain embodiments, statistically significant
differential expression may include situations wherein the
expression level of a given AspRS sequence provides at least about
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25,
30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600,
700, 800, 900, 1000 percent (%) or greater difference in expression
(i.e., differential expression that may be higher or lower) in a
suspected biological sample as compared to an appropriate control,
including all integers and decimal points in between.
[0251] As an additional example, differential expression may also
be determined by performing Z-testing, i.e., calculating an
absolute Z score, as described herein and known in the art (see
Example 1). Z-testing is typically utilized to identify significant
differences between a sample mean and a population mean. For
example, as compared to a standard normal table (e.g., a control
tissue), at a 95% confidence interval (i.e., at the 5% significance
level), a Z-score with an absolute value greater than 1.96
indicates non-randomness. For a 99% confidence interval, if the
absolute Z is greater than 2.58, it means that p<0.01, and the
difference is even more significant--the null hypothesis can be
rejected with greater confidence. In these and related embodiments,
an absolute Z-score of 1.96, 2, 2.58, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20 or more, including all decimal
points in between (e.g., 10.1, 10.6, 11.2, etc.), may provide a
strong measure of statistical significance. In certain embodiments,
an absolute Z-score of greater than 6 may provide exceptionally
high statistical significance.
[0252] Substantial similarly relates generally to the lack of a
statistically significant difference in the expression levels
between the biological sample and the reference control. Examples
of substantially similar expression levels may include situations
wherein the expression level of a given SSCIGS provides less than
about a 0.05.times., 0.1.times., 0.2.times., 0.3.times.,
0.4.times., 0.5.times., 0.6.times., 0.7.times., 0.8.times.,
0.9.times.. 1.0.times., 1.1.times., 1.2.times., 1.3.times., or
1.4.times. difference in expression (i.e., differential expression
that may be higher or lower expression) in a suspected biological
sample as compared to a reference sample, including all decimal
points in between (e.g., 0.15.times., 0.25.times., 0.35.times.,
etc.). In certain embodiments, differential expression may include
situations wherein the expression level of a given AspRS sequence
provides less than about 0.25. 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50 percent (%)
difference in expression (i.e., differential expression that may be
higher or lower) in a suspected biological sample as compared to a
reference sample, including all decimal points in between.
[0253] In certain embodiments, such as when using an Affymetrix
Microarray to measure the expression levels of an AspRS
polynucleotide or polypeptide sequence, differential expression may
also be determined by the mean expression value summarized by
Affymetrix Microarray Suite 5 software (Affymetrix, Santa Clara,
Calif.), or other similar software, typically with a scaled mean
expression value of 1000.
[0254] Embodiments of the present invention include methods of
detecting the presence or levels of an AspRS polynucleotide or
polypeptide reference sequence or a portion thereof to distinguish
between cells, tissues, or other biological samples of a different
organism or species, wherein the presence or levels of that
sequence associates with a selected organism or species. General
examples include methods of distinguishing between humans and any
combination of bacteria, fungi, plants, and other non-human
animals. Included within animals are methods of distinguishing
between humans and any combination of vertebrates and
invertebrates, including vertebrates such as fish, amphibians,
reptiles, birds, and non-human mammals, and inverterbrates such as
insects, mollusks, crustaceans, and corals. Included within
non-human mammals are methods of distinguishing between humans and
any combination of non-human mammals from the Order Afrosoricida,
Macroscelidea, Tubulidentata, Hyracoidea, Proboscidea, Sirenia,
Cingulata, Pilosa, Scandentia, Dermoptera, Primates, Rodentia,
Lagomorpha, Erinaceomorpha, Soricomorpha, Chiroptera, Pholidota,
Cetacea, Carnivora, Perissodactyla, or Artiodactyla. Included
within the Primate Order are monkeys, apes, gorillas, and
chimpanzees, among others known in the art. Accordingly, the
presence or levels of an AspRS polynucleotide or polypeptide
reference sequence or variant, as described herein, may be used to
identify the source of a given biological sample, such as a cell,
tissue, or organ, by distinguishing between any combination of
these organisms, or by distinguishing between humans and any one or
more of these organisms, such as a panel of organisms. In certain
embodiments, the source of a given biological sample may also be
determined by comparing the presence or levels of an AspRS sequence
or a portion thereof to a pre-determined value.
[0255] Embodiments of the present invention include methods of
detecting the presence or levels of an AspRS polynucleotide or
polypeptide reference sequence or a portion thereof to distinguish
between cells or other biological samples that originate from
different tissues or organs. Non-limiting examples include methods
of distinguishing between a cell or other biological sample that
originates from any combination of skin (e.g., dermis, epidermis,
subcutaneous layer), hair follicles, nervous system (e.g., brain,
spinal cord, peripheral nerves), auditory system or balance organs
(e.g., inner ear, middle ear, outer ear), respiratory system (e.g.,
nose, trachea, lungs), gastroesophogeal tissues, the
gastrointestinal system (e.g., mouth, esophagus, stomach, small
intestines, large intestines, rectum), vascular system (e.g.,
heart, blood vessels and arteries), liver, gallbladder,
lymphatic/immune system (e.g., lymph nodes, lymphoid follicles,
spleen, thymus, bone marrow), uro-genital system (e.g., kidneys,
ureter, bladder, urethra, cervix, Fallopian tubes, ovaries, uterus,
vulva, prostate, bulbourethral glands, epidiymis, prostate, seminal
vesicles, testicles), musculoskeletal system (e.g., skeletal
muscles, smooth muscles, bone, cartilage, tendons, ligaments),
adipose tissue, mammaries, and the endocrine system (e.g.,
hypothalamus, pituitary, thyroid, pancreas, adrenal glands). Hence,
based on the association of an AspRS polynucleotide or polypeptide
sequence as described herein, these methods may be used to identify
or characterize the tissue or organ from which a cell or other
biological sample is derived.
[0256] Embodiments of the present invention include methods of
detecting the presence or levels of an AspRS polynucleotide or
polypeptide reference sequence or a portion thereof to distinguish
between or characterize the developmental or differentiation state
of the cell. Also included are methods of differentiating between
germ cells, stem cells, and somatic cells. Examples of
developmental states include neonatal and adult. Examples of
cellular differentiation states include all of the discreet and
identifiable stages between a totipotent cell, a pluripotent cell,
a multipotent progenitor stem cell and a mature, fully
differentiated cell.
[0257] A totipotent cell has total potential, typically arises
during sexual and asexual reproduction, and includes and spores and
zygotes, though in certain instances cells can dedifferentiate and
regain totipotency. A pluripotent cell includes a stem cell that
has the potential to differentiate into any of the three germ
layers, including the endoderm (interior stomach lining,
gastrointestinal tract, the lungs), the mesoderm (muscle, bone,
blood, urogenital), and the ectoderm (epidermal tissues and nervous
system). Multipotent progenitor cells are typically capable of
differentiating into a limited number of tissue types. Examples of
multipotent cells include, without limitation, hematopoietic stem
cells (adult stem cells) from the bone marrow that give rise to
immune cells such as red blood cells, white blood cells, and
platelets, mesenchymal stem cells (adult stem cells) from the bone
marrow that give rise to stromal cells, fat cells, and various
types of bone cells, epithelial stem cells (progenitor cells) that
give rise to the various types of skin cells, and muscle satellite
cells (progenitor cells) that contribute to differentiated muscle
tissue. Accordingly, the presence or levels of particular AspRS
polynucleotide or polypeptide sequence can be used to distinguish
between or characterize the above-noted cellular differentiation
states, as compared to a control or a predetermined level.
[0258] Embodiments of the present invention include methods of
detecting the presence or levels of an AspRS polynucleotide or
polypeptide reference sequence to characterize or diagnose the
condition or a cell, tissue, organ, or subject, in which that
condition may be characterized as healthy, diseased, at risk for
being diseased, or treated. For such diagnostic purposes, the term
"diagnostic" or "diagnosed" includes identifying the presence or
nature of a pathologic condition, characterizing the risk of
developing such a condition, and/or measuring the change (or no
change) of a pathologic condition in response to therapy.
Diagnostic methods may differ in their sensitivity and specificity.
In certain embodiments, the "sensitivity" of a diagnostic assay
refers to the percentage of diseased cells, tissues or subjects
which test positive (percent of "true positives"). Diseased cells,
tissues or subjects not detected by the assay are typically
referred to as "false negatives." Cells, tissues or subjects that
are not diseased and which test negative in the assay may be termed
"true negatives." In certain embodiments, the "specificity" of a
diagnostic assay may be defined as one (1) minus the false positive
rate, where the "false positive" rate is defined as the proportion
of those samples or subjects without the disease and which test
positive. While a particular diagnostic method may not provide a
definitive diagnosis of a condition, it suffices if the method
provides a positive indication that aids in diagnosis.
[0259] In certain instances, the presence or risk of developing a
pathologic condition can be diagnosed by comparing the presence or
levels of one or more selected AspRS polynucleotide or polypeptide
reference sequences or portions thereof that correlate with the
condition, whether by increased or decreased levels, as compared to
a suitable control. A "suitable control" or "appropriate control"
includes a value, level, feature, characteristic, or property
determined in a cell or other biological sample of a tissue or
organism, e.g., a control or normal cell, tissue or organism,
exhibiting, for example, normal traits, such as the absence of the
condition. In certain embodiments, a "suitable control" or
"appropriate control" is a predefined value, level, feature,
characteristic, or property. Other suitable controls will be
apparent to persons skilled in the art. Examples of diseases and
conditions are described elsewhere herein.
[0260] Embodiments of the present invention include AspRS
polynucleotide or nucleic acid-based detection techniques, which
offer certain advantages due to sensitivity of detection. Hence,
certain embodiments relate to the use or detection of AspRS
polynucleotides as part of a diagnostic method or assay. The
presence and/or levels of AspRS polynucleotides may be measured by
any method known in the art, including hybridization assays such as
Northern blot, quantitative or qualitative polymerase chain
reaction (PCR), quantitative or qualitative reverse transcriptase
PCR (RT-PCR), microarray, dot or slot blots, or in situ
hybridization such as fluorescent in situ hybridization (FISH),
among others. Certain of these methods are described in greater
detail below.
[0261] AspRS polynucleotides such as DNA and RNA can be collected
and/or generated from blood, biological fluids, tissues, organs,
cell lines, or other relevant sample using techniques known in the
art, such as those described in Kingston. (2002 Current Protocols
in Molecular Biology, Greene Publ. Assoc. Inc. & John Wiley
& Sons, Inc., NY, N.Y. (see, e.g., as described by Nelson et
al. Proc Natl Acad Sci USA, 99: 11890-11895, 2002) and
elsewhere.
[0262] Complementary DNA (cDNA) libraries can be generated using
techniques known in the art, such as those described in Ausubel et
al. (2001 Current Protocols in Molecular Biology, Greene Publ.
Assoc. Inc. & John Wiley & Sons, Inc., NY, N.Y.); Sambrook
et al. (1989 Molecular Cloning, Second Ed., Cold Spring Harbor
Laboratory, Plainview, N.Y.); Maniatis et al. (1982 Molecular
Cloning, Cold Spring Harbor Laboratory, Plainview, N.Y.) and
elsewhere.
[0263] Certain embodiments may employ hybridization methods for
detecting AspRS polynucleotide sequences. Methods for conducting
polynucleotide hybridization assays have been well developed in the
art. Hybridization assay procedures and conditions will vary
depending on the application and are selected in accordance with
the general binding methods known including those referred to in:
Maniatis et al. Molecular Cloning: A Laboratory Manual (2nd Ed.
Cold Spring Harbor, N.Y., 1989); Berger and Kimmel Methods in
Enzymology, Vol. 152, Guide to Molecular Cloning Techniques
(Academic Press, Inc., San Diego, Calif., 1987); Young and Davism,
P. N. A. S, 80: 1194 (1983). Methods and apparatus for carrying out
repeated and controlled hybridization reactions have been described
in U.S. Pat. Nos. 5,871,928, 5,874,219, 6,045,996 and 6,386,749,
6,391,623 each of which are incorporated herein by reference
[0264] Certain embodiments may employ nucleic acid amplification
methods for detecting AspRS polynucleotide sequences. The term
"amplification" or "nucleic acid amplification" refers to the
production of multiple copies of a target nucleic acid that
contains at least a portion of the intended specific target nucleic
acid sequence. The multiple copies may be referred to as amplicons
or amplification products. In certain embodiments, the amplified
target contains less than the complete target gene sequence
(introns and exons) or an expressed target gene sequence (spliced
transcript of exons and flanking untranslated sequences). For
example, specific amplicons may be produced by amplifying a portion
of the target polynucleotide by using amplification primers that
hybridize to, and initiate polymerization from, internal positions
of the target polynucleotide. Preferably, the amplified portion
contains a detectable target sequence that may be detected using
any of a variety of well-known methods.
[0265] "Selective amplification" or "specific amplification," as
used herein, refers to the amplification of a target nucleic acid
sequence according to the present invention wherein detectable
amplification of the target sequence is substantially limited to
amplification of target sequence contributed by a nucleic acid
sample of interest that is being tested and is not contributed by
target nucleic acid sequence contributed by some other sample
source, e.g., contamination present in reagents used during
amplification reactions or in the environment in which
amplification reactions are performed.
[0266] By "amplification conditions" is meant conditions permitting
nucleic acid amplification according to the present invention.
Amplification conditions may, in some embodiments, be less
stringent than "stringent hybridization conditions" as described
herein. Oligonucleotides used in the amplification reactions of the
present invention hybridize to their intended targets under
amplification conditions, but may or may not hybridize under
stringent hybridization conditions. On the other hand, detection
probes of the present invention typically hybridize under stringent
hybridization conditions. Acceptable conditions to carry out
nucleic acid amplifications according to the present invention can
be easily ascertained by someone having ordinary skill in the art
depending on the particular method of amplification employed.
[0267] Many well-known methods of nucleic acid amplification
require thermocycling to alternately denature double-stranded
nucleic acids and hybridize primers; however, other well-known
methods of nucleic acid amplification are isothermal. The
polymerase chain reaction (U.S. Pat. Nos. 4,683,195; 4,683,202;
4,800,159; 4,965,188), commonly referred to as PCR, uses multiple
cycles of denaturation, annealing of primer pairs to opposite
strands, and primer extension to exponentially increase copy
numbers of the target sequence. In a variation called RT-PCR,
reverse transcriptase (RT) is used to make a complementary DNA
(cDNA) from mRNA, and the cDNA is then amplified by PCR to produce
multiple copies of DNA.
[0268] As noted above, the term "PCR" refers to multiple
amplification cycles that selectively amplify a target nucleic acid
species. Included are quantitative PCR (qPCR), real-time PCR),
reverse transcription PCR (RT-PCR) and quantitative reverse
transcription PCR (qRT-PCR) is well described in the art. The term
"pPCR" refers to quantitative polymerase chain reaction, and the
term "qRT-PCR" refers to quantitative reverse transcription
polymerase chain reaction. qPCR and qRT-PCR may be used to amplify
and simultaneously quantify a targeted cDNA molecule. It enables
both detection and quantification of a specific sequence in a cDNA
pool, such as a selected AspRS gene or transcript.
[0269] The term "real-time PCR" may use DNA-binding dye to bind to
all double-stranded (ds) DNA in PCR, causing fluorescence of the
dye. An increase in DNA product during PCR therefore leads to an
increase in fluorescence intensity and is measured at each cycle,
thus allowing DNA concentrations to be quantified. However, dsDNA
dyes such as SYBR Green will bind to all dsDNA PCR products.
Fluorescence is detected and measured in the real-time PCR
thermocycler, and its geometric increase corresponding to
exponential increase of the product is used to determine the
threshold cycle ("Ct") in each reaction.
[0270] The term "Ct Score" refers to the threshold cycle number,
which is the cycle at which PCR amplification has surpassed a
threshold level. If there is a higher quantity of mRNA for a
particular gene in a sample, it will cross the threshold earlier
than a lowly expressed gene since there is more starting RNA to
amplify. Therefore, a low Ct score indicates high gene expression
in a sample and a high Ct score is indicative of low gene
expression.
[0271] Certain embodiments may employ the ligase chain reaction
(Weiss, R. 1991, Science 254: 1292), commonly referred to as LCR,
which uses two sets of complementary DNA oligonucleotides that
hybridize to adjacent regions of the target nucleic acid. The DNA
oligonucleotides are covalently linked by a DNA ligase in repeated
cycles of thermal denaturation, hybridization and ligation to
produce a detectable double-stranded ligated oligonucleotide
product.
[0272] In certain embodiments, other techniques may be used to
evaluate RNA transcripts of the transcripts from a particular cDNA
library, including microarray analysis (Han, M., et al., Nat
Biotechnol, 19: 631-635, 2001; Bao, P., et al., Anal Chem, 74:
1792-1797, 2002; Schena et al., Proc. Natl. Acad. Sci. USA
93:10614-19, 1996; and Heller et al., Proc. Natl. Acad. Sci. USA
94:2150-55, 1997) and SAGE (serial analysis of gene expression).
Like MPSS, SAGE is digital and can generate a large number of
signature sequences. (see e.g., Velculescu, V. E., et al., Trends
Genet, 16: 423-425., 2000; Tuteja R. and Tuteja N. Bioassays. 2004
August; 26(8):916-22), although orders of magnitude fewer than that
are available from techniques such as MPSS.
[0273] In certain embodiments, the term "microarray" includes a
"nucleic acid microarray" having a substrate-bound plurality of
nucleic acids, hybridization to each of the plurality of bound
nucleic acids being separately detectable. The substrate can be
solid or porous, planar or non-planar, unitary or distributed.
Nucleic acid microarrays include all the devices so called in
Schena (ed.), DNA Microarrays: A Practical Approach (Practical
Approach Series), Oxford University Press (1999); Nature Genet.
21(1) (suppl.): 1-60 (1999); Schena (ed.), Microarray Biochip:
Tools and Technology, Eaton Publishing Company/BioTechniques Books
Division (2000). Nucleic acid microarrays may include a
substrate-bound plurality of nucleic acids in which the plurality
of nucleic acids are disposed on a plurality of beads, rather than
on a unitary planar substrate, as described, for example, in
Brenner et al., Proc. Natl. Acad. Sci. USA 97(4): 1665-1670 (2000).
Examples of nucleic acid microarrays may be found in U.S. Pat. Nos.
6,391,623, 6,383,754, 6,383,749, 6,380,377, 6,379,897, 6,376,191,
6,372,431, 6,351,712 6,344,316, 6,316,193, 6,312,906, 6,309,828,
6,309,824, 6,306,643, 6,300,063, 6,287,850, 6,284,497, 6,284,465,
6,280,954, 6,262,216, 6,251,601, 6,245,518, 6,263,287, 6,251,601,
6,238,866, 6,228,575, 6,214,587, 6,203,989, 6,171,797, 6,103,474,
6,083,726, 6,054,274, 6,040,138, 6,083,726, 6,004,755, 6,001,309,
5,958,342, 5,952,180, 5,936,731, 5,843,655, 5,814,454, 5,837,196,
5,436,327, 5,412,087, and 5,405,783, the disclosures of which are
incorporated by reference.
[0274] Additional examples include nucleic acid arrays that are
commercially available from Affymetrix (Santa Clara, Calif.) under
the brand name GeneChip.TM.. Further exemplary methods of
manufacturing and using arrays are provided in, for example, U.S.
Pat. Nos. 7,028,629; 7,011,949; 7,011,945; 6,936,419; 6,927,032;
6,924,103; 6,921,642; and 6,818,394.
[0275] The present invention as related to arrays and microarrays
also contemplates many uses for polymers attached to solid
substrates. These uses include gene expression monitoring,
profiling, library screening, genotyping and diagnostics. Gene
expression monitoring and profiling methods and methods useful for
gene expression monitoring and profiling are shown in U.S. Pat.
Nos. 5,800,992, 6,013,449, 6,020,135, 6,033,860, 6,040,138,
6,177,248 and 6,309,822. Genotyping and uses therefore are shown in
U.S. Ser. Nos. 10/442,021, 10/013,598 (U.S. Application No.
2003/0036069), and U.S. Pat. Nos. 5,925,525, 6,268,141, 5,856,092,
6,267,152, 6,300,063, 6,525,185, 6,632,611, 5,858,659, 6,284,460,
6,361,947, 6,368,799, 6,673,579 and 6,333,179. Other methods of
nucleic acid amplification, labeling and analysis that may be used
in combination with the methods disclosed herein are embodied in
U.S. Pat. Nos. 5,871,928, 5,902,723, 6,045,996, 5,541,061, and
6,197,506.
[0276] As will be apparent to persons skilled in the art, certain
embodiments may employ oligonucleotides, such as primers or probes,
for amplification or detection, as described herein. While the
design and sequence of oligonucleotides depends on their function
as described herein, several variables are generally taken into
account. Among the most relevant are: length, melting temperature
(Tm), specificity, complementarity with other oligonucleotides in
the system, G/C content, polypyrimidine (T, C) or polypurine (A, G)
stretches, and the 3'-end sequence.
[0277] Certain embodiments therefore include methods for detecting
a target AspRS polynucleotide in a sample, typically wherein the
polynucleotide comprises the sequence of a reference AspRS
polynucleotide described herein, comprising a) hybridizing the
sample with a probe comprising a sequence complementary to the
target polynucleotide in the sample, and which probe specifically
hybridizes to said target polynucleotide, under conditions whereby
a hybridization complex is formed between said probe and said
target polynucleotide or fragments thereof, and b) detecting the
presence or absence of said hybridization complex, and optionally,
if present, the amount thereof. Also included are methods for
detecting a target AspRS polynucleotide in a sample, the
polynucleotide comprising the sequence of a reference AspRS
polynucleotide, as described herein, comprising a) amplifying the
target polynucleotide or fragment thereof, and b) detecting the
presence or absence of said amplified target polynucleotide or
fragment thereof, and, optionally, if present, the amount
thereof.
[0278] Embodiments of the present invention include a variety of
AspRS polypeptide-based detection techniques, including
antibody-based detection techniques. Included in these embodiments
are the use of AspRS polypeptides to generate antibodies or other
binders, which may then be used in diagnostic methods and
compositions to detect or quantitate selected AspRS polypeptides in
a cell or other biological sample, typically from a subject.
[0279] Certain embodiments may employ standard methodologies such
as western blotting and immunoprecipitation, enzyme-linked
immunosorbent assays (ELISA), flow cytometry, and
immunofluorescence assays (IFA). These well-known methods typically
utilize one or more monoclonal or polyclonal antibodies as
described herein that specifically bind to a selected AspRS
polypeptide of the invention, or a unique region of that AspRS
polypeptide, and generally do not bind significantly to other AspRS
polypeptides, such as a full-length AspRS polypeptide. In certain
embodiments, the unique region of the AspRS polypeptide may be
encoded by a unique splice junction or a particular
three-dimensional structure of a newly identified alternate splice
variant or protein fragment, such as a proteolytic fragment.
[0280] Certain embodiments may employ "arrays," such as
"microarrays." In certain embodiments, a "microarray" may also
refer to a "peptide microarray" or "protein microarray" having a
substrate-bound collection or plurality of polypeptides, the
binding to each of the plurality of bound polypeptides being
separately detectable. Alternatively, the peptide microarray may
have a plurality of binders, including but not limited to
monoclonal antibodies, polyclonal antibodies, phage display
binders, yeast 2 hybrid binders, and aptamers, which can
specifically detect the binding of the AspRS polypeptides described
herein. The array may be based on autoantibody detection of these
AspRS polypeptides, as described, for example, in Robinson et al.,
Nature Medicine 8(3):295-301 (2002). Examples of peptide arrays may
be found in WO 02/31463, WO 02/25288, WO 01/94946, WO 01/88162, WO
01/68671, WO 01/57259, WO 00/61806, WO 00/54046, WO 00/47774, WO
99/40434, WO 99/39210, and WO 97/42507 and U.S. Pat. Nos.
6,268,210, 5,766,960, and 5,143,854, each of which are incorporated
by reference.
[0281] Certain embodiments may employ MS or other molecular
weight-based methods for diagnostically detecting AspRS polypeptide
sequences. Mass spectrometry (MS) refers generally to an analytical
technique for determining the elemental composition of a sample or
molecule. MS may also be used for determining the chemical
structures of molecules, such as peptides and other chemical
compounds.
[0282] Generally, the MS principle consists of ionizing chemical
compounds to generate charged molecules or molecule fragments, and
then measuring their mass-to-charge ratios. In an illustrative MS
procedure: a sample is loaded onto the MS instrument, and undergoes
vaporization, the components of the sample are ionized by one of a
variety of methods (e.g., by impacting them with an electron beam),
which results in the formation of positively charged particles, the
positive ions are then accelerated by a magnetic field,
computations are performed on the mass-to-charge ratio (m/z) of the
particles based on the details of motion of the ions as they
transit through electromagnetic fields, and, detection of the ions,
which in step prior were sorted according to m/z.
[0283] An illustrative MS instruments has three modules: an ion
source, which converts gas phase sample molecules into ions (or, in
the case of electrospray ionization, move ions that exist in
solution into the gas phase); a mass analyzer, which sorts the ions
by their masses by applying electromagnetic fields; and a detector,
which measures the value of an indicator quantity and thus provides
data for calculating the abundances of each ion present.
[0284] The MS technique has both qualitative and quantitative uses,
including identifying unknown compounds, determining the isotopic
composition of elements in a molecule, and determining the
structure of a compound by observing its fragmentation. Other uses
include quantifying the amount of a compound in a sample or
studying the fundamentals of gas phase ion chemistry (the chemistry
of ions and neutrals in a vacuum). Accordingly, MS techniques may
be used according to any of the methods provided herein to measure
the presence or levels of an AspRS polypeptide of the invention in
a biological sample, and to compare those levels to a control
sample or a pre-determined value.
[0285] B. Discovery of Compounds and Therapeutic Agents
[0286] Certain embodiments relate to the use of AspRS polypeptide
or AspRS polynucleotide references sequences in drug discovery,
typically to identify agents that modulate one or more of the
non-canonical activities of the reference AspRS. For example,
certain embodiments include methods of identifying one or more
"binding partners" of an AspRS reference polypeptide, or a
polypeptide that comprises an AspRS reference sequence such as a
cellular protein or other host molecule that associates with the
AspRS polypeptide and participates in its non-canonical activity or
activities. Also included are methods of identifying a compound
(e.g., polypeptide) or other agent that agonizes or antagonizes the
non-canonical activity of an AspRS reference polypeptide or active
variant thereof, such as by interacting with the AspRS polypeptide
and/or one or more of its cellular binding partners.
[0287] Certain embodiments therefore include methods of identifying
a binding partner of an AspRS reference polypeptide, comprising a)
combining the AspRS polypeptide with a biological sample under
suitable conditions, and b) detecting specific binding of the AspRS
polypeptide to a binding partner, thereby identifying a binding
partner that specifically binds to the AspRS reference polypeptide.
Also included are methods of screening for a compound that
specifically binds to an AspRS reference polypeptide or a binding
partner of the AspRS polypeptide, comprising a) combining the
polypeptide or the binding partner with at least one test compound
under suitable conditions, and b) detecting binding of the
polypeptide or the binding partner to the test compound, thereby
identifying a compound that specifically binds to the polypeptide
or its binding partner. In certain embodiments, the compound is a
polypeptide or peptide. In certain embodiments, the compound is a
small molecule or other (e.g., non-biological) chemical compound.
In certain embodiments, the compound is a peptide mimetic.
[0288] Any method suitable for detecting protein-protein
interactions may be employed for identifying cellular proteins that
interact with an AspRS reference polypeptide, interact with one or
more of its cellular binding partners, or both. Examples of
traditional methods that may be employed include
co-immunoprecipitation, cross-linking, and co-purification through
gradients or chromatographic columns of cell lysates or proteins
obtained from cell lysates, mainly to identify proteins in the
lysate that interact with the AspRS polypeptide.
[0289] In these and related embodiments, at least a portion of the
amino acid sequence of a protein that interacts with an AspRS
polypeptide or its binding partner can be ascertained using
techniques well known to those of skill in the art, such as via the
Edman degradation technique. See, e.g., Creighton Proteins:
Structures and Molecular Principles, W. H. Freeman & Co., N.Y.,
pp. 34 49, 1983. The amino acid sequence obtained may be used as a
guide for the generation of oligonucleotide mixtures that can be
used to screen for gene sequences encoding such proteins. Screening
may be accomplished, for example, by standard hybridization or PCR
techniques, as described herein and known in the art. Techniques
for the generation of oligonucleotide mixtures and the screening
are well known. See, e.g., Ausubel et al. Current Protocols in
Molecular Biology Green Publishing Associates and Wiley
Interscience, N.Y., 1989; and Innis et al., eds. PCR Protocols: A
Guide to Methods and Applications Academic Press, Inc., New York,
1990.
[0290] Additionally, methods may be employed in the simultaneous
identification of genes that encode the binding partner or other
polypeptide. These methods include, for example, probing expression
libraries, in a manner similar to the well known technique of
antibody probing of lambda-gt11 libraries, using labeled AspRS
protein, or another polypeptide, peptide or fusion protein, e.g., a
variant AspRS polypeptide or AspRS domain fused to a marker (e.g.,
an enzyme, fluor, luminescent protein, or dye), or an Ig-Fc
domain.
[0291] One method that detects protein interactions in vivo is the
two-hybrid system. An example of this system has been described
(Chien et al., PNAS USA 88:9578 9582, 1991) and is commercially
available from Clontech (Palo Alto, Calif.). In certain instances,
the two-hybrid system or other such methodology may be used to
screen activation domain libraries for proteins that interact with
the "bait" gene product. By way of example, and not by way of
limitation, an AspRS reference polypeptide or variant may be used
as the bait gene product. An AspRS binding partner may also be used
as a "bait" gene product. Total genomic or cDNA sequences are fused
to the DNA encoding an activation domain. This library and a
plasmid encoding a hybrid of a bait AspRS gene product fused to the
DNA-binding domain are co-transformed into a yeast reporter strain,
and the resulting transformants are screened for those that express
the reporter gene.
[0292] Also included are three-hybrid systems, which allow the
detection of RNA-protein interactions in yeast. See, e.g., Hook et
al., RNA. 11:227-233, 2005. Accordingly, these and related methods
can be used to identify a cellular binding partner of an AspRS
polypeptide. These and related methods can also be used to identify
other compounds such as binding agents or nucleic acids that
interact with the AspRS polypeptide, its cellular binding partner,
or both.
[0293] As noted above, once isolated, binding partners can be
identified and can, in turn, be used in conjunction with standard
techniques to identify proteins or other compounds with which it
interacts. Certain embodiments thus relate to methods of screening
for a compound that specifically binds to the binding partner of an
AspRS reference polypeptide, comprising a) combining the binding
partner with at least one test compound under suitable conditions,
and b) detecting binding of the binding partner to the test
compound, thereby identifying a compound that specifically binds to
the binding partner. In certain embodiments, the test compound is a
polypeptide. In certain embodiments, the test compound is a
chemical compound, such as a small molecule compound or peptide
mimetic.
[0294] Certain embodiments include methods of screening for a
compound that modulates the activity of an AspRS reference
polypeptide, comprising a) combining the polypeptide with at least
one test compound under conditions permissive for the activity of
the polypeptide, b) assessing the activity of the polypeptide in
the presence of the test compound, and c) comparing the activity of
the polypeptide in the presence of the test compound with the
activity of the polypeptide in the absence of the test compound,
wherein a change in the activity of the polypeptide in the presence
of the test compound is indicative of a compound that modulates the
activity of the polypeptide.
[0295] Certain embodiments include methods of screening for a
compound that modulates the activity of a binding partner of an
AspRS reference polypeptide, comprising a) combining the
polypeptide with at least one test compound under conditions
permissive for the activity of the binding partner, b) assessing
the activity of the binding partner in the presence of the test
compound, and c) comparing the activity of the binding partner in
the presence of the test compound with the activity of the binding
partner in the absence of the test compound, wherein a change in
the activity of the binding partner in the presence of the test
compound is indicative of a compound that modulates the activity of
the binding partner. Typically, these and related embodiments
include assessing a selected non-canonical activity that is
associated with the AspRS polypeptide or its binding partner.
Included are in vitro and in vivo conditions, such as cell culture
conditions.
[0296] Certain embodiments include methods of screening a compound
for effectiveness as a full or partial agonist of an AspRS
reference polypeptide or an active fragment or variant thereof,
comprising a) exposing a sample comprising the polypeptide to a
compound, and b) detecting agonist activity in the sample,
typically by measuring an increase in the non-canonical activity of
the AspRS polypeptide. Certain methods include a) exposing a sample
comprising a binding partner of the AspRS polypeptide to a
compound, and b) detecting agonist activity in the sample,
typically by measuring an increase in the selected non-canonical
activity of the AspRS polypeptide. Certain embodiments include
compositions that comprise an agonist compound identified by the
method and a pharmaceutically acceptable carrier or excipient.
[0297] Also included are methods of screening a compound for
effectiveness as a full or partial antagonist of an AspRS reference
polypeptide, comprising a) exposing a sample comprising the
polypeptide to a compound, and b) detecting antagonist activity in
the sample, typically by measuring a decrease in the non-canonical
activity of the AspRS polypeptide. Certain methods include a)
exposing a sample comprising a binding partner of the AspRS
polypeptide to a compound, and b) detecting antagonist activity in
the sample, typically by measuring a decrease in the selected
non-canonical activity of the AspRS polypeptide. Certain
embodiments include compositions that comprise an antagonist
compound identified by the method and a pharmaceutically acceptable
carrier or excipient.
[0298] In certain embodiments, in vitro systems may be designed to
identify compounds capable of interacting with or modulating an
AspRS reference sequence or its binding partner. Certain of the
compounds identified by such systems may be useful, for example, in
modulating the activity of the pathway, and in elaborating
components of the pathway itself. They may also be used in screens
for identifying compounds that disrupt interactions between
components of the pathway; or may disrupt such interactions
directly. One exemplary approach involves preparing a reaction
mixture of the AspRS polypeptide and a test compound under
conditions and for a time sufficient to allow the two to interact
and bind, thus forming a complex that can be removed from and/or
detected in the reaction mixture
[0299] In vitro screening assays can be conducted in a variety of
ways. For example, an AspRS polypeptide, a cellular binding
partner, or test compound(s) can be anchored onto a solid phase. In
these and related embodiments, the resulting complexes may be
captured and detected on the solid phase at the end of the
reaction. In one example of such a method, the AspRS polypeptide
and/or its binding partner are anchored onto a solid surface, and
the test compound(s), which are not anchored, may be labeled,
either directly or indirectly, so that their capture by the
component on the solid surface can be detected. In other examples,
the test compound(s) are anchored to the solid surface, and the
AspRS polypeptide and/or its binding partner, which are not
anchored, are labeled or in some way detectable. In certain
embodiments, microtiter plates may conveniently be utilized as the
solid phase. The anchored component (or test compound) may be
immobilized by non-covalent or covalent attachments. Non-covalent
attachment may be accomplished by simply coating the solid surface
with a solution of the protein and drying. Alternatively, an
immobilized antibody, preferably a monoclonal antibody, specific
for the protein to be immobilized may be used to anchor the protein
to the solid surface. The surfaces may be prepared in advance and
stored.
[0300] To conduct an exemplary assay, the non-immobilized component
is typically added to the coated surface containing the anchored
component. After the reaction is complete, un-reacted components
are removed (e.g., by washing) under conditions such that any
specific complexes formed will remain immobilized on the solid
surface. The detection of complexes anchored on the solid surface
can be accomplished in a number of ways. For instance, where the
previously non-immobilized component is pre-labeled, the detection
of label immobilized on the surface indicates that complexes were
formed. Where the previously non-immobilized component is not
pre-labeled, an indirect label can be used to detect complexes
anchored on the surface; e.g., using a labeled antibody specific
for the previously non-immobilized component (the antibody, in
turn, may be directly labeled or indirectly labeled with a labeled
anti-Ig antibody).
[0301] Alternatively, the presence or absence of binding of a test
compound can be determined, for example, using surface plasmon
resonance (SPR) and the change in the resonance angle as an index,
wherein an AspRS polypeptide or a cellular binding partner is
immobilized onto the surface of a commercially available sensorchip
(e.g., manufactured by Biacore.TM.) according to a conventional
method, the test compound is contacted therewith, and the
sensorchip is illuminated with a light of a particular wavelength
from a particular angle. The binding of a test compound can also be
measured by detecting the appearance of a peak corresponding to the
test compound by a method wherein an AspRS polypeptide or a
cellular binding partner is immobilized onto the surface of a
protein chip adaptable to a mass spectrometer, a test compound is
contacted therewith, and an ionization method such as MALDI-MS,
ESI-MS, FAB-MS and the like is combined with a mass spectrometer
(e.g., double-focusing mass spectrometer, quadrupole mass
spectrometer, time-of-flight mass spectrometer, Fourier
transformation mass spectrometer, ion cyclotron mass spectrometer
and the like).
[0302] In certain embodiments, cell-based assays, membrane
vesicle-based assays, or membrane fraction-based assays can be used
to identify compounds that modulate interactions in the
non-canonical pathway of the selected AspRS polypeptide. To this
end, cell lines that express an AspRS polypeptide and/or a binding
partner, or a fusion protein containing a domain or fragment of
such proteins (or a combination thereof), or cell lines (e.g., COS
cells, CHO cells, HEK293 cells, Hela cells etc.) that have been
genetically engineered to express such protein(s) or fusion
protein(s) can be used. Test compound(s) that influence the
non-canonical activity can be identified by monitoring a change
(e.g., a statistically significant change) in that activity as
compared to a control or a predetermined amount.
[0303] For embodiments that relate to antisense and RNAi agents,
for example, also included are methods of screening a compound for
effectiveness in altering expression of an AspRS reference
polynucleotide, comprising a) exposing a sample comprising the
AspRS reference polynucleotide to a compound such as a potential
antisense oligonucleotide, and b) detecting altered expression of
the AspRS polynucleotide. In certain non-limiting examples, these
and related embodiments can be employed in cell-based assays or in
cell-free translation assays, according to routine techniques in
the art. Also included are the antisense and RNAi agents identified
by such methods.
[0304] Also included are any of the above methods, or other
screening methods known in the art, which are adapted for
high-throughput screening (HTS). HTS typically uses automation to
run a screen of an assay against a library of candidate compounds,
for instance, an assay that measures an increase or a decrease in a
non-canonical activity, as described herein.
[0305] C. Methods of Treatment
[0306] In another aspect, the present invention relates to methods
of using the compositions of the present invention for treating a
cell, tissue or subject with a composition as described herein. The
cells or tissue that may be modulated by the present invention are
preferably mammalian cells, or more preferably human cells. Such
cells can be of a healthy state or of a diseased state.
[0307] Accordingly, the AspRS agents described herein, including
AspRS polypeptides, AspRS polynucleotides, AspRS
polynucleotide-based vectors, antisense oligonucleotides, RNAi
agents, as well as binding agents such as peptides, antibodies and
antigen-binding fragments, peptide mimetics and other small
molecules, can be used to treat a variety of non-limiting diseases
or conditions associated with the non-canonical activities of a
reference AspRS. Examples of such non-canonical activities include
modulation of cell proliferation, modulation of cell migration,
modulation of cell differentiation (e.g., hematopoiesis),
modulation of apoptosis or other forms of cell death, modulation of
cell signaling, modulation of angiogenesis, modulation of cell
binding, modulation of cellular metabolism, modulation of cytokine
production or activity, modulation of cytokine receptor activity,
modulation of inflammation, and the like.
[0308] Included are polynucleotide-based therapies, such as
antisense therapies and RNAi interference therapies, which
typically relate to reducing the expression of a target molecule,
such as a particular splice variant of an AspRS polypeptide or a
cellular binding partner of an AspRS polypeptide, which otherwise
contributes to its non-canonical activity. Antisense or RNAi
therapies typically antagonize the non-canonical activity, such as
by reducing expression of the AspRS reference polypeptide. Also
included are polypeptides, antibodies, peptide mimetics, or other
small molecule-based therapies, which either agonize or antagonize
the non-canonical activity of an AspRS reference polypeptide, such
as by interacting directly with the AspRS polypeptide, its cellular
binding partner(s), or both.
[0309] In certain embodiments, for example, methods are provided
for modulating therapeutically relevant cellular activities
including, but not limited to, cellular metabolism, cell
differentiation, cell proliferation, cell death, cell mobilization,
cell migration, gene transcription, mRNA translation, cell
impedance, cytokine production, and the like, comprising contacting
a cell with an AspRS composition as described herein. Accordingly,
the AspRS compositions may be employed in treating essentially any
cell or tissue or subject that would benefit from modulation of one
or more such activities.
[0310] The AspRS compositions may also be used in any of a number
of therapeutic contexts including, for example, those relating to
the treatment or prevention of neoplastic diseases, immune system
diseases (e.g., autoimmune diseases and inflammation), infectious
diseases, metabolic diseases, neuronal/neurological diseases,
muscular/cardiovascular diseases, diseases associated with aberrant
hematopoiesis, diseases associated with aberrant angiogenesis,
diseases associated with aberrant cell survival, and others.
[0311] For example, in certain illustrative embodiments, the AspRS
compositions of the invention may be used to modulate angiogenesis,
e.g., via modulation of endothelial cell proliferation and/or
signaling. Endothelial cell proliferation and/or cell signaling may
be monitored using an appropriate cell line (e.g., Human
microvascular endothelial lung cells (HMVEC-L) and Human umbilical
vein endothelial cells (HUVEC)), and using an appropriate assay
(e.g., endothelial cell migration assays, endothelial cell
proliferation assays, tube-forming assays, matrigel plug assays,
etc.), many of which are known and available in the art.
[0312] Therefore, in related embodiments, the compositions of the
invention may be employed in the treatment of essentially any cell
or tissue or subject that would benefit from modulation of
angiogenesis. For example, in some embodiments, a cell or tissue or
subject experiencing or susceptible to angiogenesis (e.g., an
angiogenic condition) may be contacted with a suitable composition
of the invention to inhibit an angiogenic condition. In other
embodiments, a cell or tissue experiencing or susceptible to
insufficient angiogenesis (e.g., an angiostatic condition) may be
contacted with an appropriate composition of the invention in order
to interfere with angiostatic activity and/or promote
angiogenesis.
[0313] Illustrative examples of angiogenic conditions include, but
are not limited to, age-related macular degeneration (AMD), cancer
(both solid and hematologic), developmental abnormalities
(organogenesis), diabetic blindness, endometriosis, ocular
neovascularization, psoriasis, rheumatoid arthritis (RA), and skin
disclolorations (e.g., hemangioma, nevus flammeus or nevus
simplex). Examples of anti-angiogenic conditions include, but are
not limited to, cardiovascular disease, restenosis, tissue damage
after reperfusion of ischemic tissue or cardiac failure, chronic
inflammation and wound healing.
[0314] The compositions of the invention may also be useful as
immunomodulators for treating anti- or pro-inflammatory indications
by modulating the cells that mediate, either directly or
indirectly, autoimmune and/or inflammatory disease, conditions and
disorders. The utility of the compositions of the invention as
immunomodulators can be monitored using any of a number of known
and available techniques in the art including, for example,
migration assays (e.g., using leukocytes or lymphocytes), cytokine
production assays, or cell viability assays (e.g., using B-cells,
T-cells, monocytes or NK cells).
[0315] "Inflammation" refers generally to the biological response
of tissues to harmful stimuli, such as pathogens, damaged cells
(e.g., wounds), and irritants. The term "inflammatory response"
refers to the specific mechanisms by which inflammation is achieved
and regulated, including, merely by way of illustration, immune
cell activation or migration, cytokine production, vasodilation,
including kinin release, fibrinolysis, and coagulation, among
others described herein and known in the art. Ideally, inflammation
is a protective attempt by the body to both remove the injurious
stimuli and initiate the healing process for the affected tissue or
tissues. In the absence of inflammation, wounds and infections
would never heal, creating a situation in which progressive
destruction of the tissue would threaten survival. On the other
hand, excessive or chronic inflammation may associate with a
variety of diseases, such as hay fever, atherosclerosis, and
rheumatoid arthritis, among others described herein and known in
the art.
[0316] Clinical signs of chronic inflammation are dependent upon
duration of the illness, inflammatory lesions, cause and anatomical
area affected. (see, e.g., Kumar et al., Robbins Basic
Pathology--8.sup.th Ed., 2009 Elsevier, London; Miller, L M,
Pathology Lecture Notes, Atlantic Veterinary College,
Charlottetown, PEI, Canada). Chronic inflammation is associated
with a variety of pathological conditions or diseases, including,
for example, allergies, Alzheimer's disease, anemia, aortic valve
stenosis, arthritis such as rheumatoid arthritis and
osteoarthritis, cancer, congestive heart failure, fibromyalgia,
fibrosis, heart attack, kidney failure, lupus, pancreatitis,
stroke, surgical complications, inflammatory lung disease,
inflammatory bowel disease, atherosclerosis, neurological
disorders, diabetes, metabolic disorders, obesity, and psoriasis,
among others described herein and known in the art. Hence, AspRS
compositions may be used to treat or manage chronic inflammation,
modulate any of one or more of the individual chronic inflammatory
responses, or treat any one or more diseases or conditions
associated with chronic inflammation.
[0317] Certain specific inflammatory responses include cytokine
production and activity, and related pathways. For instance,
certain exemplary embodiments relate to modulating cell-signaling
through nuclear factor-kB (NF-kB), such as by increasing the
downstream activities of this transcription factor. In certain
instances, increases in NF-kB activity can lead to increases in
cytokine signaling or activity, such as pro-inflammatory cytokines
(e.g., TNF-.alpha.), and anti-inflammatory cytokines (e.g.,
IL-10).
[0318] Criteria for assessing the signs and symptoms of
inflammatory and other conditions, including for purposes of making
differential diagnosis and also for monitoring treatments such as
determining whether a therapeutically effective dose has been
administered in the course of treatment, e.g., by determining
improvement according to accepted clinical criteria, will be
apparent to those skilled in the art and are exemplified by the
teachings of e.g., Berkow et al., eds., The Merck Manual, 16.sup.th
edition, Merck and Co., Rahway, N.J., 1992; Goodman et al., eds.,
Goodman and Gilman's The Pharmacological Basis of Therapeutics,
10.sup.th edition, Pergamon Press, Inc., Elmsford, N.Y., (2001);
Avery's Drug Treatment: Principles and Practice of Clinical
Pharmacology and Therapeutics, 3rd edition, ADIS Press, Ltd.,
Williams and Wilkins, Baltimore, Md. (1987); Ebadi, Pharmacology,
Little, Brown and Co., Boston, (1985); Osolci al., eds.,
Remington's Pharmaceutical Sciences, 18.sup.th edition, Mack
Publishing Co., Easton, Pa. (1990); Katzung, Basic and Clinical
Pharmacology, Appleton and Lange, Norwalk, Conn. (1992).
[0319] Also included are methods of modulating an immune response,
such as an innate immune response. As used herein, the term "immune
response" includes a measurable or observable reaction to an
antigen, vaccine composition, or immunomodulatory molecule mediated
by one or more cells of the immune system. An immune response
typically begins with an antigen or immunomodulatory molecule
binding to an immune system cell. A reaction to an antigen or
immunomodulatory molecule may be mediated by many cell types,
including a cell that initially binds to an antigen or
immunomodulatory molecule and cells that participate in mediating
an innate, humoral, cell-mediated immune response.
[0320] An "innate immune response," as used herein, may involve
binding of pathogen-associated molecular patterns (PAMPs) or an
AspRS polypeptide to cell surface receptors, such as toll-like
receptors. Activation of toll-like receptors and Ipaf-signaling
pathways in response to PAMPs or other signals leads to the
production of immunomodulatory molecules, such as cytokines and
co-stimulatory molecules, which induce and/or enhance an immune
response. Cells involved in the innate immune response include, for
example, dendritic cells, macrophages, natural killer cells, and
neutrophils, among others.
[0321] Certain embodiments relate to increasing an innate immune
response. Other embodiments relate to decreasing an innate immune
response. In certain aspects, an innate immune response is mediated
by one or more toll-like receptors (TLRs), such as TLR2 and/or
TLR4. Certain AspRS polypeptides of the invention bind to TLRS such
as TLR2 and/or TLR4. TLRs recognize PAMPs that distinguish
infectious agents from self and mediating the production of
immunomodulatory molecules, such as cytokines, necessary for the
development of effective adaptive immunity (Aderem, A. and
Ulevitch, R. J. Nature 406: 782-787 (2000) and Brightbill, H. D.,
Immunology 101: 1-10 (2000), herein incorporated by reference).
Members of the toll-like receptor family recognize a variety of
antigen types and can discriminate between pathogens. For example,
TLR2 recognizes various fungal, Gram-positive, and mycobacterial
components, TLR4 recognizes the Gram-negative product
lipopolysaccharide (LPS), and TLR9 recognizes nucleic acids such as
CpG repeats in bacterial DNA.
[0322] AspRS compositions that stimulate innate immunity (e.g., via
TLR2 and/r TLR4) can be useful in the treatment of a wide variety
of conditions, either alone or in combination with other therapies.
Specific examples of such conditions include infectious diseases,
such as bacterial, viral, and parasitic infectious diseases. AspRS
compositions that stimulate innate immunity can also be useful as
vaccine adjuvants, to enhance a subject's immune response to the
primary antigen, whether in a live, attenuated, or other type of
vaccine.
[0323] Examples of viral infectious diseases or agents (and their
corresponding vaccines) include, but are not limited to, Hepatitis
A, Hepatitis B, Hepatitis C, Hepatitis E, Caliciviruses associated
diarrhoea, Rotavirus diarrhoea, Haemophilus influenzae B pneumonia
and invasive disease, influenza, measles, mumps, rubella,
Parainfluenza associated pneumonia, Respiratory syncytial virus
(RSV) pneumonia, Severe Acute Respiratory Syndrome (SARS), Human
papillomavirus, Herpes simplex type 2 genital ulcers, HIV/AIDS,
Dengue Fever, Japanese encephalitis, Tick-borne encephalitis,
West-Nile virus associated disease, Yellow Fever, Epstein-Barr
virus, Lassa fever, Crimean-Congo haemorrhagic fever, Ebola
haemorrhagic fever, Marburg haemorrhagic fever, Rabies, Rift Valley
fever, Smallpox, leprosy, upper and lower respiratory infections,
poliomyelitis, among others described elsewhere herein.
[0324] Examples of bacterial infections disease or agents include,
but are not limited to, Bacillus antracis, Borellia burgdorferi,
Brucella abortus, Brucella canus, Brucella melitensis, Brucella
suis, Campylobacter jejuni, Chlamydia pneumoniae, Chlamydia
psitacci, Chlamydia trachomatis, Clostridium botulinum, C.
difficile, C. perfringens, C. tetani, Corynebacterium diphtheriae
(i.e., diphtheria), Enterococcus, Escherichia coli, Haemophilus
influenza, Helicobacter pylori, Legionella pneumophila, Leptospira,
Listeria monocytogenes, Mycobacterium leprae, M. tuberculosis,
Mycoplasma pneumoniae, Neisseria gonorrhea, N. meningitidis,
Pseudomonas aeruginosa, Rickettsia recketisii, Salmonella typhi, S.
typhimurium, Shigella sonnei, Staphylococcus aureus, S.
epidermidis, S. saprophyticus, Streptococcus agalactiae, S.
pneumoniae, S. pyogenes, Treponema pallidum, Vibrio cholera,
Yersinia pestis, Bordatella pertussis, and otitis media (e.g.,
often caused by Streptococcus pneumoniae, Haemophilus influenzae,
or Moraxella catarrhalis), among others described elsewhere
herein.
[0325] Examples of parasitic infectious diseases include, but are
not limited to, Amoebiasis (e.g., Entemoeba histolytica), Hookworm
Disease (e.g., nematode parasites such as Necator americanus and
Ancylostoma duodenale), Leishmaniasis, Malaria (four species of the
protozoan parasite Plasmodium; P. falciparum, P. vivax, P. ovale,
and P. malariae), Schistosomiasis (parasitic Schistosoma; S.
mansoni, S. haematobium, and S. japonicum), Onchocerca volvulus
(River blindness), Trypanosoma cruzi (Chagas disease/American
sleeping sickness), and Dracunculus medinensis, lymphatic
filariasis.
[0326] Certain AspRS compositions may be useful in the treatment or
reduction of endotoxic shock, which often results from exposure to
foreign antigens, such as lipopolysaccharide (LPS). Because
endotoxic shock can be mediated by TLR signaling, and
naturally-occurring endogenous AspRS fragments may stimulate TLRs,
certain of the binding agents, antisense agents, or RNAi agents
provided herein may render a subject more resistant to endotoxic
shock by antagonizing or otherwise reducing the endogenous AspRS
fragment-mediated stimulation of TLR2 and/or TLR4.
[0327] Also included are methods of treating immune diseases.
Illustrative immune system diseases, disorders or conditions that
may be treated according to the present invention include, but are
not limited to, primary immunodeficiencies, immune-mediated
thrombocytopenia, Kawasaki syndrome, bone marrow transplant (for
example, recent bone marrow transplant in adults or children),
chronic B cell lymphocytic leukemia, HIV infection (for example,
adult or pediatric HIV infection), chronic inflammatory
demyelinating polyneuropathy, post-transfusion purpura, and the
like.
[0328] Additionally, further diseases, disorders and conditions
include Guillain-Barre syndrome, anemia (for example, anemia
associated with parvovirus B19, patients with stable multiple
myeloma who are at high risk for infection (for example, recurrent
infection), autoimmune hemolytic anemia (for example, warm-type
autoimmune hemolytic anemia), thrombocytopenia (for example,
neonatal thrombocytopenia), and immune-mediated neutropenia),
transplantation (for example, cytomegalovirus (CMV)-negative
recipients of CMV-positive organs), hypogammaglobulinemia (for
example, hypogammaglobulinemic neonates with risk factor for
infection or morbidity), epilepsy (for example, intractable
epilepsy), systemic vasculitic syndromes, myasthenia gravis (for
example, decompensation in myasthenia gravis), dermatomyositis, and
polymyositis.
[0329] Further autoimmune diseases, disorders and conditions
include but are not limited to, autoimmune hemolytic anemia,
autoimmune neonatal thrombocytopenia, idiopathic thrombocytopenia
purpura, autoimmunocytopenia, hemolytic anemia, antiphospholipid
syndrome, dermatitis, allergic encephalomyelitis, myocarditis,
relapsing polychondritis, rheumatic heart disease,
glomerulonephritis (for example, IgA nephropathy), multiple
sclerosis, neuritis, uveitis ophthalmia, polyendocrinopathies,
purpura (for example, Henloch-Scoenlein purpura), Reiter's disease,
stiff-man syndrome, autoimmune pulmonary inflammation,
Guillain-Barre Syndrome, insulin dependent diabetes mellitus, and
autoimmune inflammatory eye disease.
[0330] Additional autoimmune diseases, disorders or conditions
include, but are not limited to, autoimmune thyroiditis;
hypothyroidism, including Hashimoto's thyroiditis and thyroiditis
characterized, for example, by cell-mediated and humoral thyroid
cytotoxicity; SLE (which is often characterized, for example, by
circulating and locally generated immune complexes); Goodpasture's
syndrome (which is often characterized, for example, by
anti-basement membrane antibodies); pemphigus (which is often
characterized, for example, by epidermal acantholytic antibodies);
receptor autoimmunities such as, for example, Graves' disease
(which is often characterized, for example, by antibodies to a
thyroid stimulating hormone receptor; myasthenia gravis, which is
often characterized, for example, by acetylcholine receptor
antibodies); insulin resistance (which is often characterized, for
example, by insulin receptor antibodies); autoimmune hemolytic
anemia (which is often characterized, for example, by phagocytosis
of antibody-sensitized red blood cells); and autoimmune
thrombocytopenic purpura (which is often characterized, for
example, by phagocytosis of antibody-sensitized platelets).
[0331] Further autoimmune diseases, disorders or conditions
include, but are not limited to, rheumatoid arthritis (which is
often characterized, for example, by immune complexes in joints);
scleroderma with anti-collagen antibodies (which is often
characterized, for example, by nucleolar and other nuclear
antibodies); mixed connective tissue disease, (which is often
characterized, for example, by antibodies to extractable nuclear
antigens, for example, ribonucleoprotein);
polymyositis/dermatomyositis (which is often characterized, for
example, by nonhistone anti-nuclear antibodies); pernicious anemia
(which is often characterized, for example, by antiparietal cell,
antimicrosome, and anti-intrinsic factor antibodies); idiopathic
Addison's disease (which is often characterized, for example, by
humoral and cell-mediated adrenal cytotoxicity); infertility (which
is often characterized, for example, by antispennatozoal
antibodies); glomerulonephritis (which is often characterized, for
example, by glomerular basement membrane antibodies or immune
complexes); by primary glomerulonephritis, by IgA nephropathy;
bullous pemphigoid (which is often characterized, for example, by
IgG and complement in the basement membrane); Sjogren's syndrome
(which is often characterized, for example, by multiple tissue
antibodies and/or the specific nonhistone antinuclear antibody
(SS-B)); diabetes mellitus (which is often characterized, for
example, by cell-mediated and humoral islet cell antibodies); and
adrenergic drug resistance, including adrenergic drug resistance
with asthma or cystic fibrosis (which is often characterized, for
example, by beta-adrenergic receptor antibodies).
[0332] Still further autoimmune diseases, disorders or conditions
include, but are not limited to chronic active hepatitis (which is
often characterized, for example by smooth muscle antibodies);
primary biliary cirrhosis (which is often characterized, for
example, by anti-mitochondrial antibodies); other endocrine gland
failure (which is characterized, for example, by specific tissue
antibodies in some cases); vitiligo (which is often characterized,
for example, by anti-melanocyte antibodies); vasculitis (which is
often characterized, for example, by immunoglobulin and complement
in vessel walls and/or low serum complement); post-myocardial
infarction conditions (which are often characterized, for example,
by anti-myocardial antibodies); cardiotomy syndrome (which is often
characterized, for example, by anti-myocardial antibodies);
urticaria (which is often characterized, for example, by IgG and
IgM antibodies to IgE); atopic dermatitis (which is often
characterized, for example, by IgG and IgM antibodies to IgE);
asthma (which is often characterized, for example, by IgG and IgM
antibodies to IgE); inflammatory myopathies; and other
inflammatory, granulomatous, degenerative, and atrophic
disorders.
[0333] Also included are methods of modulating hematopoiesis and
related conditions. Examples of hematopoietic processes that may be
modulated by the AspRS polypeptides of the invention include,
without limitation, the formation of myeloid cells (e.g., erythroid
cells, mast cells monocytes/macrophages, myeloid dendritic cells,
granulocytes such as basophils, neutrophils, and eosinophils,
megakaryocytes, platelets) and lymphoid cells (e.g., natural killer
cells, lymphoid dendritic cells, B-cells, and T-cells). Certain
specific hematopoietic processes include erythropoiesis,
granulopoiesis, lymphopoiesis, megakaryopoiesis, thrombopoiesis,
and others. Also included are methods of modulating the trafficking
or mobilization of hematopoietic cells, including hematopoietic
stem cells, progenitor cells, erythrocytes, granulocytes,
lymphocytes, megakaryocytes, and thrombocytes.
[0334] The methods of modulating hematopoiesis may be practiced in
vivo, in vitro, ex vivo, or in any combination thereof. These
methods can be practiced on any biological sample, cell culture, or
tissue that contains hematopoietic stem cells, hematopoietic
progenitor cells, or other stem or progenitor cells that are
capable of differentiating along the hematopoietic lineage (e.g.,
adipose tissue derived stem cells). For in vitro and ex vivo
methods, stem cells and progenitor cells, whether of hematopoietic
origin or otherwise, can be isolated and/or identified according to
the techniques and characteristics described herein and known in
the art.
[0335] In other embodiments, the AspRS compositions of the
invention may be used to modulate cellular proliferation and/or
survival and, accordingly, for treating or preventing diseases,
disorders or conditions characterized by abnormalities in cellular
proliferation and/or survival. For example, in certain embodiments,
the AspRS compositions may be used to modulate apoptosis and/or to
treat diseases or conditions associated with abnormal apoptosis.
Apoptosis is the term used to describe the cell signaling cascade
known as programmed cell death. Various therapeutic indications
exist for molecules that induce apoptosis (e.g. cancer), as well as
those that inhibit apoptosis (i.e. stroke, myocardial infarction,
sepsis, etc.). Apoptosis can be monitored by any of a number of
available techniques known and available in the art including, for
example, assays that measure fragmentation of DNA, alterations in
membrane asymmetry, activation of apoptotic caspases and/or release
of cytochrome C and AIF.
[0336] Illustrative diseases associated with increased cell
survival, or the inhibition of apoptosis include, but are not
limited to, cancers (such as follicular lymphomas, carcinomas, and
hormone-dependent tumors, including, but not limited to colon
cancer, cardiac tumors, pancreatic cancer, melanoma,
retinoblastoma, glioblastoma, lung cancer, intestinal cancer,
testicular cancer, stomach cancer, neuroblastoma, myxoma, myoma,
lymphoma, endothelioma, osteoblastoma, osteoclastoma, osteosarcoma,
chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposi's
sarcoma and ovarian cancer); autoimmune disorders (such as,
multiple sclerosis, Sjogren's syndrome, Graves' disease,
Hashimoto's thyroiditis, autoimmune diabetes, biliary cirrhosis,
Behcet's disease, Crohn's disease, polymyositis, systemic lupus
erythematosus and immune-related glomerulonephritis, autoimmune
gastritis, autoimmune thrombocytopenic purpura, and rheumatoid
arthritis) and viral infections (such as herpes viruses, pox
viruses and adenoviruses), inflammation, graft vs. host disease
(acute and/or chronic), acute graft rejection, and chronic graft
rejection.
[0337] Further illustrative diseases or conditions associated with
increased cell survival include, but are not limited to,
progression and/or metastases of malignancies and related disorders
such as leukemia (including acute leukemias (for example, acute
lymphocytic leukemia, acute myelocytic leukemia, including
myeloblastic, promyelocytic, myelomonocytic, monocytic, and
erythroleukemia)) and chronic leukemias (for example, chronic
myelocytic (granulocytic) leukemia and chronic lymphocytic
leukemia), myelodysplastic syndrome polycythemia vera, lymphomas
(for example, Hodgkin's disease and non-Hodgkin's disease),
multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain
diseases, and solid tumors including, but not limited to, sarcomas
and carcinomas such as fibrosarcoma, myxosarcoma, liposarcoma,
chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,
endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,
synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,
rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast
cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,
basal cell carcinoma, adenocarcinoma, sweat gland carcinoma,
sebaceous gland carcinoma, papillary carcinoma, papillary
adenocarcinomas, cystadenocarcinoma, medullary carcinoma,
bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct
carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's
tumor, cervical cancer, testicular tumor, lung carcinoma, small
cell lung carcinoma, bladder carcinoma, epithelial carcinoma,
glioma, astrocytoma, medulloblastoma, craniopharyngioma,
ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,
oligodendroglioma, menangioma, melanoma, neuroblastoma, and
retinoblastoma.
[0338] Illustrative diseases associated with increased apoptosis
include, but are not limited to, AIDS (such as HIV-induced
nephropathy and HIV encephalitis), neurodegenerative disorders
(such as Alzheimer's disease, Parkinson's disease, amyotrophic
lateral sclerosis, retinitis pigmentosa, cerebellar degeneration
and brain tumor or prior associated disease), autoimmune disorders
such as multiple sclerosis, Sjogren's syndrome, Graves' disease,
Hashimoto's thyroiditis, autoimmune diabetes, biliary cirrhosis,
Behcet's disease, Crohn's disease, polymyositis, systemic lupus
erythematosus, immune-related glomerulonephritis, autoimmune
gastritis, thrombocytopenic purpura, and rheumatoid arthritis,
myelodysplastic syndromes (such as aplastic anemia), graft vs. host
disease (acute and/or chronic), ischemic injury (such as that
caused by myocardial infarction, stroke and reperfusion injury),
liver injury or disease (for example, hepatitis related liver
injury, cirrhosis, ischemia/reperfusion injury, cholestosis (bile
duct injury) and liver cancer), toxin-induced liver disease (such
as that caused by alcohol), septic shock, ulcerative colitis,
cachexia, and anorexia.
[0339] In still further embodiments, the compositions of the
invention may be used in the treatment of neuronal/neurological
diseases or disorders, illustrative examples of which include
Parkinson's disease, Alzheimer's disease, Pick's disease,
Creutzfeldt-Jacob disease, Huntington's chorea, alternating
hemiplegia, amyotrophic lateral sclerosis, ataxia, cerebral palsy,
chronic fatigue syndrome, chronic pain syndromes, congenital
neurological anomalies, cranial nerve diseases, delirium, dementia,
demyelinating diseases, dysautonomia, epilepsy, headaches,
Huntington's disease, hydrocephalus, meningitis, movement
disorders, muscle diseases, nervous system neoplasms,
neurocutaneous syndromes, neurodegenerative diseases, neurotoxicity
syndromes, ocular motility disorders, peripheral nervous system
disorders, pituitary disorders, porencephaly, Rett syndrome, sleep
disorders, spinal cord disorders, stroke, sydenham's chorea,
tourette syndrome, nervous system trauma and injuries, etc.
[0340] Furthermore, additional embodiments relate to the use of the
compositions of the invention in the treatment of metabolic
disorders such as adrenoleukodystrophy, Krabbe's disease (globoid
cell leukodystrophy), metachromatic leukodystrophy, Alexander's
disease, Canavan's disease (spongiform leukodystrophy),
Pelizaeus-Merzbacher disease, Cockayne's syndrome, Hurler's
disease, Lowe's syndrome, Leigh's disease, Wilson's disease,
Hallervorden-Spatz disease, Tay-Sachs disease, etc. The utility of
the compositions of the invention in modulating metabolic processes
may be monitored using any of a variety of techniques known and
available in the art including, for example, assays which measure
adipocyte lipogenesis or adipocyte lipolysis.
[0341] In more specific embodiments of the invention, the AspRS
compositions of the invention may be used to modulate cellular
signaling, for example, via cell signaling proteins (e.g., Akt).
Cell signaling may be monitored using any of a number of well known
assays. For example, the induction of general cell signaling events
can be monitored through altered phosphorylation patterns of a
variety of target proteins. Detection of cell signaling activities
in response to treatment of cells with AspRS polypeptides therefore
serves as an indicator of distinct biological effects. Target
proteins used for this assay may be selected so as to encompass key
components of major cellular signaling cascades, thereby providing
a broad picture of the cell signaling landscape and its therapeutic
relevance. Generally, such assays involve cell treatment with AspRS
polypeptides followed by immunodetection with antibodies that
specifically detect the phosphorylated (activated) forms of the
target proteins.
[0342] Illustrative target proteins used for monitoring
therapeutically relevant cell signaling events may include, but are
not limited to: p38 MAPK (mitogen-activated protein kinase;
activated by cellular stress and inflammatory cytokines; involved
in cell differentiation and apoptosis); SAPK/JNK (stress-activated
protein kinase/Jun-amino-terminal kinase; activated by cellular
stresses and inflammatory cytokines); Erk1/2, p44/42 MAPK
(mitogen-activated protein kinase Erk1 and Erk2; activated by wide
variety of extracellular signals; involved in regulation of cell
growth and differentiation); and Akt (activated by insulin and
various growth or survival factors; involved in inhibition of
apoptosis, regulation of glycogen synthesis, cell cycle regulation
and cell growth). General phosphorylation of tyrosine residues may
also be monitored as a general indicator of changes in cell
signaling mediated by phosphorylation.
[0343] Of course, it will be recognized that other classes of
proteins, such as cell adhesion molecules (e.g., cadherins,
integrins, claudins, catenins, selectins, etc.) and/or ion channel
proteins may also be assayed for monitoring cellular events or
activities modulated by the compositions of the invention.
[0344] In other specific embodiments of the invention, the AspRS
compositions of the invention may be used to modulate cytokine
production by cells, for example, by immune cells such as monocytes
and/or leukocytes. Cytokine production may be monitored using any
of a number of assays known in the art (i.e., RT-PCR, ELISA,
ELISpot, flow cytometry, etc.). Generally, such assays involve cell
treatment with AspRS polypeptides followed by detection of cytokine
mRNA or polypeptides to measure changes in cytokine production.
Detection of increases and/or decreases in cytokine production in
response to treatment of cells with AspRS polypeptides therefore
serves as an indicator of distinct biological effects. AspRS
polypeptides of the invention may induce, enhance, and/or inhibit
an immune or inflammatory response by modulating cytokine
production. For example, AspRS polypeptides and compositions of the
invention may be used to alter a cytokine profile (i.e., type 1 vs.
type 2) in a subject. Illustrative cytokines that may measured for
monitoring biological effects of the AspRS compositions include,
but are not limited to IL-1ra, IL-1.alpha., IL-1.beta., IL-2, IL-3,
IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-12p40, IL-15, IL-18,
IL-23 TGF-.beta., TNF-.alpha., IFN-.alpha., IFN-.beta.,
IFN-.gamma., RANTES, MIP-1.alpha., MIP-1.beta., MCP-1, GRO-.alpha.,
GM-CSF, G-CSF, etc.
[0345] All publications and patent applications cited in this
specification are herein incorporated by reference as if each
individual publication or patent application were specifically and
individually indicated to be incorporated by reference.
[0346] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be readily apparent to one of ordinary
skill in the art in light of the teachings of this invention that
certain changes and modifications may be made thereto without
departing from the spirit or scope of the appended claims. The
following examples are provided by way of illustration only and not
by way of limitation. Those of skill in the art will readily
recognize a variety of noncritical parameters that could be changed
or modified to yield essentially similar results.
EXAMPLES
Example 1
Generation of Human Aspartyl-tRNA Synthetase (AspRS) Fragments
[0347] Full-length recombinant human AspRS having an amino acid
sequence as set forth in SEQ ID NO: 1 was expressed and purified
from E. coli using nickel IMAC chromatography. To generate
fragments of AspRS by controlled proteolysis, the full-length
protein was treated with 42 nM human neutrophil elastase for 30
minutes before separation of the fragments by SDS-PAGE run in 4-12%
MOPS or 12% MES buffer (FIGS. 1C and D). Digestions run on SDS-PAGE
gels in 4-12% MOPS revealed only a single protein fragment at
approximately 19 kDa (FIG. 1C), while digestions run on SDS-PAGE
gels in 12% MES buffer revealed at least three additional smaller
peptide fragments between 3 and 6 kDa (FIG. 1D).
Example 2
AspRS Fragments Activate Akt in Endothelial Cells
[0348] Pools of AspRS fragments were generated by adding 42 nM
neutrophil elastase to 2 ug full-length recombinant AspRS for 30
minutes at 37.degree. C. Reactions were stopped by the addition of
alpha 1-antitrypsin (Serpin A1) in 10-fold excess of the protease.
Bovine aortic endothelial cells (bAEC) were treated with pools of
50 nM full-length AspRS protein uncleaved or cleaved with
neutrophil elastase. Cells were incubated with AspRS fragments for
10 and 15 minutes, harvested and subjected to Western blotting with
an antibody that specifically recognizes only the phosphorylated
(activated) form of the signaling molecule Akt. This treatment
resulted in strong, reproducible activation of Akt via
phosphorylation (FIGS. 2A and 2B). This effect is significant due
to the role of Akt in the regulation of apoptosis, glycogen
synthesis, cell cycle regulation, and cell growth.
Example 3
Identification of Neutrophil Elastase Cleavage Sites on AspRS
[0349] Fragments generated by cleavage with neutrophil elastase
(FIG. 1D) were analyzed using LC/MS/MS to determine accurate masses
for each fragment. In addition, individual fragments were excised
from an SDS-PAGE gel run in 4-12% MOPS or 12% MES buffer and
subjected to in-gel trypsin digestion followed by LC/MS/MS analysis
to identify the portion of the full-length protein from which the
fragment was generated and to identify non-trypsin cleavage sites
that could be attributed to neutrophil elastase. The identity of
these peptide boundaries is summarized in Table 2.
TABLE-US-00002 TABLE 2 AspRS peptide boundaries Whole mass Protease
N-term. C-term Fragment (Da) used boundary boundary D1 19437
elastase 1 154 18370 D2 21590 elastase 1 174 D3 4367 elastase 1 31
4468 D4 3309 elastase 399 425 D5 2517 elastase 413 476 D6 3479
elastase 397 425
Example 4
AspRS Fragment Increases TNF-.alpha. Secretion from PBMCs
[0350] Peripheral blood mononuclear cells (PBMCs) from healthy
donors were treated with 100 nM doses of full-length AspRS protein
and a fragment of AspRS, D1 (Table 2), for 24 hours. EMAPII
(Endothelial-monocyte-activating polypeptide II), which is known to
increase TNF-.alpha. secretion from PBMCs, was used as a positive
control. An increase in TNF-.alpha. secretion was observed in
response to the full-length AspRS and this increase was similar in
magnitude to that observed for the EMAPII positive control.
Unexpectedly, however, the D1 fragment of AspRS induced TNF-.alpha.
secretion at a level nearly 6-fold higher than that observed for
either full length AspRS or the EMAPII positive control (FIG. 3).
Thus, the D1 fragment of AspRS has a novel function that is largely
masked within the full length protein.
Example 5
AspRS FRAGMENT D1 Induces In Vitro Secretion of Cytokines Distinct
from Full Length AspRS
[0351] Full length AspRS (100 nM) or a fragment of AspRS, D1 (100
nM), were incubated with 1.times.10.sup.6 Peripheral Blood
Mononuclear Cells (PBMC) for 24 hours. After 24 hours of
incubation, supernatants were harvested, snap frozen in liquid
nitrogen and then analyzed for multiple cytokines. Supernatants
were measured for 27 distinct cytokines and compared to
buffer-treated PBMC supernatants. Error bars are representative of
2 biological replicates. As shown in FIG. 4, AspRS fragment D1
showed a large stimulation of numerous cytokines above and beyond
stimulations observed with full length AspRS (e.g., IL1-13, IL-6,
IL-8, IL-10, IL-12p40, MIP1-.alpha., MIP-1.beta., GRO-.alpha.,
MCP-1, and IL-1ra).
Example 6
AspRS Fragment Induces CD71 Marker Upregulation in Monocytes
[0352] Peripheral blood mononuclear cells (PBMC's) were isolated
from a normal blood donor. 1.5.times.10.sup.6 PBMC's were treated
with a 200 nM dose of the AspRS fragment D1 (consisting of the
first 154 amino acids of the full length protein) for 24 hours.
PBMC's treated with 10 .mu.g/mL of the plant lectin
phytohemagglutinin (PHA) served as a positive control. As shown in
FIG. 5, upregulation of the CD71 proliferation marker was seen in
the D1-treated gated monocytes after staining with an anti-CD71
antibody (Beckton-Dickinson) and analyzing the samples by flow
cytometry. There was no significant increase in CD71 upregulation
in the gated lymphocyte population of the same samples. Thus, D1
has a cell type specific ability to activate monocytes in a PBMC
mixture.
Example 7
AspRS Fragment Increases TNF-.alpha. Secretion from Monocytes and
Macrophages
[0353] Both monocyte (THP-1) and macrophage (RAW 264.7) cell lines
were treated with C-terminally tagged D1 (C-D1) or full length
AspRS(C-DRS) at 100 nM. Supernatant was collected at 2, 4, 8 and
24, hours and then analyzed for TNF-.alpha. secretion. As shown in
FIG. 6, the maximal amount of TNF-.alpha. secretion after treatment
with C-D1 was seen between 2 and 4 hours, but then decreased at 8
and 24 hours. TNF-.alpha. secretion following treatment with C-DRS
was negligible at all time points examined. The increase in
TNF-.alpha. secretion following treatment with C-D1 was
dose-dependent. In addition, treatment of cells with 100 nM, 50 nM,
25 nM, 12.5 nM, and 6 nM C-D1, N-D1, and C-DRS for 4 hours
demonstrated that only C-D1 treatment increased TNF-.alpha.
secretion.
Example 8
DRS Fragment D1 Induces Chemotaxis of a Macrophage Cell Line
[0354] To assess cell migration in vitro, 24-well Transwell
chambers with polycarbonate membranes (5 .mu.m pore size, Costar)
were coated with 0.5 mg/ml gelatin in PBS and allowed to air dry.
Detached RAW 264.7 cells (mouse monocyte/macrophage cell line) were
washed once with fresh DMEM and suspended into 2.times.10.sup.7
cells/ml with 0.1% BSA/DMEM. Full-length AspRS (DRS) or D1 was
diluted with 0.1% BSA/DMEM into different concentrations. RAW 264.7
cells were added to the upper chamber at 2.times.10.sup.6 cells in
100 .mu.l per well. The lower chambers were filled with 500 .mu.l
per well of media containing DRS or D1. After 24 hours at
37.degree. C., calcein AM (Invitrogen) was added to lower chambers
at a final concentration of 8 .mu.M to stain migrated cells.
Following a 30 minute incubation, cells that had not migrated were
removed from the upper surface of the Transwell membrane with a
cotton swab. Migrating cells on the lower membrane surface were
counted under fluorescence microscope in high power fields. As
shown in FIG. 7, D1 induced migration in a dose dependent manner,
whereas little to no migration was stimulated by full length AspRS
at the same concentrations.
Example 9
AspRS Fragment Induced TNF-.alpha. Secretion Macrophages can be
Inhibited by U0126
[0355] Macrophage (RAW 264.7) cells were pre-treated with the
small-molecule inhibitors U0126 or LY294022 at 100 nM for one hour
followed by treatment with D1 at 50 nM or LPS at 1 ng/ml for an
additional 4 hours. Supernatant was collected and analyzed for
TNF-.alpha. secretion. As shown in FIG. 8, secretion of TNF-.alpha.
was inhibited by U0126 in D1 and LPS treated cells. However,
LY294022 only inhibited TNF-.alpha. secretion in LPS treated
cells.
Example 10
AspRS FRAGMENT D1 Inhibits VEGF-Induced Angiogenesis
[0356] The purpose of this experiment was to evaluate the
anti-angiogenic activity of the D1 fragment of AspRS. D1 protein
was directly incorporated into Matrigel.RTM. plugs to determine
its' ability to inhibit VEGF-induced angiogenesis in a Modified
Matrigel.RTM. Plug Assay. Briefly, female NCR Nude mice (8
mice/group) were obtained that weighed 21-25 g on Day 1 of the
experiment. Air pouches were generated in test animals by injecting
1 ml air into the subcutaneous space between the scapulae on Days
1, 4, and 6 using a 27-gauge needle. On Day 7, 0.5 ml Matrigel.RTM.
(VWR) containing VEGF (Cell Sciences)+Saline, VEGF+Sutent (Pfizer
Pharmaceuticals), or VEGF+D1 protein was injected into the
previously created air pouches. On Day 13 (6 days after implant)
animals were euthanized and the Matrigel.RTM. plugs were excised,
photographed, and weighed. The primary endpoint used to evaluate
activity was the hemoglobin content per mg of wet Matrigel.RTM.
plug weight. As Shown in FIG. 9, D1 caused an inhibition of
VEGF-induced angiogenesis.
Example 11
C-Terminally Tagged AspRS Fragment Induced TNF-.alpha. Secretion in
Monocytes
[0357] Monocyte (THP-1) cells were treated with C- or N-terminally
tagged D1 or full length AspRS at 100 nM for four hours. After
which, supernatant was collected and analyzed for TNF-.alpha.
secretion. As shown in FIG. 10, induction of TNF-.alpha. secretion
was the greatest in cells treated with C-terminally tagged D1.
N-terminally tagged D1 induced a much smaller response, indicating
the N-terminus region of the D1 fragment likely plays an important
role in its cytokine activity. All other treatment groups had
significantly lower induction of TNF-.alpha. secretion.
Example 12
AsPRS Fragment D1 Contains a Mammalian-Specific Domain of Human
DRS
[0358] As shown in FIG. 11, a 32 amino acid peptide is found only
at the N-terminus of mammalian DRS and is not found in yeast DRS.
This region of the protein is dispensable for canonical tRNA
synthetase aminoacylation activity and is predicted to contain a
putative amphiphilic helix (reported in Jacobo-Molina and Yang
(1989), Escalante and Yang, J B C (1992)). Based on the observed
importance of the N-terminus of D1 in relation to its cytokine
activity, this unique region may be an important mediator of the
cytokine activity reported here for D1.
Example 13
Identification of Endogenous D1 Fragment from Macrophages
[0359] As illustrated in FIG. 12A, a fragment of AspRS was detected
in a mouse macrophage cell line (RAW264.7) using LC/MS/MS
proteomics analysis. FIG. 12A shows the steps by which RAW264.7
mouse macrophages were subjected to SDS-PAGE analysis; protein
bands were cut out and analyzed by LC MS/MS, and an N-terminal
fragment of AspRS was identified as D1. This mass spectral analysis
revealed that the D1 fragment comprises the N-terminal portion of
the 501 residue monomer unit of the AspRS homodimer (consisting
approximately of residues 1-171 of full-length AspRS). The D1
fragment includes the anticodon-binding domain of human AspRS (see
FIG. 12B), and has structural similarity to the EMAPII-cytokine
that contains a highly similar OB-fold domain. The EMAPII cytokine
is found as a distinct domain in p43 (a protein that is bound in
the multi-tRNA synthetases complex of mammalian cells) where, under
apoptotic conditions, it is resected and secreted to serve as an
immunomodulatory cytokine. A similar EMAPII-like domain exists in
the C-terminal region of human TyrRS. However, in contrast to
EMAP-II and the homologous domains found in TyrRS and p43, D1 has a
unique 22 amino acid extension at the N-terminus that is found only
in higher eukaryotes and forms an amphiphilic helix.
Example 14
Structural Analysis of AspRS
[0360] To better understand the structure and physiological origin
of D1, native human AspRS was crystallized and its 3-dimensional
structure was determined to a resolution of 1.9 .ANG. (see FIG.
12C). The part of the structure corresponding to D1 forms a
separate OB-fold-containing domain, while the C-terminal catalytic
domain quite resembles that of yeast and bacterial AspRS. The
linker encompassing residues 154 and 182 that connect the D1
fragment and the catalytic domain was structurally disordered,
suggesting its high flexibility. The flexibility of this linker
region and its apparent accessibility to proteases, suggested that
its cleavage by endogenous proteases should liberate D1 from native
AspRS. Treatment of recombinant native human AspRS with PMN
elastase confirmed this expectation by cleavage and clean release
of D1 at residue 154 (see Example 3).
Example 15
AspRS Fragment D1 Induces In Vivo and In Vitro Secretion of
Cytokines and Binds to Immune Cells
[0361] Macrophages are key players in innate immunity, and produce
and secrete a large number of protein cytokines including those
involved in cellular metabolism and inflammation. To probe the
possible connection between the D1 fragment of AspRS and
inflammation, D1 protein (10 mg/kg) was injected intravenously into
healthy mice, and changes were measured in inflammatory cytokines
(both pro- and anti-inflammatory) secreted into the bloodstream
relative to vehicle controls. Because human and mouse AspRS and D1
have 96.8% sequence identity, recombinant human AspRS and D1 was
used for all studies.
[0362] FIG. 13A shows in vivo TNF-.alpha. and IL-10 serum levels
from mice injected intravenously with 10 mg/kg D1. Mice show an
increase in TNF-.alpha. after 2 hours that is quickly cleared by 6
hours while IL-10 levels continue to increase.
[0363] To confirm these in vivo results, PBMCs representing a
mixture of both monocytes and lymphocytes and isolated from human
donors were also exposed to the D1 protein in vitro (as well as the
full-length AspRS protein) and tested the media for the secretion
of either TNF-.alpha. or IL-10 in response to treatment. Similar to
the effects observed in vivo, treatment with D1 resulted in
secretion of both TNF-.alpha. and IL-10 from this mixed cell
population (FIG. 13B). The effects were specific to D1 and were not
seen with native AspRS, illustrating the effects of isolating this
N-terminal domain from the parent tRNA synthetase by the process of
resection.
[0364] To investigate which cells within the PBMC mixture were
targeted by D1, its binding to the different subpopulations of
cells within the mixture was analyzed using flow cytometry. As
shown in FIG. 13C, robust binding of D1 to monocytes was observed,
with almost 100% of monocytes in the mixture bound by a D1
molecule. In contrast, no binding of native AspRS to monocytes was
detected (data not shown). Binding of the D1 protein was also
observed for a subset of lymphocytes (.about.14%). Further analysis
of the bound lymphocyte population revealed that D1 binding occurs
on both B cells (.about.80% of total B cells were bound) and T
cells (.about.20% of total T cells were bound) (see FIG. 13C,
inset). For both monocytes and lymphocytes, the effects were
specific to D1 and were not seen with native AspRS (data not
shown). These data support a role for D1 in directly binding and
perhaps modulating cells involved in the immune response.
Example 16
D1 Signals Through Nuclear Factor-kB (NF-kB)
[0365] Nuclear factor-kB (NF-kB) is a transcription factor thought
to play an important role in onset of inflammation through
stimulating transcription of pro-inflammatory cytokines (like
TNF-.alpha.) and, during the resolution of inflammation, to then
stimulate expression of anti-inflammatory cytokines like IL-10.
NF-KB also plays a central role in directing cellular responses to
many stimuli, including oxidative stress, viral and bacterial
pathogens, and inflammatory cytokines. The effects of D1 on the
activation of NF-KB in macrophages were therefore investigated.
[0366] For this experiment, RAW-Blue cells encoding an
NF-KB-inducible secreted embryonic alkaline phosphatase reporter
gene were incubated with PBS, D1, or full-length AspRS. As shown in
FIG. 14A, the treated cells showed a strong dose-dependent
activation of NF-KB with D1, as compared to the lack of activation
by full-length AspRS or PBS.
Example 17
D1 Binds to and Signals through TLR2 and TLR4
[0367] NF-KB can be triggered through a number of macrophage cell
surface receptors including the pattern-recognizing toll-like
receptors (TLRs). To investigate the potential link to the TLR
receptor family, 7 different HEK293 cell lines were stably
co-transfected with NF-KB inducible reporter genes (encoding
secreted embryonic alkaline phosphatase) and genes encoding a panel
of separate toll-like receptors (TLR2, TLR3, TLR4, TLR 5, TLR 7,
TLR 8, and TLR9). As shown in FIG. 14B, D1-induced activation of
NF-KB was observed only through TLR2 or TLR4 and not TLRs 3, 5, 7,
8, or 9.
[0368] Flow cytometry experiments were utilized to establish
whether D2 binds to TLR2 and/or TLR4. In these experiments,
V5-tagged D1 (100 nM) or AspRS (100 nM) was incubated with HEK293
cells stably expressing TLR2 or TLR4. Empty vector transfected
HEK293 cells served as the null binding control. Binding was
assessed by FITC-V5 detection using flow cytometry. FIG. 14C shows
that D1 bound strongly to stably transfected HEK cells
over-expressing TLR2 or TLR4, but bound much less so to HEK cells
that were transfected with vector alone, and which did not express
TLR2 or TLR4.
Example 18
The Amphiphilic Helix in D1 Activity
[0369] Prior work established LPSs as ligands for TLR2 and 4.
Indeed, a lipid A agonist (OM174) ligand for TLR2 and 4
demonstrated similar effects in vivo to what has been observed for
D1, namely, transient release of TNF-.alpha. (1-2 hours) and
subsequent increases in IL-10 secretion. D1 encodes an EMAP-II-like
OB-fold. Like D1, the EMAP-II domain, when released by proteolytic
cleavage from p43, stimulates secretion of TNF-.alpha. from
monocytes. The EMAP-II domain also shows additional activity on
neutrophils (stimulating migration and secretion of
myeloperoxidase). D1, however, did not act on neutrophils (data not
shown). One distinction between EMAP-II and D1 is the unique
amphiphilic helix contained within the first 22 amino acids of D1.
The role of the amphiphilic helix in D1 activity was therefore
investigated.
[0370] Initially, the entire amphiphilic helix region of 22 amino
acids was deleted from D1 to give .DELTA.22 D1. Certain point
mutations were also generated. For instance, as an amphiphilic
helix, the human D1 N-terminal helix contains positively charged
residues on one side of the helix and negatively charged residues
on the other. D1 of lower eukaryotes has a slightly longer helix
that is positively charged on both sides. The positively charged
residues of this helix have been demonstrated to strengthen tRNA
binding, with the consensus sequence LSKKXLKKXXK (SEQ ID NO:6)
being particularly important. The evolution of this helix from a
positively charged to an amphiphilic helix (through the progression
from lower to higher eukaryotic AspRSs) occurs via a concerted
switch of 3 highly conserved residues to create a cluster of
negative charges on one side of the helix that is strictly
conserved in higher eukaryotes (FIGS. 4A and B). It was
hypothesized that these negatively charged residues in particular,
when in the context of the EMAP-II-like OB-fold, may be contribute
to the activity of the novel 22-amino acid helix that is appended
to D1. To explore this possibility, substitutions were made at the
3 conserved residues of the negatively charged higher eukaryotic
cluster (E12, E16, D19) (see FIG. 15B) to switch them back to the
lower eukaryotic form (E12S, E16K, D19K) with its positively
charged cluster (SKK D1).
[0371] PBMCs were then treated with 50 nm D1, full-length AspRS,
the .DELTA.22 mutant, and charge mutants AAA and SKK. Compared to
intact D1, .DELTA.22 D1 induced very little TNF-.alpha. or IL-10
release from PBMCs (see FIG. 15C). The AAK and SKK D1 mutants also
had reduced (-4-fold for SKK) activity for both TNF-.alpha. and
IL-10 secretion, as compared to D1. These observations suggest a
role for the N-terminal amphiphilic helix in receptor binding.
[0372] In further support of this conclusion, an N-V5-D1, with the
V5-tag at the N-rather than the C-terminus, was also constructed
and tested in both TNF-.alpha. release and binding assays. The
N-V5-D1 was unable to bind or induce TNF-.alpha. secretion from
PBMCs (data not shown). Thus, the activity of D1 can be reduced
with a peptide fusion at the N-terminus, further supporting the
role of the N-terminal amphiphilic region in D1 activity.
Example 19
D1 Activity is not Due to Endotoxin Contamination
[0373] The recombinant D1 used in these studies was purified from
E. coli and shown to have an LPS-containing bacterial endotoxin
level of less than 12 EU/mg. Nonetheless, LPS is a strong stimulant
of TLR2 and TLR4 signaling, and experiments were performed to
remove any possibility of trace endotoxin being responsible for the
results seen with D1. For this purpose, a gene encoding D1 with a
secretion sequence was expressed in transfected HEK293 cells.
Conditioned media containing secreted D1 was collected,
concentrated, and incubated with PBMCs.
[0374] As shown in FIG. 16A, these media stimulated secretion of
TNF-.alpha., while media from cells transfected with vector alone
did not stimulate secretion. Further, D1-stimulated TNF-.alpha.
release was unaffected by polymyxin B, a known inactivator of
endotoxin (see FIG. 16B). In contrast, as also shown in FIG. 16B,
LPS-stimulated activation of secretion of TNF-.alpha. was
completely blocked by polymyxin B. As shown in FIG. 16C, D1 treated
with proteinase K, which completely digests proteins but not
endotoxin, resulted in complete abrogation of TNF-.alpha. activity
in PBMCs. D1 activity is therefore not due to endotoxin
contamination.
Example 20
A22 AspRS In Vivo Mouse Knock-in Experiments
[0375] Because the .DELTA.22 variant of AspRS does not stimulate
TNF-.alpha. secretion through TLR2 and TLR4, as shown above, the
generation of a .DELTA.22 AspRS knock-in mouse allows examination
of the physiological effects of the N-terminus of AspRS without
compromising the canonical and essential aminoacylation activity of
AspRS. Initial experiments focus one examining the potential
protective effects of removal of the .DELTA.22 region of AspRS,
which has been shown to contribute to AspRS activity as a TLR2 and
TLR4 endogenous ligand.
[0376] An endotoxic shock experiment is performed to test whether
mice with a .DELTA.22 AspRS knock-in are more resistant to
endotoxic shock because of their lack of an endogenous ligand for
TLR2 and TLR4. In this experiment, wild-type and .DELTA.22 AspRS
knock-in mice are injected intraperitoneally with LPS (1
.mu.g/mouse) in combination with D-GalN (20 mg) in a saline
solution of 200 .mu.L per dose. This is a commonly used model for
endotoxic shock or sepsis that results in near complete lethality
in wild-type mice (see Car et al., J Exp Med, 179:1437-44, 1994).
It is believed that by removing an endogenous pro-inflammatory
toll-like receptor ligand (i.e., the AspRS region represented by
.DELTA.22) the mice should be resistant to endotoxic shock induced
lethality.
[0377] An LPS tolerance experiment is performed to test whether
macrophages from .DELTA.22 AspRS mice are less tolerant to LPS
stimulation due to the lack of a desensitization of toll-like
receptor signaling. Macrophages from these knock-in mice should not
have been exposed to the pro-inflammatory effects of an endogenous
TLR2 & TLR4 ligand (i.e., the AspRS region represented by
.DELTA.22). To examine this possibility, wild-type and .DELTA.22
AspRS knock-in mouse peritoneal macrophages are stimulated ex vivo
with LPS (100 ng/mL) for 24 hrs, which results in an activation and
production of cytokines that can be analyzed by ELISA (see Sato et
al., J Immunol. 165:7096-101, 2000). Macrophages from the .DELTA.22
AspRS mice should demonstrate a stronger response to LPS due to the
lack of a pro-inflammatory signal that otherwise contributes to the
induction of tolerance.
[0378] As noted, the disclosure above is descriptive, illustrative
and exemplary and is not to be taken as limiting the scope defined
by the appended claims which follow.
Sequence CWU 1
1
231501PRTHomo sapiens 1Met Pro Ser Ala Ser Ala Ser Arg Lys Ser Gln
Glu Lys Pro Arg Glu1 5 10 15Ile Met Asp Ala Ala Glu Asp Tyr Ala Lys
Glu Arg Tyr Gly Ile Ser 20 25 30Ser Met Ile Gln Ser Gln Glu Lys Pro
Asp Arg Val Leu Val Arg Val 35 40 45Arg Asp Leu Thr Ile Gln Lys Ala
Asp Glu Val Val Trp Val Arg Ala 50 55 60Arg Val His Thr Ser Arg Ala
Lys Gly Lys Gln Cys Phe Leu Val Leu65 70 75 80Arg Gln Gln Gln Phe
Asn Val Gln Ala Leu Val Ala Val Gly Asp His 85 90 95Ala Ser Lys Gln
Met Val Lys Phe Ala Ala Asn Ile Asn Lys Glu Ser 100 105 110Ile Val
Asp Val Glu Gly Val Val Arg Lys Val Asn Gln Lys Ile Gly 115 120
125Ser Cys Thr Gln Gln Asp Val Glu Leu His Val Gln Lys Ile Tyr Val
130 135 140Ile Ser Leu Ala Glu Pro Arg Leu Pro Leu Gln Leu Asp Asp
Ala Val145 150 155 160Arg Pro Glu Ala Glu Gly Glu Glu Glu Gly Arg
Ala Thr Val Asn Gln 165 170 175Asp Thr Arg Leu Asp Asn Arg Val Ile
Asp Leu Arg Thr Ser Thr Ser 180 185 190Gln Ala Val Phe Arg Leu Gln
Ser Gly Ile Cys His Leu Phe Arg Glu 195 200 205Thr Leu Ile Asn Lys
Gly Phe Val Glu Ile Gln Thr Pro Lys Ile Ile 210 215 220Ser Ala Ala
Ser Glu Gly Gly Ala Asn Val Phe Thr Val Ser Tyr Phe225 230 235
240Lys Asn Asn Ala Tyr Leu Ala Gln Ser Pro Gln Leu Tyr Lys Gln Met
245 250 255Cys Ile Cys Ala Asp Phe Glu Lys Val Phe Ser Ile Gly Pro
Val Phe 260 265 270Arg Ala Glu Asp Ser Asn Thr His Arg His Leu Thr
Glu Phe Val Gly 275 280 285Leu Asp Ile Glu Met Ala Phe Asn Tyr His
Tyr His Glu Val Met Glu 290 295 300Glu Ile Ala Asp Thr Met Val Gln
Ile Phe Lys Gly Leu Gln Glu Arg305 310 315 320Phe Gln Thr Glu Ile
Gln Thr Val Asn Lys Gln Phe Pro Cys Glu Pro 325 330 335Phe Lys Phe
Leu Glu Pro Thr Leu Arg Leu Glu Tyr Cys Glu Ala Leu 340 345 350Ala
Met Leu Arg Glu Ala Gly Val Glu Met Gly Asp Glu Asp Asp Leu 355 360
365Ser Thr Pro Asn Glu Lys Leu Leu Gly His Leu Val Lys Glu Lys Tyr
370 375 380Asp Thr Asp Phe Tyr Ile Leu Asp Lys Tyr Pro Leu Ala Val
Arg Pro385 390 395 400Phe Tyr Thr Met Pro Asp Pro Arg Asn Pro Lys
Gln Ser Asn Ser Tyr 405 410 415Asp Met Phe Met Arg Gly Glu Glu Ile
Leu Ser Gly Ala Gln Arg Ile 420 425 430His Asp Pro Gln Leu Leu Thr
Glu Arg Ala Leu His His Gly Ile Asp 435 440 445Leu Glu Lys Ile Lys
Ala Tyr Ile Asp Ser Phe Arg Phe Gly Ala Pro 450 455 460Pro His Ala
Gly Gly Gly Ile Gly Leu Glu Arg Val Thr Met Leu Phe465 470 475
480Leu Gly Leu His Asn Val Arg Gln Thr Ser Met Phe Pro Arg Asp Pro
485 490 495Lys Arg Leu Thr Pro 50022322DNAHomo sapiens 2atctcgagat
agccgcagct ctcgcgatct ttctggagcc gcacctccac gcggagtccg 60agcgcgtgtg
ctgagacccc agggtcggga gggcggagac tgggagggag ggagaagccc
120ctttggcctg ccttacggaa gcctgcgagg gagggtggtg tccactgccc
agttccgtgt 180cccgatgccc agcgccagcg ccagccgcaa gagtcaggag
aagccgcggg agatcatgga 240cgcggcggaa gattatgcta aagagagata
tggaatatct tcaatgatac aatcacaaga 300aaaaccagat cgagttttgg
ttcgggttag agacttgaca atacaaaaag ctgatgaagt 360tgtttgggta
cgtgcaagag ttcatacaag cagagctaaa gggaaacagt gcttcttagt
420cctacgtcag cagcagttta atgtccaggc tcttgtggcg gtgggagacc
atgcaagcaa 480gcagatggtt aaatttgctg ccaacatcaa caaagagagc
attgtggatg tagaaggtgt 540tgtgagaaaa gtgaatcaga aaattggaag
ctgtacacag caagacgttg agttacatgt 600tcagaagatt tatgtgatca
gtttggctga accccgtctg cccctgcagc tggatgatgc 660tgttcggcct
gaggcagaag gagaagagga aggaagagct actgttaacc aggatacaag
720attagacaac agagtcattg atcttaggac atcaactagt caggcagtct
tccgtctcca 780gtctggcatc tgccatctct tccgagaaac tttaattaac
aaaggttttg tggaaatcca 840aactcctaaa attatttcag ctgccagtga
aggaggagcc aatgttttta ctgtgtcata 900ttttaaaaat aatgcatacc
tggctcagtc cccacagcta tataagcaaa tgtgcatttg 960tgctgatttt
gagaaggttt tctctattgg accagtattc agagcggaag actctaatac
1020ccatagacat ctaactgagt ttgttggttt ggacattgaa atggctttta
attaccatta 1080ccacgaagtt atggaagaaa ttgctgacac catggtacaa
atattcaaag gacttcaaga 1140aaggtttcag actgaaattc aaacagtgaa
taaacagttc ccatgtgagc cattcaaatt 1200tttggagcca actctaagac
tagaatattg tgaagcattg gctatgctta gggaagctgg 1260agtcgaaatg
ggagatgaag acgatctgag cacaccaaat gaaaagctgt tgggtcattt
1320ggtaaaggaa aagtatgata cagattttta tattcttgat aaatatccat
tggctgtaag 1380acctttctat accatgcctg acccaagaaa tcccaaacag
tccaactctt acgatatgtt 1440catgagagga gaagaaatat tgtcaggagc
tcaaagaata catgatcctc aactgctaac 1500agagagagct ttacatcatg
gaattgattt ggagaaaatt aaggcttaca ttgattcctt 1560ccgctttgga
gcccctcctc atgctggtgg aggcattgga ttggaacgag ttactatgct
1620gtttctggga ttgcataatg ttcgtcagac ctccatgttc cctcgtgatc
ccaaacgact 1680cactccttaa attcacactt tgccacttaa ctccagtgtg
gatgacagag cgagaccctg 1740cctcaaaaaa aaaaaaaaaa aaagaaagcc
acacttattc ttttcagtaa cctgctagtg 1800cacaggctgt actttaggta
cttaaaatat gcactagaat aaatttgcaa ggccctaaaa 1860tatcactgtt
atttttggag taattcagta taggttcgtt taaaagagat ttttataact
1920tcagacatgc atcagtagga aataacttga gaaattcata tggttatgtt
acaaattcat 1980attctgttac tacagtaaac gttaagagtt ttaaacagtt
aagattgtac aatttttctt 2040cttttctata ttacaagggc cccagtgtta
atgtcttaga ttttcagtat ttgaacttat 2100ttttttaaat tctgtcattg
agataagaat aattcaggta gcatctgaaa ttttaatgaa 2160tgtataattg
gcatatcatg gaaaattaac cagaaagtat cagttcttaa aagttatgcc
2220tagaaattat gtaaagctaa actactggtt agaaagtatt cagtgtaata
ttgtattaat 2280ttgttaaatt ctaaacttga atttcaataa aattttaaag ct
2322332PRTHomo sapiens 3Met Pro Ser Ala Ser Ala Ser Arg Lys Ser Gln
Glu Lys Pro Arg Glu1 5 10 15Ile Met Asp Ala Ala Glu Asp Tyr Ala Lys
Glu Arg Tyr Gly Ile Ser 20 25 30432PRTRattus norvegicus 4Met Pro
Ser Ala Asn Ala Ser Arg Lys Gly Gln Glu Lys Pro Arg Glu1 5 10 15Ile
Val Asp Ala Ala Glu Asp Tyr Ala Lys Glu Arg Tyr Gly Val Ser 20 25
30511PRTArtificial SequenceConsensus sequence of the positively
charged residues of the AspRS amphiphilic helix 5Leu Ser Lys Lys
Xaa Leu Lys Lys Xaa Xaa Lys1 5 10625PRTAnopheles gambiae 6Glu Glu
Pro Val Gly Ala Gly Ala Glu Ala Thr Ser Lys Lys Ala Ala1 5 10 15Lys
Lys Ala Ala Lys Asp Ala Glu Lys 20 25727PRTIxodes scapularis 7Asp
Glu Asn Ala Ala Leu Gly Pro Asp Gly Gln Pro Leu Ser Lys Lys1 5 10
15Ala Leu Lys Lys Gln Ala Lys Glu Gln Glu Lys 20 25819PRTLottia
gigantea 8Asp Gly Lys Pro Thr Ser Lys Lys Gly Leu Lys Lys Gln Gln
Lys Glu1 5 10 15Ala Glu Lys927PRTHelobdella robusta 9Asn Val Ser
Glu Ala Val Gly Gly Asp Asp Lys Pro Thr Ser Lys Lys1 5 10 15Ala Leu
Lys Lys Gln Gln Lys Glu Ala Glu Lys 20 251023PRTXenopus trapicalis
10Ser Ala Ala Ser Glu Glu Gln Ala Gln Ser Lys Lys Ala Leu Lys Lys1
5 10 15Gln Gln Lys Glu Ala Glu Lys 201126PRTTakifugu rubripes 11Ser
Cys Phe Ser Ala Ala Glu Ala Glu Gln Ala Gln Ser Lys Lys Gly1 5 10
15Leu Lys Lys Gln Gln Lys Glu Ala Glu Lys 20 251226PRTTetraodon
nigroviridis 12Asp Val Lys Gly Ala Ala Glu Glu Glu Gln Ala Gln Ser
Lys Lys Gly1 5 10 15Leu Lys Lys Gln Gln Lys Glu Ala Glu Lys 20
251327PRTGasterosteus aculeatus 13Glu Ala Pro Gly Ala Thr Glu Glu
Glu Gln Gln Ala Gln Ser Lys Lys1 5 10 15Gly Leu Lys Lys Gln Gln Lys
Glu Ala Glu Lys 20 251423PRTGallus gallus 14Pro Ser Ala Ala Gly Arg
Gly Gln Gly Lys Asp Arg Arg Pro Asp Asn1 5 10 15Glu Glu Gln Pro Ala
Ala Asp 201521PRTBos taurus 15Pro Ser Ala Ser Ala Ser Arg Lys Ser
Gln Glu Lys Pro Arg Glu Ile1 5 10 15Met Asp Ala Ala Glu
201620PRTRattus norvegicus 16Pro Ser Ala Asn Ala Ser Arg Lys Ser
Gln Glu Lys Pro Arg Glu Ile1 5 10 15Asp Ala Ala Glu 201721PRTMus
musculus 17Pro Ser Ala Ser Ala Ser Arg Lys Ser Gln Glu Lys Pro Arg
Glu Ile1 5 10 15Met Asp Ala Ala Glu 201821PRTProcavia capensis
18Pro Ser Ala Ser Ala Gly Arg Lys Asn Gln Glu Lys Pro Arg Glu Ile1
5 10 15Met Asp Ala Ala Glu 201921PRTMonodephis domestica 19Pro Ser
Ala Ile Ala Ser Arg Lys Thr Gln Glu Lys Pro Arg Glu Ile1 5 10 15Met
Asp Ala Ala Glu 202021PRTTarius syrichta 20Pro Ser Ala Ser Ala Ser
Arg Arg Ser Gln Glu Lys Pro Arg Glu Ile1 5 10 15Met Asp Ala Ala Glu
202121PRTPongo abelii 21Pro Ser Ala Ser Ala Ser Arg Lys Ser Gln Glu
Lys Pro Arg Glu Ile1 5 10 15Met Asp Ala Ala Glu 202221PRTPan
troglodytes 22Pro Ser Ala Asp Ala Ser Arg Lys Ser Gln Glu Lys Pro
Arg Glu Ile1 5 10 15Met Asp Ala Ala Glu 202321PRTHomo sapiens 23Pro
Ser Ala Ser Ala Cys Arg Lys Ser Gln Glu Lys Pro Arg Glu Ile1 5 10
15Met Asp Ala Ala Glu 20
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