U.S. patent application number 10/444925 was filed with the patent office on 2004-01-15 for modulation of ptp1b expression and signal transduction by rna interference.
This patent application is currently assigned to CEPTYR, Inc.. Invention is credited to Klinghoffer, Richard, Lewis, Stephen Patrick, Wilson, Linda K..
Application Number | 20040009946 10/444925 |
Document ID | / |
Family ID | 29586993 |
Filed Date | 2004-01-15 |
United States Patent
Application |
20040009946 |
Kind Code |
A1 |
Lewis, Stephen Patrick ; et
al. |
January 15, 2004 |
Modulation of PTP1B expression and signal transduction by RNA
interference
Abstract
Compositions and methods relating to small interfering RNA
(siRNA) polynucleotides are provided as pertains to modulation of
biological signal transduction. Shown are siRNA polynucleotides
that interfere with expression of PTP1B, a member of the protein
tyrosine phosphatase (PTP) class of enzymes that mediate signal
transduction. In certain preferred embodiments siRNA modulate
signal transduction pathways comprising human or murine PTP-1B and,
in certain further embodiments, insulin receptor and/or Jak2.
Modulation of PTP1B-mediated biological signal transduction has
uses in diseases associated with defects in cell proliferation,
cell differentiation and/or cell survival, such as metabolic
disorders (including diabetes and obesity), cancer, autoimmune
disease, infectious and inflammatory disorders and other
conditions.
Inventors: |
Lewis, Stephen Patrick;
(Mountlake Terrace, WA) ; Klinghoffer, Richard;
(Seattle, WA) ; Wilson, Linda K.; (Seattle,
WA) |
Correspondence
Address: |
SEED INTELLECTUAL PROPERTY LAW GROUP PLLC
701 FIFTH AVE
SUITE 6300
SEATTLE
WA
98104-7092
US
|
Assignee: |
CEPTYR, Inc.
Bothell
WA
98021
|
Family ID: |
29586993 |
Appl. No.: |
10/444925 |
Filed: |
May 23, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60462942 |
Apr 14, 2003 |
|
|
|
60383249 |
May 23, 2002 |
|
|
|
Current U.S.
Class: |
514/44A ;
536/23.1 |
Current CPC
Class: |
C12N 2310/53 20130101;
A61P 3/10 20180101; A61P 29/00 20180101; A61K 38/00 20130101; A61P
3/04 20180101; C12N 15/1137 20130101; C12N 2310/14 20130101; C12N
2310/3517 20130101; C12Y 301/03048 20130101; A61P 25/28 20180101;
C12N 2310/111 20130101 |
Class at
Publication: |
514/44 ;
536/23.1 |
International
Class: |
A61K 048/00; C07H
021/02 |
Claims
What is claimed is:
1. An isolated small interfering RNA (siRNA) polynucleotide,
comprising at least one nucleotide sequence selected from the group
consisting of SEQ ID NOS: 83-86, 88-91, 93-96, 98-101, 104-105, and
108-109.
2. The small interfering RNA polynucleotide of claim 1 that
comprises at least one nucleotide sequence selected from the group
consisting of SEQ ID NOS: 83-86, 88-91, 93-96, 98-101 and the
complementary polynucleotide thereto.
3. A small interfering RNA polynucleotide of either claim 1 or
claim 2 that is capable of interfering with expression of a PTP1B
polypeptide, wherein the PTP-1B polypeptide comprises an amino acid
sequence as set forth in a sequence selected from the group
consisting of GenBank Ace. Nos. M31724, NM.sub.--002827, and
M33689.
4. The siRNA polynucleotide of either claim 1 or claim 2 wherein
the nucleotide sequence of the siRNA polynucleotide differs by one,
two, three or four nucleotides at any of positions 1-19 of a
sequence selected from the group consisting of the sequences set
forth in SEQ ID NOS: 83-86, 88-91, 93-96, and 98-101, or at any
position of a sequence selected from the group consisting of the
sequences set forth in 104, 105, 108, and 109.
5. The siRNA polynucleotide of either claim 1 or claim 2 wherein
the nucleotide sequence of the siRNA polynucleotide differs by at
least two, three or four nucleotides at any of positions 1-19 of a
sequence selected from the group consisting of the sequences set
forth in SEQ ID NOS: 83-86, 88-91, 93-96, and 98-101, or at any
position of a sequence selected from the group consisting of the
sequences set forth in 104, 105, 108, and 109.
6. An isolated siRNA polynucleotide comprising a nucleotide
sequence according to SEQ ID NO: 83, or the complement thereof.
7. An isolated siRNA polynucleotide comprising a nucleotide
sequence according to SEQ ID NO: 88, or the complement thereof.
8. An isolated siRNA polynucleotide comprising a nucleotide
sequence according to SEQ ID NO: 93, or the complement thereof.
9. An isolated siRNA polynucleotide comprising a nucleotide
sequence according to SEQ ID NO: 98, or the complement thereof.
10. An isolated siRNA polynucleotide comprising a nucleotide
sequence according to SEQ ID NO: 104 or 105.
11. An isolated siRNA polynucleotide comprising a nucleotide
sequence according to SEQ ID NO: 108 or 109.
12. The siRNA polynucleotide of claim 1 or claim 2 wherein the
polynucleotide comprises at least one synthetic nucleotide analogue
of a naturally occurring nucleotide.
13. The siRNA polynucleotide of claim 1 or claim 2 wherein the
polynucleotide is linked to a detectable label.
14. The siRNA polynucleotide of claim 13 wherein the detectable
label is a reporter molecule.
15. The siRNA of claim 14 wherein the reporter molecule is selected
from the group consisting of a dye, a radionuclide, a luminescent
group, a fluorescent group, and biotin.
16. The siRNA polynucleotide of claim 15 wherein the fluorescent
group is fluorescein isothiocyanate.
17. The siRNA polynucleotide of claim 13 wherein the detectable
label is a magnetic particle.
18. A pharmaceutical composition comprising the siRNA
polynucleotide of either claim 1 or claim 2 and a physiologically
acceptable carrier.
19. The pharmaceutical composition of claim 18 wherein the carrier
comprises a liposome.
20. A recombinant nucleic acid construct comprising a
polynucleotide that is capable of directing transcription of a
small interfering RNA (siRNA), the polynucleotide comprising: (i) a
first promoter; (ii) a second promoter; and (iii) at least one DNA
polynucleotide segment comprising at least one nucleotide sequence
selected from the group consisting of SEQ ID NOS: 83-86, 88-91,
93-96, 98-101, or a complement thereto, wherein each DNA
polynucleotide segment and its complement are operably linked to at
least one of the first and second promoters, and wherein the
promoters are oriented to direct transcription of the DNA
polynucleotide segment and its reverse complement.
21. A recombinant nucleic acid construct comprising a
polynucleotide that is capable of directing transcription of a
small interfering RNA (siRNA), the polynucleotide comprising a
promoter operably linked to at least one DNA polynucleotide segment
comprising at least one nucleotide sequence that is selected from
the group consisting of SEQ ID NOs: 102, 103, 106, and 107.
22. The recombinant nucleic acid construct of either claim 20 or
21, comprising at least one enhancer that is selected from a first
enhancer operably linked to the first promoter and a second
enhancer operably linked to the second promoter.
23. The recombinant nucleic acid construct of claim 20, comprising
at least one transcriptional terminator that is selected from (i) a
first transcriptional terminator that is positioned in the
construct to terminate transcription directed by the first promoter
and (ii) a second transcriptional terminator that is positioned in
the construct to terminate transcription directed by the second
promoter.
24. The recombinant nucleic acid construct of either claim 20 or
claim 21 wherein the siRNA is capable of interfering with
expression of a PTP1B polypeptide, wherein the PTP1B polypeptide
comprises an amino acid sequence as set forth in a sequence
selected from the group consisting of GenBank Acc. Nos. M31724,
NM.sub.--002827, and M33689.
25. A recombinant nucleic acid construct comprising a
polynucleotide that is capable of directing transcription of a
small interfering RNA (siRNA), the polynucleotide comprising at
least one promoter and a DNA polynucleotide segment, wherein the
DNA polynucleotide segment is operably linked to the promoter, and
wherein the DNA polynucleotide segment comprises (i) at least one
DNA polynucleotide that comprises at least one nucleotide sequence
selected from the group consisting of SEQ ID NOS: 83-86, 88-91,
93-96, 98-101, or a complement thereto; (ii) a spacer sequence
comprising at least 4 nucleotides operably linked to the DNA
polynucleotide of (i); and (iii) the reverse complement of the DNA
polynucleotide of (i) operably linked to the spacer sequence.
26. The recombinant nucleic acid construct of claim 25 wherein the
siRNA comprises an overhang of at least one and no more than four
nucleotides, the overhang being located immediately 3' to
(iii).
27. The recombinant nucleic acid construct of claim 25 wherein the
spacer sequence comprises at least 9 nucleotides.
28. The recombinant nucleic acid construct of claim 25 wherein the
spacer sequence comprises two uridine nucleotides that are
contiguous with (iii).
29. The recombinant nucleic acid construct of claim 25 comprising
at least one transcriptional terminator that is operably linked to
the DNA polynucleotide segment.
30. A host cell transformed or transfected with the recombinant
nucleic acid construct of any one of claims 20, 21, 23 and
25-29.
31. A pharmaceutical composition comprising an siRNA polynucleotide
and a physiologically acceptable carrier, wherein the siRNA
polynucleotide is selected from the group consisting of: (i) an RNA
polynucleotide which comprises at least one nucleotide sequence
selected from the group consisting of SEQ ID NOS: 83-86, 88-91,
93-96, 98-101, 104-105, and 108-109, (ii) an RNA polynucleotide
that comprises at least one nucleotide sequence selected from the
group consisting of SEQ ID NOS: 83-86, 88-91, 93-96, 98-101 and the
complementary polynucleotide thereto, (iii) an RNA polynucleotide
according to (i) or (ii) wherein the nucleotide sequence of the
siRNA polynucleotide differs by one, two or three nucleotides at
any of positions 1-19 of a sequence selected from the group
consisting of the sequences set forth in SEQ ID NOS: 83-86, 88-91,
93-96, and 98-101, or at any position of a sequence selected from
the group consisting of the sequences set forth in SEQ ID NOS: 104,
105, 108, and 109, and (iv) an RNA polynucleotide according to (i)
or (ii) wherein the nucleotide sequence of the siRNA polynucleotide
differs by two, three or four nucleotides at any of positions 1-19
of a sequence selected from the group consisting of the sequences
set forth in SEQ ID NOS: 83-86, 88-91, 93-96, 98-101, or at any
position of a sequence selected from the group consisting of the
sequences set forth in SEQ ID NOS: 104, 105, 108, and 109.
32. The pharmaceutical composition of claim 31 wherein the carrier
comprises a liposome.
33. A method for interfering with expression of a PTP1B
polypeptide, or variant thereof, comprising contacting a subject
that comprises at least one cell which is capable of expressing a
PTP1B polypeptide with a siRNA polynucleotide for a time and under
conditions sufficient to interfere with PTP1B polypeptide
expression, wherein: (a) the PTP1B polypeptide comprises an amino
acid sequence as set forth in a sequence selected from the group
consisting of GenBank Acc. Nos. M31724, NM.sub.--002827,
NM.sub.--011201, and M33689, (b) the siRNA polynucleotide is
selected from the group consisting of (i) an RNA polynucleotide
which comprises at least one nucleotide sequence selected from the
group consisting of SEQ ID NOS: 83-86, 88-91, 93-96, 98-101,
104-105, and 108-109, (ii) an RNA polynucleotide that comprises at
least one nucleotide sequence selected from the group consisting of
SEQ ID NOS: 83-86, 88-91, 93-96, 98-101, and the complementary
polynucleotide thereto, (iii) an RNA polynucleotide according to
(i) or (ii) wherein the nucleotide sequence of the siRNA
polynucleotide differs by one, two or three nucleotides at any of
positions 1-19 of a sequence selected from the group consisting of
the sequences set forth in SEQ ID NOS: 83-86, 88-91, 93-96, 98-101,
or at any position of a sequence selected from the group consisting
of the sequences set forth in SEQ ID NOS: 104, 105, 108, and 109,
and (iv) an RNA polynucleotide according to (i) or (ii) wherein the
nucleotide sequence of the siRNA polynucleotide differs by two,
three or four nucleotides at any of positions 1-19 of a sequence
selected from the group consisting of the sequences set forth in
SEQ ID NOS: 83-86, 88-91, 93-96, 98-101, or at any position of a
sequence selected from the group consisting of the sequences set
forth in SEQ ID NOS: 104, 105, 108, and 109.
34. A method for interfering with expression of a PTP1B polypeptide
that comprises an amino acid sequence as set forth in a sequence
selected from the group consisting of GenBank Ace. Nos. M31724,
NM.sub.--002827, and M33689, or a variant of said PTP1B
polypeptide, said method comprising contacting, under conditions
and for a time sufficient to interfere with PTP1B polypeptide
expression, (i) a subject that comprises at least one cell that is
capable of expressing the PTP1B polypeptide, and (ii) a recombinant
nucleic acid construct according to either claim 18 or claim
22.
35. A method for identifying a component of a PTP1B signal
transduction pathway comprising: A. contacting a siRNA
polynucleotide and a first biological sample comprising at least
one cell that is capable of expressing a PTP1B polypeptide, or a
variant of said PTP1B polypeptide, under conditions and for a time
sufficient for PTP1B expression when the siRNA polynucleotide is
not present, wherein (1) the PTP1B polypeptide comprises an amino
acid sequence as set forth in a sequence selected from the group
consisting of GenBank Acc. Nos. M31724, NM.sub.--002827, and
M33689, (2) the siRNA polynucleotide is selected from the group
consisting of (i) an RNA polynucleotide which comprises at least
one nucleotide sequence selected from the group consisting of SEQ
ID NOS: 83-86, 88-91, 93-96, 98-101, 104-105, and 108-109, (ii) an
RNA polynucleotide that comprises at least one nucleotide sequence
selected from the group consisting of SEQ ID NOS: 83-86, 88-91,
93-96, 98-101 and the complementary polynucleotide thereto, (iii)
an RNA polynucleotide according to (i) or (ii) wherein the
nucleotide sequence of the siRNA polynucleotide differs by one, two
or three nucleotides at any of positions 1-19 of a sequence
selected from the group consisting of the sequences set forth in
SEQ ID NOS: 83-86, 88-91, 93-96, 98-101, or at any position of a
sequence selected from the group consisting of the sequences set
forth in SEQ ID NOS: 104, 105, 108, and 109, and (iv) an RNA
polynucleotide according to (i) or (ii) wherein the nucleotide
sequence of the siRNA polynucleotide differs by two, three or four
nucleotides at any of positions 1-19 of a sequence selected from
the group consisting of the sequences set forth in SEQ ID NOS:
83-86, 88-91, 93-96, 98-101, or at any position of a sequence
selected from the group consisting of the sequences set forth in
SEQ ID NOS: 104, 105, 108, and 109; and B. comparing a level of
phosphorylation of at least one protein that is capable of being
phosphorylated in the cell with a level of phosphorylation of the
protein in a control sample that has not been contacted with the
siRNA polynucleotide, wherein an altered level of phosphorylation
of the protein in the presence of the siRNA polynucleotide relative
to the level of phosphorylation of the protein in an absence of the
siRNA polynucleotide indicates that the protein is a component of
the PTP1B signal transduction pathway.
36. The method of claim 35 wherein the signal transduction pathway
comprises a Jak2 kinase.
37. A method for modulating an insulin receptor protein
phosphorylation state in a cell, comprising contacting the cell
with a siRNA polynucleotide under conditions and for a time
sufficient to interfere with expression of a PTP1B polypeptide,
wherein (a) the PTP1B polypeptide comprises an amino acid sequence
as set forth in a sequence selected from the group consisting of
GenBank Acc. Nos. M31724, NM.sub.--002827, NM.sub.--011201, and
M33689, (b) the siRNA polynucleotide is selected from the group
consisting of (i) an RNA polynucleotide which comprises at least
one nucleotide sequence selected from the group consisting of SEQ
ID NOS: 83-86, 88-91, 93-96, 98-101 or the complements thereof and
SEQ ID NOS: 104, 105, 108, and 109, (ii) an RNA polynucleotide that
comprises at least one nucleotide sequence selected from the group
consisting of SEQ ID NOS: 83-86, 88-91, 93-96, 98-101 and the
complementary polynucleotide thereto, (iii) an RNA polynucleotide
according to (i) or (ii) wherein the nucleotide sequence of the
siRNA polynucleotide differs by one, two or three nucleotides at
any of positions 1-19 of a sequence selected from the group
consisting of the sequences set forth in SEQ ID NOS: 83-86, 88-91,
93-96, and 98-101, or at any position of a sequence selected from
the group consisting of the sequences set forth in SEQ ID NOS: 104,
105, 108, and 109, and (iv) an RNA polynucleotide according to (i)
or (ii) wherein the nucleotide sequence of the siRNA polynucleotide
differs by two, three or four nucleotides at any of positions 1-19
of a sequence selected from the group consisting of the sequences
set forth in SEQ ID NOS: 83-86, 88-91, 93-96, and 98-101, or at any
position of a sequence selected from the group consisting of the
sequences set forth in SEQ ID NOS: 104, 105, 108, and 109; and (c)
the insulin receptor protein comprises a polypeptide which
comprises an amino acid sequence selected from the group consisting
of SEQ ID NOS: __-__ or a variant thereof.
38. A method for altering Jak2 protein phosphorylation state in a
cell, comprising contacting the cell with a siRNA polynucleotide
under conditions and for a time sufficient to interfere with
expression of a PTP1B polypeptide, wherein (a) the PTP1B
polypeptide comprises an amino acid sequence as set forth in a
sequence selected from the group consisting of GenBank Ace. Nos.
M31724, NM.sub.--002827, NM.sub.--011201, and M33689, (b) the siRNA
polynucleotide is selected from the group consisting of (i) an RNA
polynucleotide which comprises at least one nucleotide sequence
selected from the group consisting of SEQ ID NOS: 83-86, 88-91,
93-96, and 98-101 or the complements thereof, and SEQ ID NOS: 104,
105, 108, and 109, (ii) an RNA polynucleotide that comprises at
least one nucleotide sequence selected from the group consisting of
SEQ ID NOS: 83-86, 88-91, 93-96, and 98-101 and the complementary
polynucleotide thereto, (iii) an RNA polynucleotide according to
(i) or (ii) wherein the nucleotide sequence of the siRNA
polynucleotide differs by one, two or three nucleotides at any of
positions 1-19 of a sequence selected from the group consisting of
the sequences set forth in SEQ ID NOS: 83-86, 88-91, 93-96, and
98-101, or at any position of a sequence selected from the group
consisting of the sequences set forth in SEQ ID NOS: 104, 105, 108,
and 109, and (iv) an RNA polynucleotide according to (i) or (ii)
wherein the nucleotide sequence of the siRNA polynucleotide differs
by two, three or four nucleotides at any of positions 1-19 of a
sequence selected from the group consisting of the sequences set
forth in SEQ ID NOS: 83-86, 88-91, 93-96, and 98-101, or at any
position of a sequence selected from the group consisting of the
sequences set forth in SEQ ID NOS: 104, 105, 108, and 109; and (c)
the Jak2 protein comprises a polypeptide which comprises an amino
acid sequence selected from the group consisting of SEQ ID NOS:
__-__, or a variant thereof.
39. A method for treating a Jak2-associated disorder comprising
administering to a subject in need thereof a pharmaceutical
composition according to claim 31, wherein the siRNA polynucleotide
inhibits expression of a PTP1B polypeptide, or a variant
thereof.
40. The method of claim 39 wherein the Jak2-associated disorder is
selected from the group consisting of diabetes, obesity,
hyperglycemia-induced apoptosis, inflammation, and a
neurodegenerative disorder.
41. An isolated small interfering RNA (siRNA) polynucleotide,
comprising an RNA polynucleotide which comprises at least one
nucleotide sequence selected from the group consisting of SEQ ID
NOS: 18-21, 33-36, 43-46, 53-56, and 58-61.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims the benefit of U.S. Provisional
Patent Application No. 60/383,249 filed May 23, 2002, and U.S.
Provisional Patent Application No. 60/462,942 filed Apr. 14, 2003,
which are incorporated herein by reference in their entirety.
BACKGROUND OF THE INVENTION
[0002] 1. Technical Field
[0003] The present invention relates generally to compositions and
methods useful for treating conditions associated with defects in
cell proliferation, cell differentiation, and cell survival. The
invention is more particularly related to double-stranded RNA
polynucleotides that interfere with expression of a protein
tyrosine phosphatase, PTP1B, and polypeptide variants thereof. The
present invention is also related to the use of such RNA
polynucleotides to alter activation of signal transduction pathway
components or to alter cellular metabolic processes that lead to
proliferative responses, cell differentiation and development, and
cell survival.
[0004] 2. Description of the Related Art
[0005] Reversible protein tyrosine phosphorylation, coordinated by
the action of protein tyrosine kinases (PTKs) that phosphorylate
certain tyrosine residues in polypeptides, and protein tyrosine
phosphatases (PTPs) that dephosphorylate certain phosphotyrosine
residues, is a key mechanism in regulating many cellular
activities. It is becoming apparent that the diversity and
complexity of the PTPs and PTKs are comparable, and that PTPs are
equally important in delivering both positive and negative signals
for proper function of cellular machinery. Regulated tyrosine
phosphorylation contributes to specific pathways for biological
signal transduction, including those associated with cell division,
cell survival, apoptosis, proliferation and differentiation.
Defects and/or malfunctions in these pathways may underlie certain
disease conditions for which effective means for intervention
remain elusive, including for example, malignancy, autoimmune
disorders, diabetes, obesity, and infection.
[0006] The protein tyrosine phosphatase (PTP) family of enzymes
consists of more than 100 structurally diverse proteins in
vertebrates, including almost 40 human PTPs that have in common the
conserved 250 amino acid PTP catalytic domain, but which display
considerable variation in their non-catalytic segments (Charbonneau
and Tonks, 1992 Annu. Rev. Cell Biol. 8:463-493; Tonks, 1993 Semin.
Cell Biol. 4:373-453; Andersen et al., Mol Cell Biol. 21:7117-36
(2001)). This structural diversity presumably reflects the
diversity of physiological roles of individual PTP family members,
which in certain cases have been demonstrated to have specific
functions in growth, development and differentiation (Desai et al.,
1996 Cell 84:599-609; Kishihara et al., 1993 Cell 74:143-156;
Perkins et al., 1992 Cell 70:225-236; Pingel and Thomas, 1989 Cell
58:1055-1065; Schultz et al., 1993 Cell 73:1445-1454). The PTP
family includes receptor-like and non-transmembrane enzymes that
exhibit exquisite substrate specificity in vivo and that are
involved in regulating a wide variety of cellular signaling
pathways (Andersen et al., Mol. Cell. Biol. 21:7117 (2001); Tonks
and Neel, Curr. Opin. Cell Biol. 13:182 (2001)). PTPs thus
participate in a variety of physiologic functions, providing a
number of opportunities for therapeutic intervention in physiologic
processes through alteration (i.e., a statistically significant
increase or decrease) or modulation (e.g., up-regulation or
down-regulation) of PTP activity.
[0007] Although recent studies have also generated considerable
information regarding the structure, expression and regulation of
PTPs, the nature of many tyrosine phosphorylated substrates through
which the PTPs exert their effects remains to be determined.
Studies with a limited number of synthetic phosphopeptide
substrates have demonstrated some differences in the substrate
selectivities of different PTPs (Cho et al., 1993 Protein Sci. 2:
977-984; Dechert et al., 1995 Eur. J. Biochem. 231:673-681).
Analyses of PTP-mediated dephosphorylation of PTP substrates
suggest that catalytic activity may be favored by the presence of
certain amino acid residues at specific positions in the substrate
polypeptide relative to the phosphorylated tyrosine residue
(Salmeen et al., 2000 Molecular Cell 6:1401; Myers et al., 2001 J.
Biol. Chem. 276:47771; Myers et al., 1997 Proc. Natl. Acad. Sci.
USA 94:9052; Ruzzene et al., 1993 Eur. J. Biochem. 211:289-295;
Zhang et al., 1994 Biochemistry 33:2285-2290). Thus, although the
physiological relevance of the substrates used in these studies is
unclear, PTPs display a certain level of substrate selectivity in
vitro.
[0008] The PTP family of enzymes contains a common evolutionarily
conserved segment of approximately 250 amino acids known as the PTP
catalytic domain. Within this conserved domain is a unique
signature sequence motif, CX.sub.5R (SEQ ID NO: __, that is
invariant among all PTPs. In a majority of PTPs, an 11 amino acid
conserved sequence ([I/V]HCXAGXXR[S/T)G (SEQ ID NO: 1)) containing
the signature sequence motif is found. The cysteine residue in this
motif is invariant in members of the family and is essential for
catalysis of the phosphotyrosine dephosphorylation reaction. It
functions as a nucleophile to attack the phosphate moiety present
on a phosphotyrosine residue of the incoming substrate. If the
cysteine residue is altered by site-directed mutagenesis to serine
(e.g., in cysteine-to-serine or "CS" mutants) or alanine (e.g.,
cysteine-to-alanine or "CA" mutants), the resulting PTP is
catalytically deficient but retains the ability to complex with, or
bind, its substrate, at least in vitro.
[0009] CS mutants of certain PTP family members, for example, MKP-1
(Sun et al., 1993 Cell 75:487), may effectively bind phosphotyrosyl
polypeptide substrates in vitro to form stable enzyme-substrate
complexes, thereby functioning as "substrate trapping" mutant PTPs.
Such complexes can be isolated from cells in which both the mutant
PTP and the phosphotyrosyl polypeptide substrates are present.
According to non-limiting theory, expression of such a CS mutant
PTP can thus antagonize the normal function of the corresponding
wildtype PTP (and potentially other PTPs and/or other components of
a PTP signaling pathway) via a mechanism whereby the CS mutant
binds to and sequesters the substrate, precluding substrate
interaction with catalytically active, wildtype enzyme (e.g., Sun
et al., 1993).
[0010] CS mutants of certain other PTP family members, however, may
bind phosphotyrosyl polypeptide substrates and form complexes that
exist transiently and are not stable when the CS mutant is
expressed in cells, i.e., in vivo. The CS mutant of one PTP, PTP1B
(PTP-1B), is an example of such a PTP. Catalytically deficient
mutants of such enzymes that are capable of forming stable
complexes with phosphotyrosyl polypeptide substrates may be derived
by mutating a wildtype protein tyrosine phosphatase catalytic
domain invariant aspartate residue and replacing it with an amino
acid that does not cause significant alteration of the Km of the
enzyme but that results in a reduction in Kcat, as disclosed, for
example, in U.S. Pat. Nos. 5,912,138 and 5,951,979, in U.S.
application Ser. No. 09/323,426 and in PCT/US97/13016 and
PCT/US00/14211. For instance, mutation of Asp 181 in PTP1B to
alanine to create the aspartate-to-alanine (D to A or DA) mutant
PTP1B-D181A results in a PTP1B "substrate trapping" mutant enzyme
that forms a stable complex with its phosphotyrosyl polypeptide
substrate (e.g., Flint et al., 1997 Proc. Natl. Acad. Sci.
94:1680). Substrates of other PTPs can be identified using a
similar substrate trapping approach, for example substrates of the
PTP family members PTP-PEST (Garton et al., 1996 J. Mol. Cell.
Biol. 16:6408), TCPTP (Tiganis et al., 1998 Mol. Cell Biol.
18:1622), PTP-HSCF (Spencer et al., 1997 J. Cell Biol. 138:845),
and PTP-H1 (Zhang et al., 1999 J. Biol. Chem. 274:17806).
[0011] One non-transmembrane PTP, PTP1B, recognizes several
tyrosine-phosphorylated proteins as substrates, many of which are
involved in human disease. For example, therapeutic inhibition of
PTP1B in the insulin signaling pathway may serve to augment insulin
action, thereby ameliorating the state of insulin resistance common
in Type II diabetes patients. PTP1B acts as a negative regulator of
signaling that is initiated by several growth factor/hormone
receptor PTKs, including p210 Bcr-Abl (LaMontagne et al., Mol.
Cell. Biol. 18:2965-75 (1998); LaMontagne et al., Proc. Natl. Acad.
Sci. USA 95:14094-99 (1998)), receptor tyrosine kinases, such as
EGF receptor, PDGF receptor, and insulin receptor (IR) (Tonks et
al., Curr. Opin. Cell Biol. 13:182-95 (2001)), and JAK family
members such as Jak2 and others (Myers et al., J. Biol. Chem.
276:47771-74 (2001)), as well as signaling events induced by
cytokines (Tonks and Neel, 2001). Activity of PTP1B is regulated by
modifications of several amino acid residues, such as
phosphorylation of Ser residues (Brautigan and Pinault, 1993; Dadke
et al., 2001; Flint et al., 1993), and oxidation of the active Cys
residue in its catalytic motif (Lee et al., 1998; Meng et al.,
2002) which is evolutionary conserved among protein tyrosine
phosphatases and dual phosphatase family members (Andersen et al.,
2001). In addition, changes in the expression levels of PTP1B have
been noted in several human diseases, particularly those associated
with disruption of the normal patterns of tyrosine phosphorylation.
For example, therapeutic inhibition of PTPs such as PTP1B in the
insulin signaling pathway may serve to augment insulin action,
thereby ameliorating the state of insulin resistance common in
patients with type 2 diabetes.
[0012] Diabetes mellitus is a common, degenerative disease
affecting 5-10% of the human population in developed countries, and
in many countries, it may be one of the five leading causes of
death. Approximately 2% of the world's population has diabetes, the
overwhelming majority of cases (>97%) being type 2 diabetes and
the remainder being type 1. In type 1 diabetes, which is frequently
diagnosed in children or young adults, insulin production by
pancreatic islet beta cells is destroyed. Type 2 diabetes, or "late
onset" or "adult onset" diabetes, is a complex metabolic disorder
in which cells and tissues cannot effectively use available
insulin; in some cases insulin production is also inadequate. At
the cellular level, the degenerative phenotype that may be
characteristic of late onset diabetes mellitus includes, for
example, impaired insulin secretion and decreased insulin
sensitivity, i.e., an impaired response to insulin.
[0013] Studies have shown that diabetes mellitus may be preceded by
or is associated with certain related disorders. For example, an
estimated forty million individuals in the U.S. suffer from late
onset impaired glucose tolerance (IGT). IGT patients fail to
respond to glucose with increased insulin secretion. Each year a
small percentage (5-10%) of IGT individuals progress to insulin
deficient non-insulin dependent diabetes (NIDDM). Some of these
individuals further progress to insulin dependent diabetes mellitus
(IDDM). NIDDM and IDDM are associated with decreased release of
insulin by pancreatic beta cells and/or a decreased response to
insulin by cells and tissues that normally exhibit insulin
sensitivity. Other symptoms of diabetes mellitus and conditions
that precede or are associated with diabetes mellitus include
obesity, vascular pathologies, and various neuropathies, including
blindness and deafness.
[0014] Type 1 diabetes is treated with lifelong insulin therapy,
which is often associated with undesirable side effects such as
weight gain and an increased risk of hypoglycemia. Current
therapies for type 2 diabetes (NIDDM) include altered diet,
exercise therapy, and pharmacological intervention with injected
insulin or oral agents that are designed to lower blood glucose
levels. Examples of such presently available oral agents include
sulfonylureas, biguanides, thiazolidinediones, repaglinide, and
acarbose, each of which alters insulin and/or glucose levels. None
of the current pharmacological therapies, however, controls the
disease over its full course, nor do any of the current therapies
correct all of the physiological abnormalities in type 2 NIDDM,
such as impaired insulin secretion, insulin resistance, and
excessive hepatic glucose output. In addition, treatment failures
are common with these agents, such that multi-drug therapy is
frequently necessary.
[0015] In certain metabolic diseases or disorders, one or more
biochemical processes, which may be either anabolic or catabolic
(e.g., build-up or breakdown of substances, respectively), are
altered (e.g., increased or decreased in a statistically
significant manner) or modulated (e.g., up- or down-regulated to a
statistically significant degree) relative to the levels at which
they occur in a disease-free or normal subject such as an
appropriate control individual. The alteration may result from an
increase or decrease in a substrate, enzyme, cofactor, or any other
component in any biochemical reaction involved in a particular
process. Altered (i.e., increased or decreased in a statistically
significant manner relative to a normal state) PTP activity can
underlie certain disorders and suggests a PTP role in certain
metabolic diseases.
[0016] For example, disruption of the murine PTP1B gene homolog in
a knock-out mouse model results in PTP1B.sup.-/- mice exhibiting
enhanced insulin sensitivity, decreased levels of circulating
insulin and glucose, and resistance to weight gain even on a
high-fat diet, relative to control animals having at least one
functional PTP1B gene (Elchebly et al., Science 283:1544 (1999)).
Insulin receptor hyperphosphorylation has also been detected in
certain tissues of PTP1B deficient mice, consistent with a PTP1B
contribution to the physiologic regulation of insulin and glucose
metabolism (Id.). PTP-1B-deficient mice exhibit decreased adiposity
(reduced fat cell mass but not fat cell number), increased basal
metabolic rate and energy expenditure, and enhanced
insulin-stimulated glucose utilization (Klaman et al., 2000 Mol.
Cell. Biol. 20:5479). Additionally, altered PTP activity has been
correlated with impaired glucose metabolism in other biological
systems (e.g., McGuire et al., Diabetes 40:939 (1991); Myerovitch
et al., J. Clin. Invest. 84:976 (1989); Sredy et al., Metabolism
44:1074 (1995)), including PTP involvement in biological signal
transduction via the insulin receptor (see, e.g., WO 99/46268 and
references cited therein).
[0017] An integration of crystallographic, kinetic, and
PTP1B-peptide binding assays illustrated the interaction of PTP1B
and insulin receptor (IR) (Salmeen et al., Mol. Cell 6:1401-12
(2000)). The insulin receptor (IR) comprises two extracellular a
subunits and two transmembrane .beta. subunits. Activation of the
receptor results in autophosphorylation of tyrosine residues in
both .beta. subunits, each of which contains a protein kinase
domain. Extensive interactions that form between PTP1B and insulin
receptor kinase (IRK) encompass tandem pTyr residues at 1162 and
1163 of IRK, such that pTyr-1 162 is located in the active site of
PTP1B (id.). The Asp/Glu-pTyr-pTyr-Arg/Lys motif has been
implicated for optimal recognition by PTP1B for IRK. This motif is
also present in other receptor PTKs, including Trk, FGFR, and Axl.
In addition, this motif is found in the JAK family of PTKs, members
of which transmit signals from cytokine receptors, including a
classic cytokine receptor that is recognized by the satiety hormone
leptin (Touw et al., Mol. Cell. Endocrinol. 160:1-9 (2000)).
[0018] Changes in the expression levels of PTP1B have been observed
in several human diseases, particularly in diseases associated with
disruption of the normal patterns of tyrosine phosphorylation. For
example, the expression of PTP1B is induced specifically by the
p210 Bcr-Abl oncoprotein, a PTK that is directly responsible for
the initial manifestations of chronic myelogenous leukemia (CML)
(LaMontagne et al., Mol. Cell. Biol. 18:2965-75 (1998); LaMontagne
et al., Proc. Natl. Acad. Sci. USA 95:14094-99 (1998)). Expression
of PTPB1 in response to this oncoprotein is regulated, in part, by
transcription factors Sp1, Sp3, and Egr-1 (Fukada et al., J. Biol.
Chem. 276:25512-19 (2001)). These transcription factors have been
shown to bind to a p210 Bcr-Abl responsive sequence (PRS) in the
human PTP1B promoter, located between -49 to -37 base pairs from
the transcription start site, but do not appear to mediate certain
additional, independent PTP1B transcriptional events, for which
neither transcription factor(s) nor transcription factor
recognition element(s) have been defined (id.).
[0019] RNA interference (RNAi) is a polynucleotide
sequence-specific, post-transcriptional gene silencing mechanism
effected by double-stranded RNA that results in degradation of a
specific messenger RNA (mRNA), thereby reducing the expression of a
desired target polypeptide encoded by the mRNA (see, e.g., WO
99/32619; WO 01/75164; U.S. Pat. No. 6,506,559; Fire et al., Nature
391:806-11 (1998); Sharp, Genes Dev. 13:139-41 (1999); Elbashir et
al. Nature 411:494-98 (2001); Harborth et al., J. Cell Sci.
114:4557-65 (2001)). RNAi is mediated by double-stranded
polynucleotides as also described hereinbelow, for example,
double-stranded RNA (dsRNA), having sequences that correspond to
exonic sequences encoding portions of the polypeptides for which
expression is compromised. RNAi reportedly is not effected by
double-stranded RNA polynucleotides that share sequence identity
with intronic or promoter sequences (Elbashir et al., 2001). RNAi
pathways have been best characterized in Drosophila and
Caenorhabditis elegans, but "small interfering RNA" (siRNA)
polynucleotides that interfere with expression of specific
polypeptides in higher eukaryotes such as mammals (including
humans) have also been considered (e.g., Tuschl, 2001 Chembiochem.
2:239-245; Sharp, 2001 Genes Dev. 15:485; Bernstein et al., 2001
RNA 7:1509; Zamore, 2002 Science 296:1265; Plasterk, 2002 Science
296:1263; Zamore 2001 Nat. Struct. Biol. 8:746; Matzke et al., 2001
Science 293:1080; Scadden et al., 2001 EMBO Rep. 2:1107).
[0020] According to a current non-limiting model, the RNAi pathway
is initiated by ATP-dependent, processive cleavage of long dsRNA
into double-stranded fragments of about 18-27 (e.g., 19, 20, 21,
22, 23, 24, 25, 26, etc.) nucleotide base pairs in length, called
small interfering RNAs (siRNAs) (see review by Hutvagner et al.,
Curr. Opin. Gen. Dev. 12:225-32 (2002); Elbashir et al., 2001;
Nyknen et al., Cell 107:309-21 (2001); Bass, Cell 101:235-38
(2000)); Zamore et al., Cell 101:25-33 (2000)). In Drosophila, an
enzyme known as "Dicer" cleaves the longer double-stranded RNA into
siRNAs; Dicer belongs to the RNase III family of dsRNA-specific
endonucleases (WO 01/68836; Bernstein et al., Nature 409:363-66
(2001)). Further according to this non-limiting model, the siRNA
duplexes are incorporated into a protein complex, followed by
ATP-dependent unwinding of the siRNA, which then generates an
active RNA-induced silencing complex (RISC) (WO 01/68836). The
complex recognizes and cleaves a target RNA that is complementary
to the guide strand of the siRNA, thus interfering with expression
of a specific protein (Hutvagner et al., supra).
[0021] In C. elegans and Drosophila, RNAi may be mediated by long
double-stranded RNA polynucleotides (WO 99/32619; WO 01/75164; Fire
et al., 1998; Clemens et al., Proc. Natl. Acad. Sci. USA
97:6499-6503 (2000); Kisielow et al., Biochem. J. 363:1-5 (2002);
see also WO 01/92513 (RNAi-mediated silencing in yeast)). In
mammalian cells, however, transfection with long dsRNA
polynucleotides (i.e., greater than 30 base pairs) leads to
activation of a non-specific sequence response that globally blocks
the initiation of protein synthesis and causes mRNA degradation
(Bass, Nature 411:428-29 (2001)). Transfection of human and other
mammalian cells with double-stranded RNAs of about 18-27 nucleotide
base pairs in length interferes in a sequence-specific manner with
expression of particular polypeptides encoded by messenger RNAs
(mRNA) containing corresponding nucleotide sequences (WO 01/75164;
Elbashir et al., 2001; Elbashir et al., Genes Dev. 15:188-200
(2001)); Harborth et al., J. Cell Sci. 114:4557-65 (2001); Carthew
et al., Curr. Opin. Cell Biol. 13:244-48 (2001); Mailand et al.,
Nature Cell Biol. Advance Online Publication (Mar. 18, 2002);
Mailand et al. 2002 Nature Cell Biol. 4:317).
[0022] siRNA polynucleotides may offer certain advantages over
other polynucleotides known to the art for use in sequence-specific
alteration or modulation of gene expression to yield altered levels
of an encoded polypeptide product. These advantages include lower
effective siRNA polynucleotide concentrations, enhanced siRNA
polynucleotide stability, and shorter siRNA polynucleotide
oligonucleotide lengths relative to such other polynucleotides
(e.g., antisense, ribozyme or triplex polynucleotides). By way of a
brief background, "antisense" polynucleotides bind in a
sequence-specific manner to target nucleic acids, such as mRNA or
DNA, to prevent transcription of DNA or translation of the mRNA
(see, e.g., U.S. Pat. No. 5,168,053; U.S. Pat. No. 5,190,931; U.S.
Pat. No. 5,135,917; U.S. Pat. No. 5,087,617; see also, e.g., Clusel
et al., 1993 Nucl. Acids Res. 21:3405-11, describing "dumbbell"
antisense oligonucleotides). "Ribozyme" polynucleotides can be
targeted to any RNA transcript and are capable of catalytically
cleaving such transcripts, thus impairing translation of mRNA (see,
e.g., U.S. Pat. No. 5,272,262; U.S. Pat. No. 5,144,019; and U.S.
Pat. Nos. 5,168,053, 5,180,818, 5,116,742 and 5,093,246; U.S.
2002/193579). "Triplex" DNA molecules refers to single DNA strands
that bind duplex DNA to form a colinear triplex molecule, thereby
preventing transcription (see, e.g., U.S. Pat. No. 5,176,996,
describing methods for making synthetic oligonucleotides that bind
to target sites on duplex DNA). Such triple-stranded structures are
unstable and form only transiently under physiological conditions.
Because single-stranded polynucleotides do not readily diffuse into
cells and are therefore susceptible to nuclease digestion,
development of single-stranded DNA for antisense or triplex
technologies often requires chemically modified nucleotides to
improve stability and absorption by cells. siRNAs, by contrast, are
readily taken up by intact cells, are effective at interfering with
the expression of specific polypeptides at concentrations that are
several orders of magnitude lower than those required for either
antisense or ribozyme polynucleotides, and do not require the use
of chemically modified nucleotides.
[0023] Importantly, despite a number of attempts to devise
selection criteria for identifying oligonucleotide sequences that
will be effective in siRNA based on features of the desired target
mRNA sequence (e.g., percent GC content, position from the
translation start codon, or sequence similarities based on an in
silico sequence database search for homologues of the proposed
siRNA) it is presently not possible to predict with any degree of
confidence which of myriad possible candidate siRNA sequences that
can be generated as nucleotide sequences that correspond to a
desired target mRNA (e.g., dsRNA of about 18-27 nucleotide base
pairs) will in fact exhibit siRNA activity (i.e., interference with
expression of the polypeptide encoded by the mRNA). Instead,
individual specific candidate siRNA polynucleotide or
oligonucleotide sequences must be generated and tested to determine
whether interference with expression of a desired polypeptide
target can be effected. Accordingly, no routine method exists in
the art for designing a siRNA polynucleotide that is, with
certainty, capable of specifically altering the expression of a
given PTP polypeptide, and thus for the overwhelming majority of
PTPs no effective siRNA polynucleotide sequences are presently
known.
[0024] Currently, therefore, desirable goals for therapeutic
regulation of biological signal transduction include modulation of
PTP1B-mediated cellular events include, inter alia, inhibition or
potentiation of interactions among PTP1B-binding molecules,
substrates and binding partners, or of other agents that regulate
PTP1B activities. Accordingly, a need exists in the art for an
improved ability to intervene in the regulation of phosphotyrosine
signaling, including regulating PTP1B by altering PTP1B catalytic
activity, PTP1B binding to PTP1B substrate molecules, and/or PTP1B
-encoding gene expression. An increased ability to so regulate
PTP1B may facilitate the development of methods for modulating the
activity of proteins involved in phosphotyrosine signaling pathways
and for treating conditions associated with such pathways. The
present invention fulfills these needs and further provides other
related advantages.
SUMMARY OF THE INVENTION
[0025] The present invention relates to compositions and methods,
including specific siRNA polynucleotides comprising nucleotide
sequences disclosed herein, for modulating PTP1B. It is therefore
an aspect of the invention to provide an isolated small interfering
RNA (siRNA) polynucleotide, comprising in certain embodiments at
least one nucleotide sequence selected from SEQ ID NOS: 83-86,
88-91, 93-96, 98-101, 104-105, and 108-109, and in certain further
embodiments at least one nucleotide sequence selected from SEQ ID
NOS: 83-86, 88-91, 93-96, 98-101 and the complementary
polynucleotide thereto. In another embodiment the small interfering
RNA polynucleotide of either is capable of interfering with
expression of a PTP1B polypeptide, wherein the PTP-1B polypeptide
comprises an amino acid sequence as set forth in GenBank Ace. Nos.
M31724, NM.sub.--002827, or M33689. In another embodiment the
nucleotide sequence of the siRNA polynucleotide differs by one,
two, three or four nucleotides at any of positions 1-19 of a
sequence selected from SEQ ID NOS: 83-86, 88-91, 93-96, and 98-101,
or at any position of a sequence selected from SEQ ID NOS: 104,
105, 108, and 109. In other embodiments the nucleotide sequence of
the siRNA polynucleotide differs by at least two, three or four
nucleotides at any of positions 1-19 of a sequence selected from
SEQ ID NOS: 83-86, 88-91, 93-96, and 98-101, or at any position of
a sequence selected from SEQ ID NOS: 104, 105, 108, and 109.
[0026] In certain preferred embodiments the invention provides an
isolated siRNA polynucleotide comprising a nucleotide sequence
according to SEQ ID NO: 83, or the complement thereof, or an
isolated siRNA polynucleotide comprising a nucleotide sequence
according to SEQ ID NO: 88, or the complement thereof, or an
isolated siRNA polynucleotide comprising a nucleotide sequence
according to SEQ ID NO: 93, or the complement thereof, or an
isolated siRNA polynucleotide comprising a nucleotide sequence
according to SEQ ID NO: 98, or the complement thereof, or an
isolated siRNA polynucleotide comprising a nucleotide sequence
according to SEQ ID NO: 104 or 105, or an isolated siRNA
polynucleotide comprising a nucleotide sequence according to SEQ ID
NO: 108 or 109.
[0027] According to certain further embodiments of the above
described invention, the polynucleotide comprises at least one
synthetic nucleotide analogue of a naturally occurring nucleotide.
In another embodiment the polynucleotide is linked to a detectable
label, which in certain further embodiments is a reporter molecule
that may in certain still further embodiments be selected from a
dye, a radionuclide, a luminescent group, a fluorescent group, and
biotin, wherein in a still further embodiment the fluorescent group
is fluorescein isothiocyanate. In another embodiment the detectable
label is a magnetic particle. According to related embodiments
there is provided a pharmaceutical composition comprising any of
the above described siRNA polynucleotides and a physiologically
acceptable carrier, which in certain further embodiments comprises
a liposome.
[0028] The invention also provides a recombinant nucleic acid
construct comprising a polynucleotide that is capable of directing
transcription of a small interfering RNA (siRNA), the
polynucleotide comprising: (i) a first promoter; (ii) a second
promoter; and (iii) at least one DNA polynucleotide segment
comprising at least one nucleotide sequence selected from SEQ ID
NOS: 83-86, 88-91, 93-96, 98-101, or a complement thereto, wherein
each DNA polynucleotide segment and its complement are operably
linked to at least one of the first and second promoters, and
wherein the promoters are oriented to direct transcription of the
DNA polynucleotide segment and its reverse complement. In another
embodiment there is provided a recombinant nucleic acid construct
comprising a polynucleotide that is capable of directing
transcription of a small interfering RNA (siRNA), the
polynucleotide comprising a promoter operably linked to at least
one DNA polynucleotide segment comprising at least one nucleotide
sequence that is selected from SEQ ID NOs: 102, 103, 106, and 107.
In a further embodiment the recombinant nucleic acid construct
comprises at least one enhancer that is selected from a first
enhancer operably linked to the first promoter and a second
enhancer operably linked to the second promoter. In certain
embodiments the recombinant nucleic acid construct comprises at
least one transcriptional terminator that is selected from (i) a
first transcriptional terminator that is positioned in the
construct to terminate transcription directed by the first promoter
and (ii) a second transcriptional terminator that is positioned in
the construct to terminate transcription directed by the second
promoter. In certain other embodiments the siRNA is capable of
interfering with expression of a PTP1B polypeptide, wherein the
PTP1B polypeptide comprises an amino acid sequence as set forth in
a sequence selected from the group consisting of GenBank Acc. Nos.
M31724, NM.sub.--002827, and M33689. In another embodiment the
invention provides a recombinant nucleic acid construct comprising
a polynucleotide that is capable of directing transcription of a
small interfering RNA (siRNA), the polynucleotide comprising at
least one promoter and a DNA polynucleotide segment, wherein the
DNA polynucleotide segment is operably linked to the promoter, and
wherein the DNA polynucleotide segment comprises (i) at least one
DNA polynucleotide that comprises at least one nucleotide sequence
selected from SEQ ID NOS: 83-86, 88-91, 93-96, 98-101, or a
complement thereto; (ii) a spacer sequence comprising at least 4
nucleotides operably linked to the DNA polynucleotide of (i); and
(iii) the reverse complement of the DNA polynucleotide of (i)
operably linked to the spacer sequence. In certain further
embodiments the siRNA comprises an overhang of at least one and no
more than four nucleotides, the overhang being located immediately
3' to (iii). In a further embodiment the spacer sequence comprises
at least 9 nucleotides. In other further embodiments the spacer
sequence comprises two uridine nucleotides that are contiguous with
(iii). In another embodiment the recombinant nucleic acid construct
comprises at least one transcriptional terminator that is operably
linked to the DNA polynucleotide segment. According to related
embodiments, the invention provides a host cell transformed or
transfected with the above described recombinant nucleic acid
constructs.
[0029] Certain embodiments of the invention provide a
pharmaceutical composition comprising an siRNA polynucleotide and a
physiologically acceptable carrier, wherein the siRNA
polynucleotide is selected from (i) an RNA polynucleotide which
comprises at least one nucleotide sequence selected from SEQ ID
NOS: 83-86, 88-91, 93-96, 98-101, 104-105, and 108-109, (ii) an RNA
polynucleotide that comprises at least one nucleotide sequence
selected from SEQ ID NOS: 83-86, 88-91, 93-96, 98-101 and the
complementary polynucleotide thereto, (iii) an RNA polynucleotide
according to (i) or (ii) wherein the nucleotide sequence of the
siRNA polynucleotide differs by one, two or three nucleotides at
any of positions 1-19 of a sequence selected from the sequences set
forth in SEQ ID NOS: 83-86, 88-91, 93-96, and 98-101, or at any
position of a sequence selected from the sequences set forth in SEQ
ID NOS: 104, 105, 108, and 109, and (iv) an RNA polynucleotide
according to (i) or (ii) wherein the nucleotide sequence of the
siRNA polynucleotide differs by two, three or four nucleotides at
any of positions 1-19 of a sequence selected from the sequences set
forth in SEQ ID NOS: 83-86, 88-91, 93-96, 98-101, or at any
position of a sequence selected from the sequences set forth in SEQ
ID NOS: 104, 105, 108, and 109. In a further embodiment the carrier
comprises a liposome.
[0030] Turning to another aspect of the invention, there is
provided a method for interfering with expression of a PTP1B
polypeptide, or variant thereof, comprising contacting a subject
that comprises at least one cell which is capable of expressing a
PTP1B polypeptide with a siRNA polynucleotide for a time and under
conditions sufficient to interfere with PTP1B polypeptide
expression, wherein (a) the PTP1B polypeptide comprises an amino
acid sequence as set forth in a sequence selected from GenBank Ace.
Nos. M31724, NM.sub.--002827, NM.sub.--011201, and M33689, (b) the
siRNA polynucleotide is selected from (i) an RNA polynucleotide
which comprises at least one nucleotide sequence selected from SEQ
ID NOS: 83-86, 88-91, 93-96, 98-101, 104-105, and 108-109, (ii) an
RNA polynucleotide that comprises at least one nucleotide sequence
selected from SEQ ID NOS: 83-86, 88-91, 93-96, 98-101, and the
complementary polynucleotide thereto, (iii) an RNA polynucleotide
according to (i) or (ii) wherein the nucleotide sequence of the
siRNA polynucleotide differs by one, two or three nucleotides at
any of positions 1-19 of a sequence selected from the sequences set
forth in SEQ ID NOS: 83-86, 88-91, 93-96, 98-101, or at any
position of a sequence selected from the sequences set forth in SEQ
ID NOS: 104, 105, 108, and 109, and (iv) an RNA polynucleotide
according to (i) or (ii) wherein the nucleotide sequence of the
siRNA polynucleotide differs by two, three or four nucleotides at
any of positions 1-19 of a sequence selected from the sequences set
forth in SEQ ID NOS: 83-86, 88-91, 93-96, 98-101, or at any
position of a sequence selected from sequences set forth in SEQ ID
NOS: 104, 105, 108, and 109.
[0031] The invention also provides a method for interfering with
expression of a PTP1B polypeptide that comprises an amino acid
sequence as set forth in a sequence selected from GenBank Acc. Nos.
M31724, NM.sub.--002827, and M33689, or a variant of said PTP1B
polypeptide, said method comprising contacting, under conditions
and for a time sufficient to interfere with PTP1B polypeptide
expression, (i) a subject that comprises at least one cell that is
capable of expressing the PTP1B polypeptide, and (ii) a recombinant
nucleic acid construct as described above. In another embodiment
there is provided a method for identifying a component of a PTP1B
signal transduction pathway comprising: A. contacting a siRNA
polynucleotide and a first biological sample comprising at least
one cell that is capable of expressing a PTP1B polypeptide, or a
variant of said PTP1B polypeptide, under conditions and for a time
sufficient for PTP1B expression when the siRNA polynucleotide is
not present, wherein (1) the PTP1B polypeptide comprises an amino
acid sequence as set forth in a sequence selected from GenBank Ace.
Nos. M31724, NM.sub.--002827, and M33689, (2) the siRNA
polynucleotide is selected from (i) an RNA polynucleotide which
comprises at least one nucleotide sequence selected from SEQ ID
NOS: 83-86, 88-91, 93-96, 98-101, 104-105, and 108-109, (ii) an RNA
polynucleotide that comprises at least one nucleotide sequence
selected from SEQ ID NOS: 83-86, 88-91, 93-96, 98-101 and the
complementary polynucleotide thereto, (iii) an RNA polynucleotide
according to (i) or (ii) wherein the nucleotide sequence of the
siRNA polynucleotide differs by one, two or three nucleotides at
any of positions 1-19 of a sequence selected from the sequences set
forth in SEQ ID NOS: 83-86, 88-91, 93-96, 98-101, or at any
position of a sequence selected from the sequences set forth in SEQ
ID NOS: 104, 105, 108, and 109, and (iv) an RNA polynucleotide
according to (i) or (ii) wherein the nucleotide sequence of the
siRNA polynucleotide differs by two, three or four nucleotides at
any of positions 1-19 of a sequence selected from the sequences set
forth in SEQ ID NOS: 83-86, 88-91, 93-96, 98-101, or at any
position of a sequence selected from the sequences set forth in SEQ
ID NOS: 104, 105, 108, and 109; and B. comparing a level of
phosphorylation of at least one protein that is capable of being
phosphorylated in the cell with a level of phosphorylation of the
protein in a control sample that has not been contacted with the
siRNA polynucleotide, wherein an altered level of phosphorylation
of the protein in the presence of the siRNA polynucleotide relative
to the level of phosphorylation of the protein in an absence of the
siRNA polynucleotide indicates that the protein is a component of
the PTP1B signal transduction pathway. In certain further
embodiments the signal transduction pathway comprises a Jak2
kinase.
[0032] In another aspect the present invention provides a method
for modulating an insulin receptor protein phosphorylation state in
a cell, comprising contacting the cell with a siRNA polynucleotide
under conditions and for a time sufficient to interfere with
expression of a PTP1B polypeptide, wherein (a) the PTP1B
polypeptide comprises an amino acid sequence as set forth in a
sequence selected from f GenBank Acc. Nos. M31724, NM.sub.--002827,
NM.sub.--011201, and M33689, (b) the siRNA polynucleotide is
selected from (i) an RNA polynucleotide which comprises at least
one nucleotide sequence selected from SEQ ID NOS: 83-86, 88-91,
93-96, 98-101 or the complements thereof and SEQ ID NOS: 104, 105,
108, and 109, (ii) an RNA polynucleotide that comprises at least
one nucleotide sequence selected from f SEQ ID NOS: 83-86, 88-91,
93-96, 98-101 and the complementary polynucleotide thereto, (iii)
an RNA polynucleotide according to (i) or (ii) wherein the
nucleotide sequence of the siRNA polynucleotide differs by one, two
or three nucleotides at any of positions 1-19 of a sequence
selected from the sequences set forth in SEQ ID NOS: 83-86, 88-91,
93-96, and 98-101, or at any position of a sequence selected from
the sequences set forth in SEQ ID NOS: 104, 105, 108, and 109, and
(iv) an RNA polynucleotide according to (i) or (ii) wherein the
nucleotide sequence of the siRNA polynucleotide differs by two,
three or four nucleotides at any of positions 1-19 of a sequence
selected from the sequences set forth in SEQ ID NOS: 83-86, 88-91,
93-96, and 98-101, or at any position of a sequence selected from
the sequences set forth in SEQ ID NOS: 104, 105, 108, and 109; and
(c) the insulin receptor protein comprises a polypeptide which
comprises an amino acid sequence selected from the group consisting
of SEQ ID NOS: __-__, or a variant thereof.
[0033] In another embodiment there is provided a method for
altering Jak2 protein phosphorylation state in a cell, comprising
contacting the cell with a siRNA polynucleotide under conditions
and for a time sufficient to interfere with expression of a PTP1B
polypeptide, wherein (a) the PTP1B polypeptide comprises an amino
acid sequence as set forth in a sequence selected from GenBank Acc.
Nos. M31724, NM.sub.--002827, NM.sub.--011201, and M33689, (b) the
siRNA polynucleotide is selected from (i) an RNA polynucleotide
which comprises at least one nucleotide sequence selected from SEQ
ID NOS: 83-86, 88-91, 93-96, and 98-101 or the complements thereof,
and SEQ ID NOS: 104, 105, 108, and 109, (ii) an RNA polynucleotide
that comprises at least one nucleotide sequence selected from SEQ
ID NOS: 83-86, 88-91, 93-96, and 98-101 and the complementary
polynucleotide thereto, (iii) an RNA polynucleotide according to
(i) or (ii) wherein the nucleotide sequence of the siRNA
polynucleotide differs by one, two or three nucleotides at any of
positions 1-19 of a sequence selected from the sequences set forth
in SEQ ID NOS: 83-86, 88-91, 93-96, and 98-101, or at any position
of a sequence selected from the sequences set forth in SEQ ID NOS:
104, 105, 108, and 109, and (iv) an RNA polynucleotide according to
(i) or (ii) wherein the nucleotide sequence of the siRNA
polynucleotide differs by two, three or four nucleotides at any of
positions 1-19 of a sequence selected from the sequences set forth
in SEQ ID NOS: 83-86, 88-91, 93-96, and 98-101, or at any position
of a sequence selected from the sequences set forth in SEQ ID NOS:
104, 105, 108, and 109; and (c) the Jak2 protein comprises a
polypeptide which comprises an amino acid sequence selected from
the group consisting of SEQ ID NOS: __-__, or a variant thereof.
Another embodiment of the invention provides a method for treating
a Jak2-associated disorder comprising administering to a subject in
need thereof a pharmaceutical composition as described above,
wherein the siRNA polynucleotide inhibits expression of a PTP1B
polypeptide, or a variant thereof. In certain embodiments the
Jak2-associated disorder is diabetes, obesity,
hyperglycemia-induced apoptosis, inflammation, or a
neurodegenerative disorder. In another aspect the invention
provides a small interfering RNA (siRNA) polynucleotide, comprising
in certain embodiments at least one nucleotide sequence selected
from SEQ ID NOS: 83-86, 88-91, 93-96, 98-101, 104-105, and 108-109,
and in certain further embodiments at least one nucleotide sequence
selected from SEQ ID NOS: 83-86, 88-91, 93-96, 98-101 and the
complementary polynucleotide thereto. The invention also provides a
small interfering RNA (siRNA) polynucleotide, comprising an RNA
polynucleotide which comprises at least one nucleotide sequence
selected from SEQ ID NOS: 18-21, 33-36, 43-46, 53-56, and 58-61.
Certain further embodiments relate to isolated siRNA
polynucleotides that comprise nucleotide sequences having the above
recited SEQ ID NOS, including compositions and methods for
producing and therapeutically using such siRNA.
[0034] These and other embodiments of the present invention will
become apparent upon reference to the following detailed
description and attached drawings. All references disclosed herein
are hereby incorporated by reference in their entireties as if each
was incorporated individually. Also incorporated by reference are
co-pending applications, Ser. No. ______ and Ser. No. ______
(attorney docket numbers 200125.441D1 and 200125.448,
respectively), which have been filed concurrently.
BRIEF DESCRIPTION OF THE DRAWINGS
[0035] FIG. 1 depicts an immunoblot of the effect on endogenous
expression of murine PTP1B by siRNAs specific for the murine PTP1B
or the human PTP1B polynucleotide sequences. Expression was
detected using a murine anti-PTP1B monoclonal antibody. Data are
presented for two different clones of C57B16 #3 murine cells. Both
clones were transfected with mPTP1B1.1 siRNA (lanes 3 and 8);
mPTP1B1.2 (lanes 4 and 9); mPTP1B1.3 (lanes 5 and 10). One clone,
C57B16 #3 clone 3, was transfected with hPTP1B1.1 (lane 6). Lane 2:
untransfected C57B16 #3, clone 3 (NT); lane 7: untransfected C57B16
#3, clone 10.
[0036] FIG. 2 depicts an immunoblot analysis of the expression of
human PTP-1B co-transfected into 1 BKO+HIR murine fibroblasts with
human PTP-1B siRNA hairpin vectors. Expression was detected with an
anti-human PTP1B antibody (h1B) (lower portion of immunoblot). As a
protein expression control, cell lysates were probed with an
anti-human insulin receptor (IR) antibody (upper portion of
immunoblot).
[0037] FIG. 3 presents the results of an ELISA in which the level
of insulin receptor (IR) phosphorylated tyrosine was measured in
293-HEK HIR cells transfected with 0, 0.5, 3, or 10 nM hPTP1B1.3
(H1.3, SEQ ID NO: __) (FIG. 3A) or mPTP1B1.1b (M1.1, SEQ ID NO: __)
(FIG. 3B) siRNAs. The level of expression of human PTP1B in the
cells was compared by immunoblot (see tables to right of each
figure).
[0038] FIG. 4 depicts the results of an ELISA in which the level of
insulin receptor (IR) phosphorylated tyrosine was measured in
293-HEK HIR cells transfected with 0, 0.5, 3, or 10 nM siRNAs. The
siRNA polynucleotides transfected into the cells included
mPTP1B1.1b (M1.1, SEQ ID NO: __) (FIG. 4A); hPTP1B1.2 (H1.2, SEQ ID
NO: __) (FIG. 4B); hPTP1B1.3 (H1.3, SEQ ID NO: __) (FIG. 4C); and
rPTP1B1.2 (R1.2, SEQ ID NO: __) (FIG. 4D). Seventy-two hours after
transfection, cells were exposed to insulin for 7 minutes at the
designated concentrations. Cell lysates were prepared and coated
onto 96-well plates and probed with an anti-pY-IR-.beta.
antibody.
[0039] FIG. 5 represents ELISA data from three separate experiments
(Exp. 1, 2, 3) that represent the level of insulin receptor
phosphorylation in cells transfected with hPTP1B1.3 and stimulated
with 50 nM insulin (Ins). Each data point represents the average
optical density measured in duplicate wells.
DETAILED DESCRIPTION OF THE INVENTION
[0040] The present invention is directed in part to the unexpected
discovery of short RNA polynucleotide sequences that are capable of
specifically modulating expression of a desired PTP1B polypeptide,
such as a human or murine PTP1B polypeptide (e.g., GenBank Acc.
Nos. M31724, NM.sub.--002827, NM.sub.--011201, M33689,
NM.sub.--012637, NM.sub.--012637, M33962; SEQ ID NOS: __-__;
Andersen et al., 2001 Mol. Cell. Biol. 21:7117), or variant
thereof. Without wishing to be bound by theory, the RNA
polynucleotides of the present invention specifically reduce
expression of a desired target polypeptide through recruitment of
small interfering RNA (siRNA) mechanisms. In particular, and as
described in greater detail herein, according to the present
invention there are provided compositions and methods that relate
to the surprising identification of certain specific RNAi
oligonucleotide sequences of 19, 20, 21, 22, 23, 24, 25, 26 or 27
nucleotides that can be derived from corresponding polynucleotide
sequences encoding the desired PTP1B target polypeptide. These
sequences cannot be predicted through any algorithm, sequence
alignment routine, or other systematic paradigm, but must instead
be obtained through generation and functional testing for RNAi
activity of actual candidate oligonucleotides, such as those
disclosed for the first time herein.
[0041] In preferred embodiments of the invention, the siRNA
polynucleotide interferes with expression of a PTP1B target
polypeptide or a variant thereof, and comprises a RNA
oligonucleotide or RNA polynucleotide uniquely corresponding in its
nucleotide base sequence to the sequence of a portion of a target
polynucleotide encoding the target polypeptide, for instance, a
target mRNA sequence or an exonic sequence encoding such mRNA. The
invention relates in preferred embodiments to siRNA polynucleotides
that interfere with expression of specific polypeptides in mammals,
which in certain particularly preferred embodiments are humans and
in certain other particularly preferred embodiments are non-human
mammals. Hence, according to non-limiting theory, the siRNA
polynucleotides of the present invention direct sequence-specific
degradation of mRNA encoding a desired PTP1B.
[0042] SiRNA Polynucleotides
[0043] As used herein, the term "siRNA" means either: (i) a double
stranded RNA oligonucleotide, or polynucleotide, that is 18 base
pairs, 19 base pairs, 20 base pairs, 21 base pairs, 22 base pairs,
23 base pairs, 24 base pairs, 25 base pairs, 26 base pairs, 27 base
pairs, 28 base pairs, 29 base pairs or 30 base pairs in length and
that is capable of interfering with expression and activity of a
PTP-1B polypeptide, or a variant of the PTP-1B polypeptide, wherein
a single strand of the siRNA comprises a portion of a RNA
polynucleotide sequence that encodes the PTP-1B polypeptide, its
variant, or a complementary sequence thereto; (ii) a single
stranded oligonucleotide, or polynucleotide of 18 nucleotides, 19
nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23
nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27
nucleotides, 28 nucleotides, 29 nucleotides or 30 nucleotides in
length and that is either capable of interfering with expression
and/or activity of a target PTP-1B polypeptide, or a variant of the
PTP-1B polypeptide, or that anneals to a complementary sequence to
result in a dsRNA that is capable of interfering with target
polypeptide expression, wherein such single stranded
oligonucleotide comprises a portion of a RNA polynucleotide
sequence that encodes the PTP-1B polypeptide, its variant, or a
complementary sequence thereto; or (iii) an oligonucleotide, or
polynucleotide, of either (i) or (ii) above wherein such
oligonucleotide, or polynucleotide, has one, two, three or four
nucleic acid alterations or substitutions therein. Certain RNAi
oligonucleotide sequences described below are complementary to the
3' non-coding region of target mRNA that encodes the PTP1B
polypeptide.
[0044] A siRNA polynucleotide is a RNA nucleic acid molecule that
mediates the effect of RNA interference, a post-transcriptional
gene silencing mechanism. A siRNA polynucleotide preferably
comprises a double-stranded RNA (dsRNA) but is not intended to be
so limited and may comprise a single-stranded RNA (see, e.g.,
Martinez et al. Cell 110:563-74 (2002)). A siRNA polynucleotide may
comprise other naturally occurring, recombinant, or synthetic
single-stranded or double-stranded polymers of nucleotides
(ribonucleotides or deoxyribonucleotides or a combination of both)
and/or nucleotide analogues as provided herein (e.g., an
oligonucleotide or polynucleotide or the like, typically in 5' to
3' phosphodiester linkage). Accordingly it will be appreciated that
certain exemplary sequences disclosed herein as DNA sequences
capable of directing the transcription of the subject invention
siRNA polynucleotides are also intended to describe the
corresponding RNA sequences and their complements, given the well
established principles of complementary nucleotide base-pairing. A
siRNA may be transcribed using as a template a DNA (genomic, cDNA,
or synthetic) that contains a RNA polymerase promoter, for example,
a U6 promoter or the H1 RNA polymerase III promoter, or the siRNA
may be a synthetically derived RNA molecule. In certain embodiments
the subject invention siRNA polynucleotide may have blunt ends,
that is, each nucleotide in one strand of the duplex is perfectly
complementary (e.g., by Watson-Crick base-pairing) with a
nucleotide of the opposite strand. In certain other embodiments, at
least one strand of the subject invention siRNA polynucleotide has
at least one, and preferably two nucleotides that "overhang" (i.e.,
that do not base pair with a complementary base in the opposing
strand) at the 3' end of either strand, or preferably both strands,
of the siRNA polynucleotide. In a preferred embodiment of the
invention, each strand of the siRNA polynucleotide duplex has a
two-nucleotide overhang at the 3' end. The two-nucleotide overhang
is preferably a thymidine dinucleotide (TT) but may also comprise
other bases, for example, a TC dinucleotide or a TG dinucleotide,
or any other dinucleotide. For a discussion of 3' ends of siRNA
polynucleotides see, e.g., WO 01/75164.
[0045] Preferred siRNA polynucleotides comprise double-stranded
oligomeric nucleotides of about 18-30 nucleotide base pairs,
preferably about 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 base
pairs, and in other preferred embodiments about 19, 20, 21, 22 or
23 base pairs, or about 27 base pairs, whereby the use of "about"
indicates, as described above, that in certain embodiments and
under certain conditions the processive cleavage steps that may
give rise to functional siRNA polynucleotides that are capable of
interfering with expression of a selected polypeptide may not be
absolutely efficient. Hence, siRNA polynucleotides, for instance,
of "about" 18, 19, 20, 21, 22, 23, 24, or 25 base pairs may include
one or more siRNA polynucleotide molecules that may differ (e.g.,
by nucleotide insertion or deletion) in length by one, two, three
or four base pairs, by way of non-limiting theory as a consequence
of variability in processing, in biosynthesis, or in artificial
synthesis. The contemplated siRNA polynucleotides of the present
invention may also comprise a polynucleotide sequence that exhibits
variability by differing (e.g., by nucleotide substitution,
including transition or transversion) at one, two, three or four
nucleotides from a particular sequence, the differences occurring
at any of positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, or 19 of a particular siRNA polynucleotide
sequence, or at positions 20, 21, 22, 23, 24, 25, 26, or 27 of
siRNA polynucleotides depending on the length of the molecule,
whether situated in a sense or in an antisense strand of the
double-stranded polynucleotide. The nucleotide substitution may be
found only in one strand, by way of example in the antisense
strand, of a double-stranded polynucleotide, and the complementary
nucleotide with which the substitute nucleotide would typically
form hydrogen bond base pairing may not necessarily be
correspondingly substituted in the sense strand. In preferred
embodiments, the siRNA polynucleotides are homogeneous with respect
to a specific nucleotide sequence. As described herein, preferred
siRNA polynucleotides interfere with expression of a PTP-1B
polypeptide. These polynucleotides may also find uses as probes or
primers.
[0046] Polynucleotides that are siRNA polynucleotides of the
present invention may in certain embodiments be derived from a
single-stranded polynucleotide that comprises a single-stranded
oligonucleotide fragment (e.g., of about 18-30 nucleotides, which
should be understood to include any whole integer of nucleotides
including and between 18 and 30) and its reverse complement,
typically separated by a spacer sequence. According to certain such
embodiments, cleavage of the spacer provides the single-stranded
oligonucleotide fragment and its reverse complement, such that they
may anneal to form (optionally with additional processing steps
that may result in addition or removal of one, two, three or more
nucleotides from the 3' end and/or the 5' end of either or both
strands) the double-stranded siRNA polynucleotide of the present
invention. In certain embodiments the spacer is of a length that
permits the fragment and its reverse complement to anneal and form
a double-stranded structure (e.g., like a hairpin polynucleotide)
prior to cleavage of the spacer (and, optionally, subsequent
processing steps that may result in addition or removal of one,
two, three, four, or more nucleotides from the 3' end and/or the 5'
end of either or both strands). A spacer sequence may therefore be
any polynucleotide sequence as provided herein that is situated
between two complementary polynucleotide sequence regions which,
when annealed into a double-stranded nucleic acid, comprise a siRNA
polynucleotide. Preferably a spacer sequence comprises at least 4
nucleotides, although in certain embodiments the spacer may
comprise 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20,
21-25, 26-30, 31-40, 41-50, 51-70, 71-90, 91-110, 111-150, 151-200
or more nucleotides. Examples of siRNA polynucleotides derived from
a single nucleotide strand comprising two complementary nucleotide
sequences separated by a spacer have been described (e.g.,
Brummelkamp et al., 2002 Science 296:550; Paddison et al., 2002
Genes Develop. 16:948; Paul et al. Nat. Biotechnol. 20:505-508
(2002); Grabarek et al., BioTechniques 34:734-44 (2003)).
[0047] Polynucleotide variants may contain one or more
substitutions, additions, deletions, and/or insertions such that
the activity of the siRNA polynucleotide is not substantially
diminished, as described above. The effect on the activity of the
siRNA polynucleotide may generally be assessed as described herein
or using conventional methods. Variants preferably exhibit at least
about 75%, 78%, 80%, 85%, 87%, 88% or 89% identity and more
preferably at least about 90%, 92%, 95%, 96%, 97%, 98%, or 99%
identity to a portion of a polynucleotide sequence that encodes a
native PTP1B. The percent identity may be readily determined by
comparing sequences of the polynucleotides to the corresponding
portion of a full-length PTP1B polynucleotide such as those known
to the art and cited herein, using any method including using
computer algorithms well known to those having ordinary skill in
the art, such as Align or the BLAST algorithm (Altschul, J. Mol.
Biol. 219:555-565, 1991; Henikoff and Henikoff, Proc. Natl. Acad.
Sci. USA 89:10915-10919, 1992), which is available at the NCBI
website (see [online] Internet:<URL:
http://www/ncbi.nlm.nih.gov/cgi-bin/BLAST). Default parameters may
be used.
[0048] Certain siRNA polynucleotide variants are substantially
homologous to a portion of a native PTP1B gene. Single-stranded
nucleic acids derived (e.g., by thermal denaturation) from such
polynucleotide variants are capable of hybridizing under moderately
stringent conditions to a naturally occurring DNA or RNA sequence
encoding a native PTP1B polypeptide (or a complementary sequence).
A polynucleotide that detectably hybridizes under moderately
stringent conditions may have a nucleotide sequence that includes
at least 10 consecutive nucleotides, more preferably 11, 12, 13,
14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or
30 consecutive nucleotides complementary to a particular
polynucleotide. In certain preferred embodiments such a sequence
(or its complement) will be unique to a PTP1B polypeptide for which
interference with expression is desired, and in certain other
embodiments the sequence (or its complement) may be shared by PTP1B
and one or more PTPs for which interference with polypeptide
expression is desired.
[0049] Suitable moderately stringent conditions include, for
example, pre-washing in a solution of 5.times.SSC, 0.5% SDS, 1.0 mM
EDTA (pH 8.0); hybridizing at 50.degree. C.-70.degree. C.,
5.times.SSC for 1-16 hours (e.g., overnight); followed by washing
once or twice at 22-65.degree. C. for 20-40 minutes with one or
more each of 2.times., 0.5.times. and 0.2.times.SSC containing
0.05-0.1% SDS. For additional stringency, conditions may include a
wash in 0.1.times.SSC and 0.1% SDS at 50-60.degree. C. for 15-40
minutes. As known to those having ordinary skill in the art,
variations in stringency of hybridization conditions may be
achieved by altering the time, temperature, and/or concentration of
the solutions used for pre-hybridization, hybridization, and wash
steps. Suitable conditions may also depend in part on the
particular nucleotide sequences of the probe used, and of the
blotted, proband nucleic acid sample. Accordingly, it will be
appreciated that suitably stringent conditions can be readily
selected without undue experimentation when a desired selectivity
of the probe is identified, based on its ability to hybridize to
one or more certain proband sequences while not hybridizing to
certain other proband sequences.
[0050] Sequence specific siRNA polynucleotides of the present
invention may be designed using one or more of several criteria.
For example, to design a siRNA polynucleotide that has 19
consecutive nucleotides identical to a sequence encoding a
polypeptide of interest (e.g., PTP1B and other polypeptides
described herein), the open reading frame of the polynucleotide
sequence may be scanned for 21-base sequences that have one or more
of the following characteristics: (1) an A+T/G+C ratio of
approximately 1:1 but no greater than 2:1 or 1:2; (2) an AA
dinucleotide or a CA dinucleotide at the 5' end; (3) an internal
hairpin loop melting temperature less than 55.degree. C.; (4) a
homodimer melting temperature of less than 37.degree. C. (melting
temperature calculations as described in (3) and (4) can be
determined using computer software known to those skilled in the
art); (5) a sequence of at least 16 consecutive nucleotides not
identified as being present in any other known polynucleotide
sequence (such an evaluation can be readily determined using
computer programs available to a skilled artisan such as BLAST to
search publicly available databases). Alternatively, an siRNA
polynculeotide sequence may be designed and chosen using a computer
software available commercially from various vendors (e.g.,
OligoEngine.TM. (Seattle, Wash.); Dharmacon, Inc. (Lafayette,
Colo.); Ambion Inc. (Austin, Tex.); and QIAGEN, Inc. (Valencia,
Calif.)). (See also Elbashir et al., Genes & Development
15:188-200 (2000); Elbashir et al., Nature 411:494-98 (2001); and
[online] Internet:URL<http://www.mp-
ibpc.gwdg.de/abteilungen/100/105/Tuschl_MIV2(3).sub.--2002.p df.)
The siRNA polynucleotides may then be tested for their ability to
interfere with the expression of the target polypeptide according
to methods known in the art and described herein. The determination
of the effectiveness of an siRNA polynucleotide includes not only
consideration of its ability to interfere with polypeptide
expression but also includes consideration of whether the siRNA
polynucleotide manifests undesirably toxic effects, for example,
apoptosis of a cell for which cell death is not a desired effect of
RNA interference (e.g., interference of PTP1B expression in a
cell).
[0051] It should be appreciated that not all siRNAs designed using
the above methods will be effective at silencing or interfering
with expression of a PTP1B target polypeptide. And further, that
the siRNAs will effect silencing to different degrees. Such siRNAs
must be tested for their effectiveness, and selections made
therefrom based on the ability of a given siRNA to interfere with
or modulate (e.g., decrease in a statistically significant manner)
the expression of PTP1B. Accordingly, identification of specific
siRNA polynucleotide sequences that are capable of interfering with
expression of a PTP1B polypeptide requires production and testing
of each siRNA, as demonstrated in greater detail below (see
Examples).
[0052] Furthermore, not all siRNAs that interfere with protein
expression will have a physiologically important effect. The
inventors here have designed, and describe herein, physiologically
relevant assays for measuring the influence of modulated target
polypeptide expression, for instance, cellular proliferation,
induction of apoptosis, and/or altered levels of protein tyrosine
phosphorylation (e.g., insulin receptor phosphorylation), to
determine if the levels of interference with target protein
expression that were observed using the siRNAs of the invention
have clinically relevant significance. Additionally, and according
to non-limiting theory, the invention contemplates altered (e.g.,
decreased or increased in a statistically significant manner)
expression levels of one or more polypeptides of interest, and/or
altered (i.e., increased or decreased) phosphorylation levels of
one or more phosphoproteins of interest, which altered levels may
result from impairment of PTP1B protein expression and/or cellular
compensatory mechanisms that are induced in response to
RNAi-mediated inhibition of a specific target polypeptide
expression.
[0053] Persons having ordinary skill in the art will also readily
appreciate that as a result of the degeneracy of the genetic code,
many nucleotide sequences may encode a polypeptide as described
herein. That is, an amino acid may be encoded by one of several
different codons and a person skilled in the art can readily
determine that while one particular nucleotide sequence may differ
from another (which may be determined by alignment methods
disclosed herein and known in the art), the sequences may encode
polypeptides with identical amino acid sequences. By way of
example, the amino acid leucine in a polypeptide may be encoded by
one of six different codons (TTA, TTG, CTT, CTC, CTA, and CTG) as
can serine (TCT, TCC, TCA, TCG, AGT, and AGC). Other amino acids,
such as proline, alanine, and valine, for example, may be encoded
by any one of four different codons (CCT, CCC, CCA, CCG for
proline; GCT, GCC, GCA, GCG for alanine; and GTT, GTC, GTA, GTG for
valine). 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.
[0054] Polynucleotides, including target polynucleotides (e.g.,
polynucleotides capable of encoding a target polypeptide of
interest), may be prepared using any of a variety of techniques,
which will be useful for the preparation of specifically desired
siRNA polynucleotides and for the identification and selection of
desirable sequences to be used in siRNA polynucleotides. For
example, a polynucleotide may be amplified from cDNA prepared from
a suitable cell or tissue type. Such polynucleotides may be
amplified via polymerase chain reaction (PCR). For this approach,
sequence-specific primers may be designed based on the sequences
provided herein and may be purchased or synthesized. An amplified
portion may be used to isolate a full-length gene, or a desired
portion thereof, from a suitable library (e.g., human skeletal
muscle cDNA) using well known techniques. Within such techniques, a
library (cDNA or genomic) is screened using one or more
polynucleotide probes or primers suitable for amplification.
Preferably, a library is size-selected to include larger molecules.
Random primed libraries may also be preferred for identifying 5'
and upstream regions of genes. Genomic libraries are preferred for
obtaining introns and extending 5' sequences. Suitable sequences
for a siRNA polynucleotide contemplated by the present invention
may also be selected from a library of siRNA polynucleotide
sequences.
[0055] For hybridization techniques, a partial sequence may be
labeled (e.g., by nick-translation or end-labeling with .sup.32P)
using well known techniques. A bacterial or bacteriophage library
may then be screened by hybridizing filters containing denatured
bacterial colonies (or lawns containing phage plaques) with the
labeled probe (see, e.g., Sambrook et al., Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring
Harbor, N.Y., 2001). Hybridizing colonies or plaques are selected
and expanded, and the DNA is isolated for further analysis. Clones
may be analyzed to determine the amount of additional sequence by,
for example, PCR using a primer from the partial sequence and a
primer from the vector. Restriction maps and partial sequences may
be generated to identify one or more overlapping clones. A
full-length cDNA molecule can be generated by ligating suitable
fragments, using well known techniques.
[0056] Alternatively, numerous amplification techniques are known
in the art for obtaining a full-length coding sequence from a
partial cDNA sequence. Within such techniques, amplification is
generally performed via PCR. One such technique is known as "rapid
amplification of cDNA ends" or RACE. This technique involves the
use of an internal primer and an external primer, which hybridizes
to a polyA region or vector sequence, to identify sequences that
are 5' and 3' of a known sequence. Any of a variety of commercially
available kits may be used to perform the amplification step.
Primers may be designed using, for example, software well known in
the art. Primers (or oligonucleotides for other uses contemplated
herein, including, for example, probes and antisense
oligonucleotides) are preferably 15, 16, 17, 18, 19, 20, 21, 22,
23, 24, 25, 26, 27, 28, 29, 30, 31 or 32 nucleotides in length,
have a GC content of at least 40% and anneal to the target sequence
at temperatures of about 54.degree. C. to 72.degree. C. The
amplified region may be sequenced as described above, and
overlapping sequences assembled into a contiguous sequence. Certain
oligonucleotides contemplated by the present invention may, for
some preferred embodiments, have lengths of 18, 19, 20, 21, 22, 23,
24, 25, 26, 27, 28, 29, 30, 31, 32, 33-35, 35-40, 41-45, 46-50,
56-60, 61-70, 71-80, 81-90 or more nucleotides.
[0057] A number of specific siRNA polynucleotide sequences useful
for interfering with PTP1B polypeptide expression are presented in
the Examples, the Drawings, and the Sequence Listing. SiRNA
polynucleotides may generally be prepared by any method known in
the art, including, for example, solid phase chemical synthesis.
Modifications in a polynucleotide sequence may also be introduced
using standard mutagenesis techniques, such as
oligonucleotide-directed site-specific mutagenesis. Further, siRNAs
may be chemically modified or conjugated to improve their serum
stability and/or delivery properties. Included as an aspect of the
invention are the siRNAs described herein wherein the ribose has
been removed therefrom. Alternatively, siRNA polynucleotide
molecules may be generated by in vitro or in vivo transcription of
suitable DNA sequences (e.g., polynucleotide sequences encoding a
PTP, or a desired portion thereof), provided that the DNA is
incorporated into a vector with a suitable RNA polymerase promoter
(such as T7, U6, H1, or SP6). In addition, a siRNA polynucleotide
may be administered to a patient, as may be a DNA sequence (e.g, a
recombinant nucleic acid construct as provided herein) that
supports transcription (and optionally appropriate processing
steps) such that a desired siRNA is generated in vivo.
[0058] Accordingly, a siRNA polynucleotide that is complementary to
at least a portion of a PTP1B coding sequence may be used to
modulate gene expression, or as a probe or primer. Identification
of siRNA polynucleotide sequences and DNA encoding genes for their
targeted delivery involves techniques described herein with regard
to PTP1B. Identification of such siRNA polynucleotide sequences and
DNA encoding genes for their targeted delivery involves techniques
that are also described herein. As discussed above, siRNA
polynucleotides exhibit desirable stability characteristics and
may, but need not, be further designed to resist degradation by
endogenous nucleolytic enzymes by using such linkages as
phosphorothioate, methylphosphonate, sulfone, sulfate, ketyl,
phosphorodithioate, phosphoramidate, phosphate esters, and other
such linkages (see, e.g., Agrwal et al., Tetrahedron Lett.
28:3539-3542 (1987); Miller et al., J. Am. Chem. Soc. 93:6657-6665
(1971); Stec et al., Tetrahedron Lett. 26:2191-2194 (1985); Moody
et al., Nucleic Acids Res. 12:4769-4782 (1989); Uznanski et al.,
Nucleic Acids Res. (1989); Letsinger et al., Tetrahedron 40:137-143
(1984); Eckstein, Annu. Rev. Biochem. 54:367-402 (1985); Eckstein,
Trends Biol. Sci. 14:97-100 (1989); Stein, In:
Oligodeoxynucleotides. Antisense Inhibitors of Gene Expression,
Cohen, ed., Macmillan Press, London, pp. 97-117 (1989); Jager et
al., Biochemistry 27:7237-7246 (1988)).
[0059] Any polynucleotide of the invention 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 phosphodiester linkages in the backbone; and/or the inclusion
of nontraditional bases such as inosine, queosine, and wybutosine
and the like, as well as acetyl- methyl-, thio- and other modified
forms of adenine, cytidine, guanine, thymine, and uridine.
[0060] Nucleotide sequences as described herein may be joined to a
variety of other nucleotide sequences using established recombinant
DNA techniques. For example, a polynucleotide may be cloned into
any of a variety of cloning vectors, including plasmids, phagemids,
lambda phage derivatives, and cosmids. Vectors of particular
interest include expression vectors, replication vectors, probe
generation vectors, and sequencing vectors. In general, a suitable
vector contains an origin of replication functional in at least one
organism, convenient restriction endonuclease sites, and one or
more selectable markers. (See, e.g., WO 01/96584; WO 01/29058; U.S.
Pat. No. 6,326,193; U.S. 2002/0007051). Other elements will depend
upon the desired use, and will be apparent to those having ordinary
skill in the art. For example, the invention contemplates the use
of siRNA polynucleotide sequences in the preparation of recombinant
nucleic acid constructs including vectors for interfering with the
expression of a desired target polypeptide such as a PTP1B
polypeptide in vivo; the invention also contemplates the generation
of siRNA transgenic or "knock-out" animals and cells (e.g., cells,
cell clones, lines or lineages, or organisms in which expression of
one or more desired polypeptides (e.g., a target polypeptide) is
fully or partially compromised). An siRNA polynucleotide that is
capable of interfering with expression of a desired polypeptide
(e.g., a target polypeptide) as provided herein thus includes any
siRNA polynucleotide that, when contacted with a subject or
biological source as provided herein under conditions and for a
time sufficient for target polypeptide expression to take place in
the absence of the siRNA polynucleotide, results in a statistically
significant decrease (alternatively referred to as "knockdown" of
expression) in the level of target polypeptide expression that can
be detected. Preferably the decrease is greater than 10%, more
preferably greater than 20%, more preferably greater than 30%, more
preferably greater than 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%
or 98% relative to the expression level of the polypeptide detected
in the absence of the siRNA, using conventional methods for
determining polypeptide expression as known to the art and provided
herein. Preferably, the presence of the siRNA polynucleotide in a
cell does not result in or cause any undesired toxic effects, for
example, apoptosis or death of a cell in which apoptosis is not a
desired effect of RNA interference.
[0061] Within certain embodiments, siRNA polynucleotides may be
formulated so as to permit entry into a cell of a mammal, and
expression therein. Such formulations are particularly useful for
therapeutic purposes, as described below. Those having ordinary
skill in the art will appreciate that there are many ways to
achieve expression of a polynucleotide in a target cell, and any
suitable method may be employed. For example, a polynucleotide may
be incorporated into a viral vector using well known techniques
(see also, e.g., U.S. 2003/0068821). A viral vector may
additionally transfer or incorporate a gene for a selectable marker
(to aid in the identification or selection of transduced cells)
and/or a targeting moiety, such as a gene that encodes a ligand for
a receptor on a specific target cell, to render the vector target
specific. Targeting may also be accomplished using an antibody, by
methods known to those having ordinary skill in the art.
[0062] Other formulations for therapeutic purposes include
colloidal dispersion systems, such as macromolecule complexes,
nanocapsules, microspheres, beads, and lipid-based systems
including oil-in-water emulsions, micelles, mixed micelles, and
liposomes. A preferred colloidal system for use as a delivery
vehicle in vitro and in vivo is a liposome (i.e., an artificial
membrane vesicle). The preparation and use of such systems is well
known in the art.
[0063] Within other embodiments, one or more promoters may be
identified, isolated and/or incorporated into recombinant nucleic
acid constructs of the present invention, using standard
techniques. The present invention provides nucleic acid molecules
comprising such a promoter sequence or one or more cis- or
trans-acting regulatory elements thereof. Such regulatory elements
may enhance or suppress expression of a siRNA. A 5' flanking region
may be generated using standard techniques, based on the genomic
sequence provided herein. If necessary, additional 5' sequences may
be generated using PCR-based or other standard methods. The 5'
region may be subcloned and sequenced using standard methods.
Primer extension and/or RNase protection analyses may be used to
verify the transcriptional start site deduced from the cDNA.
[0064] To define the boundary of the promoter region, putative
promoter inserts of varying sizes may be subcloned into a
heterologous expression system containing a suitable reporter gene
without a promoter or enhancer. Suitable reporter genes may include
genes encoding luciferase, beta-galactosidase, chloramphenicol
acetyl transferase, secreted alkaline phosphatase, or the Green
Fluorescent Protein gene (see, e.g., Ui-Tei et al., FEBS Lett.
479:79-82 (2000). Suitable expression systems are well known and
may be prepared using well known techniques or obtained
commercially. Internal deletion constructs may be generated using
unique internal restriction sites or by partial digestion of
non-unique restriction sites. Constructs may then be transfected
into cells that display high levels of siRNA polynucleotide and/or
polypeptide expression. In general, the construct with the minimal
5' flanking region showing the highest level of expression of
reporter gene is identified as the promoter. Such promoter regions
may be linked to a reporter gene and used to evaluate agents for
the ability to modulate promoter-driven transcription.
[0065] Once a functional promoter is identified, cis- and
trans-acting elements may be located. Cis-acting sequences may
generally be identified based on homology to previously
characterized transcriptional motifs. Point mutations may then be
generated within the identified sequences to evaluate the
regulatory role of such sequences. Such mutations may be generated
using site-specific mutagenesis techniques or a PCR-based strategy.
The altered promoter is then cloned into a reporter gene expression
vector, as described above, and the effect of the mutation on
reporter gene expression is evaluated.
[0066] In general, polypeptides and polynucleotides as described
herein 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. A "gene" includes the segment of DNA involved
in producing a polypeptide chain; it further includes regions
preceding and following the coding region "leader and trailer," for
example promoter and/or enhancer and/or other regulatory sequences
and the like, as well as intervening sequences (introns) between
individual coding segments (exons).
[0067] The effect of siRNA interference with expression of a
component in the signal transduction pathway induced by insulin,
for example, may be evaluated by determining the level of tyrosine
phosphorylation of insulin receptor beta (IR-.beta.) and/or of the
downstream signaling molecule PKB/Akt and/or of any other
downstream polypeptide that may be a component of a particular
signal transduction pathway as provided herein.
[0068] As noted above, regulated tyrosine phosphorylation
contributes to specific pathways for biological signal
transduction, including those associated with cell division, cell
survival, apoptosis, proliferation and differentiation, and
"biological signal transduction pathways," or "inducible signaling
pathways" in the context of the present invention include transient
or stable associations or interactions among molecular components
involved in the control of these and similar processes in cells.
Depending on the particular pathway of interest, an appropriate
parameter for determining induction of such pathway may be
selected. For example, for signaling pathways associated with cell
proliferation, a variety of well known methodologies are available
for quantifying proliferation, including, for example,
incorporation of tritiated thymidine into cellular DNA, monitoring
of detectable (e.g., fluorimetric or calorimetric) indicators of
cellular respiratory activity (for example, conversion of the
tetrazolium salts (yellow)
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
or
3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl-
)-2H-tetrazolium (MTS) to formazan dyes (purple) in metabolically
active cells), or cell counting, or the like. Similarly, in the
cell biology arts, multiple techniques are known for assessing cell
survival (e.g., vital dyes, metabolic indicators, etc.) and for
determining apoptosis (for example, annexin V binding, DNA
fragmentation assays, caspase activation, marker analysis, e.g.,
poly(ADP-ribose) polymerase (PARP), etc.). Other signaling pathways
will be associated with particular cellular phenotypes, for example
specific induction of gene expression (e.g., detectable as
transcription or translation products, or by bioassays of such
products, or as nuclear localization of cytoplasmic factors),
altered (e.g., statistically significant increases or decreases)
levels of intracellular mediators (e.g., activated kinases or
phosphatases, altered levels of cyclic nucleotides or of
physiologically active ionic species, etc.), altered cell cycle
profiles, or altered cellular morphology, and the like, such that
cellular responsiveness to a particular stimulus as provided herein
can be readily identified to determine whether a particular cell
comprises an inducible signaling pathway.
[0069] PTP1B
[0070] The sequence of PTP-1B as used herein, means any sequence,
as the context requires, selected from the following group (e.g.,
GenBank Accession Nos. M31724 (SEQ ID NOS: __-__); NM.sub.--002827
(SEQ ID NOS: __-__); NM.sub.--011201 (SEQ ID NOS: __-__; M31724
(SEQ ID NOS: __-__); M33689 (SEQ ID NOS: __-__); M33962 (SEQ ID
NOS: __-__) The invention also includes variants or mutated forms
of PTP1B that contain single nucleotide polymorphisms (SNPs), or
allelic forms.
[0071] Specific substitutions of individual amino acids through
introduction of site-directed mutations are well-known and may be
made according to methodologies with which those having ordinary
skill in the art will be familiar. The effects on catalytic
activity of the resulting mutant PTP may be determined empirically
by testing the resulting modified protein for the preservation of
the Km and reduction of Kcat to less than 1 per minute as provided
herein and as previously disclosed (e.g., WO98/04712; Flint et al.,
1997 Proc. Nat. Acad. Sci. USA 94:1680). In addition, the effect on
the ability of the resulting mutant PTP molecule to phosphorylate
one or more tyrosine residues can also be determined empirically
merely by testing such a mutant for the presence of
phosphotyrosine, as also provided herein, for example, following
exposure of the mutant to conditions in vitro or in vivo where it
may act as a phosphate acceptor for a protein tyrosine kinase.
[0072] In particular, portions of two PTP1B polypeptide sequences
are regarded as "corresponding" amino acid sequences, regions,
fragments or the like, based on a convention of numbering one PTP1B
sequence according to amino acid position number, and then aligning
the sequence to be compared in a manner that maximizes the number
of amino acids that match or that are conserved residues, for
example, that remain polar (e.g., D, E, K, R, H, S, T, N, Q),
hydrophobic (e.g, A, P, V, L, I, M, F, W, Y) or neutral (e.g, C, G)
residues at each position. Similarly, a DNA sequence encoding a
candidate PTP that is to be mutated as provided herein, or a
portion, region, fragment or the like, may correspond to a known
wildtype PTP1B-encoding DNA sequence according to a convention for
numbering nucleic acid sequence positions in the known wildtype
PTP1B DNA sequence, whereby the candidate PTP DNA sequence is
aligned with the known PTP1B DNA such that at least 70%, preferably
at least 80% and more preferably at least 90% of the nucleotides in
a given sequence of at least 20 consecutive nucleotides of a
sequence are identical. In certain preferred embodiments, a
candidate PTP DNA sequence is greater than 95% identical to a
corresponding known PTP1B DNA sequence. In certain particularly
preferred embodiments, a portion, region or fragment of a candidate
PTP DNA sequence is identical to a corresponding known PTP1B DNA
sequence. As is well known in the art, an individual whose DNA
contains no irregularities (e.g, a common or prevalent form) in a
particular gene responsible for a given trait may be said to
possess a wildtype genetic complement (genotype) for that gene,
while the presence of irregularities known as mutations in the DNA
for the gene, for example, substitutions, insertions or deletions
of one or more nucleotides, indicates a mutated or mutant
genotype.
[0073] Modification of DNA may be performed by a variety of
methods, including site-specific or site-directed mutagenesis of
DNA encoding the polypeptide of interest (e.g., a siRNA target
polypeptide) and the use of DNA amplification methods using primers
to introduce and amplify alterations in the DNA template, such as
PCR splicing by overlap extension (SOE). Site-directed mutagenesis
is typically effected using a phage vector that has single- and
double-stranded forms, such as M13 phage vectors, which are
well-known and commercially available. Other suitable vectors that
contain a single-stranded phage origin of replication may be used
(see, e.g., Veira et al., Meth. Enzymol. 15:3, 1987). In general,
site-directed mutagenesis is performed by preparing a
single-stranded vector that encodes the protein of interest (e.g.,
PTP1B). An oligonucleotide primer that contains the desired
mutation within a region of homology to the DNA in the
single-stranded vector is annealed to the vector followed by
addition of a DNA polymerase, such as E. coli DNA polymerase I
(Klenow fragment), which uses the double stranded region as a
primer to produce a heteroduplex in which one strand encodes the
altered sequence and the other the original sequence. Additional
disclosure relating to site-directed mutagenesis may be found, for
example, in Kunkel et al. (Methods in Enzymol. 154:367, 1987) and
in U.S. Pat. Nos. 4,518,584 and 4,737,462. The heteroduplex is
introduced into appropriate bacterial cells, and clones that
include the desired mutation are selected. The resulting altered
DNA molecules may be expressed recombinantly in appropriate host
cells to produce the modified protein.
[0074] SiRNAs of the invention may be fused to other nucleotide
molecules, or to polypeptides, in order to direct their delivery or
to accomplish other functions. Thus, for example, fusion proteins
comprising a siRNA oligonucleotide that is capable of specifically
interfering with expression of PTP1B may comprise affinity tag
polypeptide sequences, which refers to polypeptides or peptides
that facilitate detection and isolation of the such polypeptide via
a specific affinity interaction with a ligand. The ligand may be
any molecule, receptor, counterreceptor, antibody or the like with
which the affinity tag may interact through a specific binding
interaction as provided herein. Such peptides include, for example,
poly-His or "FLAG.RTM." or the like, e.g., the antigenic
identification peptides described in U.S. Pat. No. 5,011,912 and in
Hopp et al., (1988 Bio/Technology 6:1204), or the XPRESS.TM.
epitope tag (Invitrogen, Carlsbad, Calif.). The affinity sequence
may be a hexa-histidine tag as supplied, for example, by a pBAD/His
(Invitrogen) or a pQE-9 vector to provide for purification of the
mature polypeptide fused to the marker in the case of a bacterial
host, or, for example, the affinity sequence may be a hemagglutinin
(HA) tag when a mammalian host, e.g., COS-7 cells, is used. The HA
tag corresponds to an antibody defined epitope derived from the
influenza hemagglutinin protein (Wilson et al., 1984 Cell
37:767).
[0075] The present invention also relates to vectors and to
constructs that include or encode siRNA polynucleotides of the
present invention, and in particular to "recombinant nucleic acid
constructs" that include any nucleic acid such as a DNA
polynculeotide segment that may be transcribed to yield PTP1B
polynucleotide-specific siRNA polynucleotides according to the
invention as provided above; to host cells which are genetically
engineered with vectors and/or constructs of the invention and to
the production of siRNA polynucleotides, polypeptides, and/or
fusion proteins of the invention, or fragments or variants thereof,
by recombinant techniques. SiRNA sequences disclosed herein as RNA
polynucleotides may be engineered to produce corresponding DNA
sequences using well-established methodologies such as those
described herein. Thus, for example, a DNA polynucleotide may be
generated from any siRNA sequence described herein (including in
the Sequence Listing), such that the present siRNA sequences will
be recognized as also providing corresponding DNA polynucleotides
(and their complements). These DNA polynucleotides are therefore
encompassed within the contemplated invention, for example, to be
incorporated into the subject invention recombinant nucleic acid
constructs from which siRNA may be transcribed.
[0076] According to the present invention, a vector may comprise a
recombinant nucleic acid construct containing one or more promoters
for transcription of an RNA molecule, for example, the human U6
snRNA promoter (see, e.g., Miyagishi et al, Nat. Biotechnol.
20:497-500 (2002); Lee et al., Nat. Biotechnol. 20:500-505 (2002);
Paul et al., Nat. Biotechnol. 20:505-508 (2002); Grabarek et al.,
BioTechniques 34:73544 (2003); see also Sui et al., Proc. Natl.
Acad. Sci. USA 99:5515-20 (2002)). Each strand of a siRNA
polynucleotide may be transcribed separately each under the
direction of a separate promoter and then may hybridize within the
cell to form the siRNA polynucleotide duplex. Each strand may also
be transcribed from separate vectors (see Lee et al., supra).
Alternatively, the sense and antisense sequences specific for a
PTP1B sequence may be transcribed under the control of a single
promoter such that the siRNA polynucleotide forms a hairpin
molecule (Paul et al., supra). In such an instance, the
complementary strands of the siRNA specific sequences are separated
by a spacer that comprises at least four nucleotides, but may
comprise at least 5, 6, 7, 8, 9, 10, 11, 12, 14, 16, 94 18
nucleotides or more nucleotides as described herein. In addition,
siRNAs transcribed under the control of a U6 promoter that form a
hairpin may have a stretch of about four uridines at the 3' end
that act as the transcription termination signal (Miyagishi et al.,
supra; Paul et al., supra). By way of illustration, if the target
sequence is 19 nucleotides, the siRNA hairpin polynucleotide
(beginning at the 5' end) has a 19-nucleotide sense sequence
followed by a spacer (which as two uridine nucleotides adjacent to
the 3' end of the 19-nucleotide sense sequence), and the spacer is
linked to a 19 nucleotide antisense sequence followed by a
4-uridine terminator sequence, which results in an overhang. SiRNA
polynucleotides with such overhangs effectively interfere with
expression of the target polypeptide (see id.). A recombinant
construct may also be prepared using another RNA polymerase III
promoter, the HI RNA promoter, that may be operatively linked to
siRNA polynucleotide specific sequences, which may be used for
transcription of hairpin structures comprising the siRNA specific
sequences or separate transcription of each strand of a siRNA
duplex polynucleotide (see, e.g., Brummelkamp et al., Science
296:550-53 (2002); Paddison et al., supra). DNA vectors useful for
insertion of sequences for transcription of an siRNA polynucleotide
include pSUPER vector (see, e.g., Brummelkamp et al., supra); pAV
vectors derived from pCWRSVN (see, e.g., Paul et al., supra); and
pIND (see, e.g., Lee et al., supra), or the like.
[0077] PTP1B polypeptides can be expressed in mammalian cells,
yeast, bacteria, or other cells under the control of appropriate
promoters, providing ready systems for determination of siRNA
polynucleotides that are capable of interfering with polypeptide
expression as provided herein. Appropriate cloning and expression
vectors for use with prokaryotic and eukaryotic hosts are
described, for example, by Sambrook, et al., Molecular Cloning: A
Laboratory Manual, Third Edition, Cold Spring Harbor, N.Y.,
(2001).
[0078] Generally, recombinant expression vectors for use in the
preparation of recombinant nucleic acid constructs or vectors of
the invention will include origins of replication and selectable
markers permitting transformation of the host cell, e.g., the
ampicillin resistance gene of E. coli and S. cerevisiae TRP1 gene,
and a promoter derived from a highly-expressed gene to direct
transcription of a downstream structural sequence (e.g., a siRNA
polynucleotide sequence). Such promoters can be derived from
operons encoding glycolytic enzymes such as 3-phosphoglycerate
kinase (PGK), .alpha.-factor, acid phosphatase, or heat shock
proteins, among others. For PTP polypeptide expression (including
PTP fusion proteins and substrate trapping mutant PTPs), and for
other expression of other polypeptides of interest, the
heterologous structural sequence is assembled in appropriate phase
with translation initiation and termination sequences. Optionally,
the heterologous sequence can encode a fusion protein including an
N-terminal identification peptide imparting desired
characteristics, e.g., stabilization or simplified purification of
expressed recombinant product.
[0079] Useful expression constructs for bacterial use are
constructed by inserting into an expression vector a structural DNA
sequence encoding a desired siRNA polynucleotide, together with
suitable transcription initiation and termination signals in
operable linkage, for example, with a functional promoter. The
construct may comprise one or more phenotypic selectable markers
and an origin of replication to ensure maintenance of the vector
construct and, if desirable, to provide amplification within the
host. Suitable prokaryotic hosts for transformation include E.
coli, Bacillus subtilis, Salmonella typhimurium and various species
within the genera Pseudomonas, Streptomyces, and Staphylococcus,
although others may also be employed as a matter of choice. Any
other plasmid or vector may be used as long as they are replicable
and viable in the host.
[0080] As a representative but nonlimiting example, useful
expression vectors for bacterial use can comprise a selectable
marker and bacterial origin of replication derived from
commercially available plasmids comprising genetic elements of the
well known cloning vector pBR322 (ATCC 37017). Such commercial
vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals,
Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, Wis., USA).
These pBR322 "backbone" sections are combined with an appropriate
promoter and the structural sequence to be expressed.
[0081] Following transformation of a suitable host strain and
growth of the host strain to an appropriate cell density, the
selected promoter, if it is a regulated promoter as provided
herein, is induced by appropriate means (e.g., temperature shift or
chemical induction) and cells are cultured for an additional
period. Cells are typically harvested by centrifugation, disrupted
by physical or chemical means, and the resulting crude extract
retained for further purification. Microbial cells employed in
expression of proteins can be disrupted by any convenient method,
including freeze-thaw cycling, sonication, mechanical disruption,
or use of cell lysing agents; such methods are well know to those
skilled in the art.
[0082] Thus, for example, the nucleic acids of the invention as
described herein (e.g., DNA sequences from which siRNA may be
transcribed) may be included in any one of a variety of expression
vector constructs as a recombinant nucleic acid construct for
expressing a PTP1B polynucleotide-specific siRNA polynucleotide as
provided herein. Such vectors and constructs include chromosomal,
nonchromosomal and synthetic DNA sequences, e.g., derivatives of
SV40; bacterial plasmids; phage DNA; baculovirus; yeast plasmids;
vectors derived from combinations of plasmids and phage DNA, viral
DNA, such as vaccinia, adenovirus, fowl pox virus, and
pseudorabies. However, any other vector may be used for preparation
of a recombinant nucleic acid construct as long as it is replicable
and viable in the host.
[0083] The appropriate DNA sequence(s) may be inserted into the
vector by a variety of procedures. In general, the DNA sequence is
inserted into an appropriate restriction endonuclease site(s) by
procedures known in the art. Standard techniques for cloning, DNA
isolation, amplification and purification, for enzymatic reactions
involving DNA ligase, DNA polymerase, restriction endonucleases and
the like, and various separation techniques are those known and
commonly employed by those skilled in the art. A number of standard
techniques are described, for example, in Ausubel et al. (1993
Current Protocols in Molecular Biology, Greene Publ. Assoc. Inc.
& John Wiley & Sons, Inc., Boston, Mass.); Sambrook et al.
(2001 Molecular Cloning, Third Ed., Cold Spring Harbor Laboratory,
Plainview, N.Y.); Maniatis et al. (1982 Molecular Cloning, Cold
Spring Harbor Laboratory, Plainview, N.Y.); and elsewhere.
[0084] The DNA sequence in the expression vector is operatively
linked to at least one appropriate expression control sequences
(e.g., a promoter or a regulated promoter) to direct mRNA
synthesis. Representative examples of such expression control
sequences include LTR or SV40 promoter, the E. coli lac or trp, the
phage lambda P.sub.L promoter and other promoters known to control
expression of genes in prokaryotic or eukaryotic cells or their
viruses. Promoter regions can be selected from any desired gene
using CAT (chloramphenicol transferase) vectors or other vectors
with selectable markers. Two appropriate vectors are pKK232-8 and
pCM7. Particular named bacterial promoters include lacd, lacZ, T3,
T7, gpt, lambda P.sub.R, P.sub.L and trp. Eukaryotic promoters
include CMV immediate early, HSV thymidine kinase, early and late
SV40, LTRs from retrovirus, and mouse metallothionein-I. Selection
of the appropriate vector and promoter is well within the level of
ordinary skill in the art, and preparation of certain particularly
preferred recombinant expression constructs comprising at least one
promoter or regulated promoter operably linked to a nucleic acid
encoding a PTP1B polypeptide is described herein.
[0085] As noted above, in certain embodiments the vector may be a
viral vector such as a retroviral vector. For example, retroviruses
from which the retroviral plasmid vectors may be derived include,
but are not limited to, Moloney Murine Leukemia Virus, spleen
necrosis virus, retroviruses such as Rous Sarcoma Virus, Harvey
Sarcoma virus, avian leukosis virus, gibbon ape leukemia virus,
human immunodeficiency virus, adenovirus, Myeloproliferative
Sarcoma Virus, and mammary tumor virus.
[0086] The viral vector includes one or more promoters. Suitable
promoters which may be employed include, but are not limited to,
the retroviral LTR; the SV40 promoter; and the human
cytomegalovirus (CMV) promoter described in Miller, et al.,
Biotechniques 7:980-990 (1989), or any other promoter (e.g.,
cellular promoters such as eukaryotic cellular promoters including,
but not limited to, the histone, pol III, and .beta.-actin
promoters). Other viral promoters that may be employed include, but
are not limited to, adenovirus promoters, thymidine kinase (TK)
promoters, and B19 parvovirus promoters. The selection of a
suitable promoter will be apparent to those skilled in the art from
the teachings contained herein, and may be from among either
regulated promoters or promoters as described above.
[0087] The retroviral plasmid vector is employed to transduce
packaging cell lines to form producer cell lines. Examples of
packaging cells which may be transfected include, but are not
limited to, the PE501, PA317, .psi.-2, .psi.-AM, PA12, T19-14X,
VT-19-17-H2, .psi.CRE, .psi.CRIP, GP+E-86, GP+envAm12, and DAN cell
lines as described in Miller, Human Gene Therapy, 1:5-14 (1990),
which is incorporated herein by reference in its entirety. The
vector may transduce the packaging cells through any means known in
the art. Such means include, but are not limited to,
electroporation, the use of liposomes, and calcium phosphate
precipitation. In one alternative, the retroviral plasmid vector
may be encapsulated into a liposome, or coupled to a lipid, and
then administered to a host.
[0088] The producer cell line generates infectious retroviral
vector particles that include the nucleic acid sequence(s) encoding
the PTP1B polypeptide and variants and fusion proteins thereof.
Such retroviral vector particles then may be employed, to transduce
eukaryotic cells, either in vitro or in vivo. The transduced
eukaryotic cells will express the nucleic acid sequence(s) encoding
the siRNA polynucleotide that is capable of specifically
interfering with expression of a polypeptide or fusion protein.
Eukaryotic cells which may be transduced include, but are not
limited to, embryonic stem cells, embryonic carcinoma cells, as
well as hematopoietic stem cells, hepatocytes, fibroblasts,
myoblasts, keratinocytes, endothelial cells, bronchial epithelial
cells and various other culture-adapted cell lines.
[0089] In another aspect, the present invention relates to host
cells containing the above described recombinant PTP1B expression
constructs. Host cells are genetically engineered (transduced,
transformed or transfected) with the vectors and/or expression
constructs of this invention that may be, for example, a cloning
vector, a shuttle vector, or an expression construct. The vector or
construct may be, for example, in the form of a plasmid, a viral
particle, a phage, etc. The engineered host cells can be cultured
in conventional nutrient media modified as appropriate for
activating promoters, selecting transformants or amplifying
particular genes such as genes encoding siRNA polynucleotides or
fusion proteins thereof. The culture conditions for particular host
cells selected for expression, such as temperature, pH and the
like, will be readily apparent to the ordinarily skilled
artisan.
[0090] The host cell can be a higher eukaryotic cell, such as a
mammalian cell, or a lower eukaryotic cell, such as a yeast cell,
or the host cell can be a prokaryotic cell, such as a bacterial
cell. Representative examples of appropriate host cells according
to the present invention include, but need not be limited to,
bacterial cells, such as E. coli, Streptomyces, Salmonella
typhimurium; fungal cells, such as yeast; insect cells, such as
Drosophila S2 and Spodoptera S19; animal cells, such as CHO, COS or
293 cells; adenoviruses; plant cells, or any suitable cell already
adapted to in vitro propagation or so established de novo. The
selection of an appropriate host is deemed to be within the scope
of those skilled in the art from the teachings herein.
[0091] Various mammalian cell culture systems can also be employed
to produce siRNA polynucleotides from recombinant nucleic acid
constructs of the present invention. The invention is therefore
directed in part to a method of producing a siRNA polynucleotide,
by culturing a host cell comprising a recombinant nucleic acid
construct that comprises at least one promoter operably linked to a
nucleic acid sequence encoding a siRNA polynucleotide specific for
a PTP1B polypeptide. In certain embodiments, the promoter may be a
regulated promoter as provided herein, for example a
tetracylcine-repressible promoter. In certain embodiments the
recombinant expression construct is a recombinant viral expression
construct as provided herein. Examples of mammalian expression
systems include the COS-7 lines of monkey kidney fibroblasts,
described by Gluzman, Cell 23:175 (1981), and other cell lines
capable of expressing a compatible vector, for example, the C127,
3T3, CHO, HeLa, HEK, and BHK cell lines. Mammalian expression
vectors will comprise an origin of replication, a suitable promoter
and enhancer, and also any necessary ribosome binding sites,
polyadenylation site, splice donor and acceptor sites,
transcriptional termination sequences, and 5' flanking
nontranscribed sequences, for example as described herein regarding
the preparation of recombinant siRNA polynucleotide constructs. DNA
sequences derived from the SV40 splice, and polyadenylation sites
may be used to provide the required nontranscribed genetic
elements. Introduction of the construct into the host cell can be
effected by a variety of methods with which those skilled in the
art will be familiar, including but not limited to, for example,
liposomes including cationic liposomes, calcium phosphate
transfection, DEAE-Dextran mediated transfection, or
electroporation (Davis et al., 1986 Basic Methods in Molecular
Biology), or other suitable technique.
[0092] The expressed recombinant siRNA polynucleotides may be
useful in intact host cells; in intact organelles such as cell
membranes, intracellular vesicles or other cellular organelles; or
in disrupted cell preparations including but not limited to cell
homogenates or lysates, microsomes, uni- and multilamellar membrane
vesicles or other preparations. Alternatively, expressed
recombinant siRNA polynucleotides can be recovered and purified
from recombinant cell cultures by methods including ammonium
sulfate or ethanol precipitation, acid extraction, anion or cation
exchange chromatography, phosphocellulose chromatography,
hydrophobic interaction chromatography, affinity chromatography,
hydroxylapatite chromatography and lectin chromatography. Finally,
high performance liquid chromatography (HPLC) can be employed for
final purification steps.
[0093] Samples
[0094] According to the present invention, a method is provided for
interfering with expression of a PTP1B polypeptide as provided
herein. A method is also provided for interfering with expression
of a PTP1B polypeptide, comprising contacting a siRNA
polynucleotide with a cell that is capable of expressing PTP1B,
typically in a biological sample or in a subject or biological
source. A "sample" as used herein refers to a biological sample
containing PTP1B, and may be provided by obtaining a blood sample,
biopsy specimen, tissue explant, organ culture or any other tissue
or cell preparation from a subject or a biological source. A sample
may further refer to a tissue or cell preparation in which the
morphological integrity or physical state has been disrupted, for
example, by dissection, dissociation, solubilization,
fractionation, homogenization, biochemical or chemical extraction,
pulverization, lyophilization, sonication or any other means for
processing a sample derived from a subject or biological source. In
certain preferred embodiments, the sample is a cell that comprises
at least one PTP1B polypeptide, and in certain particularly
preferred embodiments the cell comprises an inducible biological
signaling pathway, at least one component of which is PTP1B. In
particularly preferred embodiments the cell is a mammalian cell,
for example, Rat-1 fibroblasts, COS cells, CHO cells, HEK-293
cells, HepG2, HII4E-C3, L6, and 3T3-L1, or other well known model
cell lines, which are available from the American Type Culture
Collection (ATCC, Manassas, Va.). In other preferred embodiments,
the cell line is derived from PTP-1B knockout animals and which may
be transfected with human insulin receptor (HIR), for example, 1BKO
mouse embryo fibroblasts.
[0095] The subject or biological source may be a human or non-human
animal, a primary cell culture or culture adapted cell line
including but not limited to genetically engineered cell lines that
may contain chromosomally integrated or episomal recombinant
nucleic acid sequences, immortalized or immortalizable cell lines,
somatic cell hybrid cell lines, differentiated or differentiatable
cell lines, transformed cell lines and the like. Optionally, in
certain situations it may be desirable to treat cells in a
biological sample with hydrogen peroxide and/or with another agent
that directly or indirectly promotes reactive oxygen species (ROS)
generation, including biological stimuli as described herein; in
certain other situations it may be desirable to treat cells in a
biological sample with a ROS scavenger, such as N-acetyl cysteine
(MAC) or superoxide dismutase (SOD) or other ROS scavengers known
in the art; in other situations cellular glutathione (GSH) may be
depleted by treating cells with L-buthionine-SR-sulfoximine (Bso);
and in other circumstances cells may be treated with pervanadate to
enrich the sample in tyrosine phosphorylated proteins. Other means
may also be employed to effect an increase in the population of
tyrosine phosphorylated proteins present in the sample, including
the use of a subject or biological source that is a cell line that
has been transfected with at least one gene encoding a protein
tyrosine kinase.
[0096] Additionally or alternatively, a biological signaling
pathway may be induced in subject or biological source cells by
contacting such cells with an appropriate stimulus, which may vary
depending upon the signaling pathway under investigation, whether
known or unknown. For example, a signaling pathway that, when
induced, results in protein tyrosine phosphorylation and/or protein
tyrosine dephosphorylation may be stimulated in subject or
biological source cells using any one or more of a variety of well
known methods and compositions known in the art to stimulate
protein tyrosine kinase (PTK) and/or PTP activity. These stimuli
may include, without limitation, exposure of cells to cytokines,
growth factors, hormones, peptides, small molecule mediators, cell
stressors (e.g., ultraviolet light; temperature shifts; osmotic
shock; ROS or a source thereof, such as hydrogen peroxide,
superoxide, ozone, etc. or any agent that induces or promotes ROS
production (see, e.g., Halliwell and Gutteridge, Free Radicals in
Biology and Medicine (3.sup.rd Ed.) 1999 Oxford University Press,
Oxford, UK); heavy metals; alcohol) or other agents that induce
PTK-mediated protein tyrosine phosphorylation and/or PTP-mediated
phosphoprotein tyrosine dephosphorylation. Such agents may include,
for example, interleukins (e.g., IL-1, IL-3), interferons (e.g.,
IFN-.gamma.), human growth hormone, insulin, epidermal growth
factor (EGF), platelet derived growth factor (PDGF), granulocyte
colony stimulating factor (G-CSF), granulocyte-megakaryocyte colony
stimulating factor (GM-CSF), transforming growth factor (e.g.,
TGF-.beta.1), tumor necrosis factor (e.g., TNF-.alpha.) and
fibroblast growth factor (FGF; e.g., basic FGF (bFGF)), any agent
or combination of agents capable of triggering T lymphocyte
activation via the T cell receptor for antigen (TCR; TCR-inducing
agents may include superantigens, specifically recognized antigens
and/or MHC-derived peptides, MHC peptide tetramers (e.g., Altman et
al., 1996 Science 274:94-96); TCR-specific antibodies or fragments
or derivatives thereof), lectins (e.g., PHA, PWM, ConA, etc.),
mitogens, G-protein coupled receptor agonists such as
angiotensin-2, thrombin, thyrotropin, parathyroid hormone,
lysophosphatidic acid (LPA), sphingosine-1-phosphate, serotonin,
endothelin, acetylcholine, platelet activating factor (PAF) or
bradykinin, as well as other agents with which those having
ordinary skill in the art will be familiar (see, e.g., Rhee et al.,
[online] Oct. 10, 2000 Science's stke,
Internet:URL<www.stke.org/cgl/content/full/OC-
_sigtrans;2000/53/pe1>), and references cited therein).
[0097] As noted above, regulated tyrosine phosphorylation
contributes to specific pathways for biological signal
transduction, including those associated with cell division, cell
survival, apoptosis, proliferation and differentiation, and
"inducible signaling pathways" in the context of the present
invention include transient or stable associations or interactions
among molecular components involved in the control of these and
similar processes in cells. Depending on the particular pathway of
interest, an appropriate parameter for determining induction of
such pathway may be selected. For example, for signaling pathways
associated with cell proliferation, a variety of well known
methodologies are available for quantifying proliferation,
including, for example, incorporation of tritiated thymidine into
cellular DNA, monitoring of detectable (e.g., fluorimetric or
calorimetric) indicators of cellular respiratory activity, (e.g.,
MTT assay) or cell counting, or the like. Similarly, in the cell
biology arts there are known multiple techniques for assessing cell
survival (e.g., vital dyes, metabolic indicators, etc.) and for
determining apoptosis (e.g., annexin V binding, DNA fragmentation
assays, caspase activation, PARP cleavage, etc.). Other signaling
pathways will be associated with particular cellular phenotypes,
for example specific induction of gene expression (e.g., detectable
as transcription or translation products, or by bioassays of such
products, or as nuclear localization of cytoplasmic factors),
altered (e.g., statistically significant increases or decreases)
levels of intracellular mediators (e.g., activated kinases or
phosphatases, altered levels of cyclic nucleotides or of
physiologically active ionic species, etc.), altered cell cycle
profiles, or altered cellular morphology, and the like, such that
cellular responsiveness to a particular stimulus as provided herein
can be readily identified to determine whether a particular cell
comprises an inducible signaling pathway.
[0098] In preferred embodiments, a PTP1B substrate may be any
naturally or non-naturally occurring phosphorylated peptide,
polypeptide or protein that can specifically bind to and/or be
dephosphorylated by PTP1B.
[0099] Identification and selection of PTP1B substrates as provided
herein, for use in the present invention, may be performed
according to procedures with which those having ordinary skill in
the art will be familiar, or may, for example, be conducted
according to the disclosures of WO 00/75339, U.S. application Ser.
No. 09/334,575, or U.S. application Ser. No. 10/366,547, and
references cited therein. The phosphorylated protein/PTP complex
may be isolated, for example, by conventional isolation techniques
as described in U.S. Pat. No. 5,352,660, including salting out,
chromatography, electrophoresis, gel filtration, fractionation,
absorption, polyacrylamide gel electrophoresis, agglutination,
combinations thereof or other strategies. PTP1B substrates that are
known may also be prepared according to well known procedures that
employ principles of molecular biology and/or peptide synthesis
(e.g., Ausubel et al., Current Protocols in Molecular Biology,
Greene Publ. Assoc. Inc. & John Wiley & Sons, Inc., Boston,
Mass. (1993); Sambrook et al., Molecular Cloning, Third Ed., Cold
Spring Harbor Laboratory, Plainview, N.Y. (2001); Fox, Molec.
Biotechnol. 3:249 (1995); Maeji et al., Pept. Res. 8:33
(1995)).
[0100] The PTP1B substrate peptides of the present invention may
therefore be derived from PTP1B substrate proteins, polypeptides
and peptides as provided herein having amino acid sequences that
are identical or similar to tyrosine phosphorylated PTP1B substrate
sequences known in the art. For example by way of illustration and
not limitation, peptide sequences derived from the known PTP1B
substrate proteins referred to above are contemplated for use
according to the instant invention, as are peptides having at least
70% similarity (preferably 70% identity), more preferably 80%
similarity (more preferably 80% identity), more preferably 90%
similarity (more preferably 90% identity) and still more preferably
95% similarity (still more preferably 95% identity) to the
polypeptides described in references cited herein and in the
Examples and to portions of such polypeptides as disclosed herein.
As known in the art "similarity" between two polypeptides is
determined by comparing the amino acid sequence and conserved amino
acid substitutes thereto of the polypeptide to the sequence of a
second polypeptide (e.g., using GENEWORKS, Align or the BLAST
algorithm, or another algorithm, as described above).
[0101] In certain preferred embodiments of the present invention,
the siRNA polynucleotide and/or the PTP1B substrate is detectably
labeled, and in particularly preferred embodiments the siRNA
polynucleotide and/or PTP substrate is capable of generating a
radioactive or a fluorescent signal. The siRNA polynucleotide
and/or PTP substrate can be detectably labeled by covalently or
non-covalently attaching a suitable reporter molecule or moiety,
for example a radionuclide such as .sup.32P (e.g., Pestka et al.,
1999 Protein Expr. Purif. 17:203-14), a radiohalogen such as iodine
[.sup.125I or .sup.131I] (e.g., Wilbur, 1992 Bioconjug. Chem.
3:433-70), or tritium [.sup.3H]; an enzyme; or any of various
luminescent (e.g., chemiluminescent) or fluorescent materials
(e.g., a fluorophore) selected according to the particular
fluorescence detection technique to be employed, as known in the
art and based upon the present disclosure. Fluorescent reporter
moieties and methods for labeling siRNA polynucleotides and/or PTP
substrates as provided herein can be found, for example in Haugland
(1996 Handbook of Fluorescent Probes and Research Chemicals--Sixth
Ed., Molecular Probes, Eugene, Oreg.; 1999 Handbook of Fluorescent
Probes and Research Chemicals--Seventh Ed., Molecular Probes,
Eugene, Oreg., [Internet]<: http://www.probes.com/lit/>) and
in references cited therein. Particularly preferred for use as such
a fluorophore in the subject invention methods are fluorescein,
rhodamine, Texas Red, AlexaFluor-594, AlexaFluor-488, Oregon Green,
BODIPY-FL, umbelliferone, dichlorotriazinylamine fluorescein,
dansyl chloride, phycoerythrin or Cy-5. Examples of suitable
enzymes include, but are not limited to, horseradish peroxidase,
biotin, alkaline phosphatase, .beta.-galactosidase and
acetylcholinesterase. Appropriate luminescent materials include
luminol, and suitable radioactive materials include radioactive
phosphorus [.sup.32P]. In certain other preferred embodiments of
the present invention, a detectably labeled siRNA polynucleotide
comprises a magnetic particle, for example a paramagnetic or a
diamagnetic particle or other magnetic particle or the like
(preferably a microparticle) known to the art and suitable for the
intended use. Without wishing to be limited by theory, according to
certain such embodiments there is provided a method for selecting a
cell that has bound, adsorbed, absorbed, internalized or otherwise
become associated with a siRNA polynucleotide that comprises a
magnetic particle. For example, selective isolation of a population
or subpopulation of cells containing one or more PTP1B-specific
siRNA polynucleotide-magnetic particle conjugates may offer certain
advantages in the further characterization or regulation of PTP
signaling pathways.
[0102] In certain embodiments of the present invention, particular
PTP1B-specific siRNA polynucleotides of interest may be identified
by contacting a candidate siRNA polynucleotide with a sample
comprising a cell that comprises a PTP1B gene and that is capable
of PTP1B gene transcription or expression (e.g., translation),
under conditions and for a time sufficient to detect PTP1B gene
transcription or expression, and comparing PTP1B transcription
levels, PTP1B polypeptide expression and/or PTP1B functional
expression (e.g., PTP1B catalytic activity) in the absence and
presence of the candidate siRNA polynucleotide. Preferably PTP1B
transcription or expression is decreased in the presence of the
siRNA polynucleotide, thereby providing an alternative to PTP
active site directed approaches to modulating PTP1B activity. (The
invention need not be so limited, however, and contemplates other
embodiments wherein transcription and/or expression levels of a
signal transduction component other than that which is specifically
targeted by the siRNA may be increased in the presence of a certain
PTP1B-specific siRNA polynucleotide. By way of non-limiting theory,
such an increase may result from a cellular compensatory mechanism
that is induced as a result of the siRNA.)
[0103] For a cell that expresses PTP1B and comprises an insulin
receptor, such as IR-.beta., and the siRNA polynucleotide effects
an increase in insulin receptor phosphorylation, presumably (and
according to non-binding theory) by decreasing PTP1B levels through
interference with PTP-1B expression. Methods for determining
insulin receptor phosphorylation are known in the art (e.g.,
Cheatham et al., 1995 Endocr. Rev. 16:117-142) and are described in
greater detail below. In certain other further embodiments wherein
the cell comprises an insulin receptor, any of a variety of
cellular insulin responses may be monitored according to
art-established methodologies, including but not limited to glucose
uptake (e.g., Elchebly et al., 1999 Science 283:1544; McGuire et
al., 1991 Diabetes 40:939; Myerovitch et al., 1989 J. Clin. Invest.
84:976; Sredy et al. 1995 Metabolism 44:1074; WO 99/46268);
glycogen synthesis (e.g., Berger et al., 1998 Anal. Biochem.
261:159), Glut4 recruitment to a plasma membrane (Robinson et al.,
1992 J. Cell Biol. 117:1181); liver transcription events, or amino
acid import (Hyde et al., 2002 J. Biol. Chem. 277:13628-34 (2002)).
In certain other further embodiments wherein the cell comprises an
insulin receptor, cellular insulin responses that may be monitored
include MAP kinase phosphorylation, AKT phosphorylation, and other
insulin-stimulated phosphorylation events downstream of the insulin
receptor, such as P13 kinase, perk, pSTAT5, and IRS1, and
inhibition of phosphoenolpyruvate carboxykinase transcription
(Forest et al., 1990 Molec. Endocrinol. 4:1302),
phosphatidylinositoltriphosphate kinase activation (Endeman et al.,
1990 J. Biol. Chem. 265:396), lipogenesis (Moody et al., 1974 Horm.
Metab. Res. 6:12), lipolysis (Hess et al., 1991 J. Cell. Biochem.
45:374), TYK2 dephosphorylation and JAK2 (see GenBank Nos.
NM.sub.--004972, AF058925, AF005216, NM.sub.--031514, and
NM.sub.--008261) dephosphorylation (Myers et al., 2001 J. Biol.
Chem. 276:47771), interferon-stimulated pSTAT1 and pSTAT3, and EGF
or PDGRF phosphorylation (Ullrich et al., 1990 Cell 61:203). In
addition, phosphorylation of the insulin receptor, such as at
positions tyr1162/tyr1163 and at position tyr972, may be detected
with anti-phosphotyrosine antibodies that are site-specific for
tyr1162/tyr1163 or tyr972.
[0104] PTP1B activity may also be measured in whole cells
transfected with a reporter gene whose expression is dependent upon
the activation of an appropriate substrate. For example,
appropriate cells (i.e., cells that are capable of expressing PTP1B
and that have been transfected with a PTP1B-specific siRNA
polynucleotide that is either known or suspected of being capable
of interfering with PTP-1B polypeptide expression) may be
transfected with a substrate-dependent promoter linked to a
reporter gene. In such a system, expression of the reporter gene
(which may be readily detected using methods well known to those of
ordinary skill in the art) depends upon activation of substrate.
Dephosphorylation of substrate may be detected based on a decrease
in reporter activity. Candidate siRNA polynucleotides specific for
PTP1B may be added to such a system, as described above, to
evaluate their effect on PTP1B activity.
[0105] Within other aspects, the present invention provides animal
models in which an animal, by virtue of introduction of an
appropriate PTP1B-specific siRNA polynucleotide, for example, as a
transgene, does not express (or expresses a significantly reduced
amount of) a functional PTP1B. Such animals may be generated, for
example, using standard homologous recombination strategies, or
alternatively, for instance, by oocyte microinjection with a
plasmid comprising the siRNA-encoding sequence that is regulated by
a suitable promoter (e.g., ubiquitous or tissue-specific) followed
by implantation in a surrogate mother. Animal models generated in
this manner may be used to study activities of PTP signaling
pathway components and modulating agents in vivo.
[0106] Therapeutic Methods
[0107] One or more siRNA polynucleotides capable of interfering
with PTP1B polypeptide expression and identified according to the
above-described methods may also be used to modulate (e.g., inhibit
or potentiate) PTP1B activity in a patient. As used herein, a
"patient" may be any mammal, including a human, and may be
afflicted with a condition associated with undesired PTP1B activity
or may be free of detectable disease. Accordingly, the treatment
may be of an existing disease or may be prophylactic. Conditions
associated with signal transduction and/or PTP1B activity include
any disorder associated with cell proliferation, including cancer,
graft-versus-host disease (GVHD), autoimmune diseases, allergy or
other conditions in which unregulated PTP1B activity may be
involved.
[0108] For administration to a patient, one or more specific siRNA
polynucleotides, either alone, with or without chemical
modification or removal of ribose, or comprised in an appropriate
vector as described herein (e.g., including a vector which
comprises a DNA sequence from which a specific siRNA can be
transcribed) are generally formulated as a pharmaceutical
composition. A pharmaceutical composition may be a sterile aqueous
or non-aqueous solution, suspension or emulsion, which additionally
comprises a physiologically acceptable carrier (i.e., a non-toxic
material that does not interfere with the activity of the active
ingredient). Such compositions may be in the form of a solid,
liquid or gas (aerosol). Alternatively, compositions of the present
invention may be formulated as a lyophilizate or compounds may be
encapsulated within liposomes using well known technology.
Pharmaceutical compositions within the scope of the present
invention may also contain other components, which may be
biologically active or inactive. Such components include, but are
not limited to, buffers (e.g., neutral buffered saline or phosphate
buffered saline), carbohydrates (e.g., glucose, mannose, sucrose or
dextrans), mannitol, proteins, polypeptides or amino acids such as
glycine, antioxidants, chelating agents such as EDTA or
glutathione, stabilizers, dyes, flavoring agents, and suspending
agents and/or preservatives.
[0109] Any suitable carrier known to those of ordinary skill in the
art may be employed in the pharmaceutical compositions of the
present invention. Carriers for therapeutic use are well known, and
are described, for example, in Remingtons Pharmaceutical Sciences,
Mack Publishing Co. (A. R. Gennaro ed. 1985). In general, the type
of carrier is selected based on the mode of administration.
Pharmaceutical compositions may be formulated for any appropriate
manner of administration, including, for example, topical, oral,
nasal, intrathecal, rectal, vaginal, sublingual or parenteral
administration, including subcutaneous, intravenous, intramuscular,
intrasternal, intracavernous, intrameatal or intraurethral
injection or infusion. For parenteral administration, the carrier
preferably comprises water, saline, alcohol, a fat, a wax or a
buffer. For oral administration, any of the above carriers or a
solid carrier, such as mannitol, lactose, starch, magnesium
stearate, sodium saccharine, talcum, cellulose, kaolin, glycerin,
starch dextrins, sodium alginate, carboxymethylcellulose, ethyl
cellulose, glucose, sucrose and/or magnesium carbonate, may be
employed.
[0110] A pharmaceutical composition (e.g., for oral administration
or delivery by injection) may be in the form of a liquid (e.g., an
elixir, syrup, solution, emulsion or suspension). A liquid
pharmaceutical composition may include, for example, one or more of
the following: sterile diluents such as water for injection, saline
solution, preferably physiological saline, Ringer's solution,
isotonic sodium chloride, fixed oils such as synthetic mono or
diglycerides which may serve as the solvent or suspending medium,
polyethylene glycols, glycerin, propylene glycol or other solvents;
antibacterial agents such as benzyl alcohol or methyl paraben;
antioxidants such as ascorbic acid or sodium bisulfite; chelating
agents such as ethylenediaminetetraacetic acid; buffers such as
acetates, citrates or phosphates and agents for the adjustment of
tonicity such as sodium chloride or dextrose. A parenteral
preparation can be enclosed in ampoules, disposable syringes or
multiple dose vials made of glass or plastic. The use of
physiological saline is preferred, and an injectable pharmaceutical
composition is preferably sterile.
[0111] The compositions described herein may be formulated for
sustained release (i.e., a formulation such as a capsule or sponge
that effects a slow release of compound following administration).
Such compositions may generally be prepared using well known
technology and administered by, for example, oral, rectal or
subcutaneous implantation, or by implantation at the desired target
site. Sustained-release formulations may contain an agent dispersed
in a carrier matrix and/or contained within a reservoir surrounded
by a rate controlling membrane. Carriers for use within such
formulations are biocompatible, and may also be biodegradable;
preferably the formulation provides a relatively constant level of
active component release. The amount of active compound contained
within a sustained release formulation depends upon the site of
implantation, the rate and expected duration of release and the
nature of the condition to be treated or prevented.
[0112] Within a pharmaceutical composition, a therapeutic agent
comprising a polypeptide-directed siRNA polynucleotide as described
herein (or, e.g., a recombinant nucleic acid construct encoding a
siRNA polynucleotide) may be linked to any of a variety of
compounds. For example, such an agent may be linked to a targeting
moiety (e.g., a monoclonal or polyclonal antibody, a protein or a
liposome) that facilitates the delivery of the agent to the target
site. As used herein, a "targeting moiety" may be any substance
(such as a compound or cell) that, when linked to an agent enhances
the transport of the agent to a target cell or tissue, thereby
increasing the local concentration of the agent. Targeting moieties
include antibodies or fragments thereof, receptors, ligands and
other molecules that bind to cells of, or in the vicinity of, the
target tissue. An antibody targeting agent may be an intact (whole)
molecule, a fragment thereof, or a functional equivalent thereof.
Examples of antibody fragments are F(ab').sub.2, Fab', Fab and
F.sub.[v] fragments, which may be produced by conventional methods
or by genetic or protein engineering. Linkage is generally covalent
and may be achieved by, for example, direct condensation or other
reactions, or by way of bi- or multi-functional linkers. Targeting
moieties may be selected based on the cell(s) or tissue(s) toward
which the agent is expected to exert a therapeutic benefit.
[0113] Pharmaceutical compositions may be administered in a manner
appropriate to the disease to be treated (or prevented). An
appropriate dosage and a suitable duration and frequency of
administration will be determined by such factors as the condition
of the patient, the type and severity of the patient's disease, the
particular form of the active ingredient and the method of
administration. In general, an appropriate dosage and treatment
regimen provides the agent(s) in an amount sufficient to provide
therapeutic and/or prophylactic benefit (e.g., an improved clinical
outcome, such as more frequent complete or partial remissions, or
longer disease-free and/or overall survival, or a lessening of
symptom severity). For prophylactic use, a dose should be
sufficient to prevent, delay the onset of or diminish the severity
of a disease associated with cell proliferation.
[0114] Optimal dosages may generally be determined using
experimental models and/or clinical trials. In general, the amount
of siRNA polynucleotide present in a dose, or produced in situ by
DNA present in a dose (e.g, from a recombinant nucleic acid
construct comprising a siRNA polynucleotide), ranges from about
0.01 .mu.g to about 100 .mu.g per kg of host, typically from about
0.1 .mu.g to about 10 .mu.g. The use of the minimum dosage that is
sufficient to provide effective therapy is usually preferred.
Patients may generally be monitored for therapeutic or prophylactic
effectiveness using assays suitable for the condition being treated
or prevented, which will be familiar to those having ordinary skill
in the art. Suitable dose sizes will vary with the size of the
patient, but will typically range from about 10 mL to about 500 mL
for 10-60 kg animal.
[0115] The following Examples are offered by way of illustration
and not by way of limitation.
EXAMPLE 1
[0116] Interferences of PTP1B Expression by Specific siRNA
[0117] This Example describes the effect on expression of PTP-1B
expression in cells transfected with sequence-specific siRNA
polynucleotides.
[0118] Interference of Endogenous Expression of Murine PTP1B in
Mouse Fibroblasts by Sequence Specific siRNA Polynucleotides
[0119] Three siRNA sequences that were specific for murine PTP1B
polynucleotide (GenBank Ace. No. NM.sub.--011201, SEQ ID NO: __)
encoding a murine PTP1B polypeptide (GenBank Ace. No.
NM.sub.--011201, SEQ ID NO: __) and one siRNA sequence specific for
human PTP1B polynucleotide (GenBank Ace. No. NM.sub.--002827, SEQ
ID NO: __) encoding a human PTP1B polypeptide (GenBank Ace. No.
NM.sub.--002827, SEQ ID NO: __) were designed as follows. The siRNA
nucleotide sequences specific for each PTP1B were chosen by first
scanning the open reading frame of the target cDNA for 21-base
sequences that were flanked on the 5' end by two adenine bases (AA)
and that had A+T/G+C ratios that were nearly 1:1. Twenty-one-base
sequences with an A+T/G+C ratio greater than 2:1 or 1:2 were
excluded. If no 21-base sequences were identified that met this
criteria, the polynucleotide sequence encoding the PTP1B was
searched for a 21-base sequence having the bases CA at the 5' end.
The specificity of each 21-mer was determined by performing a BLAST
search of public databases. Sequences that contained at least 16 of
21 consecutive nucleotides with 100% identity with a polynucleotide
sequence other than the target sequence were not used in the
experiments.
[0120] Sense and antisense oligonucleotides for TCPTP analysis were
synthesized according to the standard protocol of the vendor
(Dharmacon Research, Inc., Lafayette, Colo.). For some experiments
described in this and other examples, the vendor gel-purified the
double-stranded siRNA polynucleotide, which was then used. In the
instances when the vendor did not prepare double-stranded siRNA,
just before transfection, double-stranded siRNAs were prepared by
annealing the sense and anti-sense oligonucleotides in annealing
buffer (100 mM potassium acetate, 30 mM HEPES-KOH, pH 7.4, 2 mM
magnesium acetate) for 1 minute at 90.degree. C., followed by a
60-minute incubation at 37.degree. C.
[0121] In each of the examples, each siRNA sequence represents the
sense strand of the siRNA polynucleotide and its corresponding
sequence identifier. Unless otherwise stated, it is to be
understood that the siRNA transfected into a cell is composed of
the sense strand and its complementary antisense strand, which form
a duplex siRNA polynucleotide.
[0122] Mouse C57B16 #3 cells, clones 3 and 10, were maintained in
cell culture according to standard cell culture methods. Each
C57B16 #3 clone was transfected with 200 nM of the following
siRNAs: mPTP1B.1 (SEQ ID NO: __), mPTP1B.2 (SEQ ID NO: __, mPTP1B.3
(SEQ ID NO: __), and hPTP1B.1 (SEQ ID NO: __). Each siRNA was
diluted in 50 .mu.l O.sub.PTIMEM.RTM. to provide a final
concentration of 200 nM per well. In a separate tube, 3 .mu.l of
Lipofectamine.TM. was combined with 10 .mu.l O.sub.PTIMEM.RTM..
Each solution was incubated for 7 minutes. The two solutions were
then mixed and incubated at room temperature for 22 minutes. The
final volume of the mixed solution was adjusted to 100 .mu.l and
then the C57B16 #3 cells were added. Cells were transfected with
the specific siRNAs, the human PTP1B siRNA, or annealing buffer
alone. The transfected cells were incubated with siRNAs for six
days.
[0123] Cell lysates were prepared by extracting the cells in ELISA
extraction buffer (50 mM Tris-HCl, pH 7.5 (room temperature); 2 mM
EDTA, pH 7-8; 1 mM phosphate (polyphosphate); 1 mM NaVO4
(monomeric), pH 10; 0.1% Triton X-100; Protease Inhibitor Cocktail
set III, (Calbiochem, San Diego, Calif., catalog #539134)). The
lysates were separated by SDS-PAGE gel and analyzed by immunoblot.
The lysates were centrifuged and aliquots of supernatant (10 .mu.l)
from each transfected cell culture sample were combined with 10
.mu.l of SDS-PAGE reducing sample buffer. The samples were heated
at 95.degree. C. for five minutes, and then applied to a 14%
Tris-glycine SDS-PAGE gel (NOVEX.RTM. from Invitrogen Life
Technologies, Carlsbad, Calif.). After electrophoresis, the
separated proteins were electrophoretically transferred from the
gel onto an Immobilon-P polyvinylidene fluoride (PVDF) membrane
(Millipore, Bedford, Mass.). The PVDF membrane was blocked in 5%
milk in TBST (20 mM Tris pH 7.5, 150 mM NaCl, 0.05% Tween-20);
incubated with an anti-murine PTP1B monoclonal antibody (Dr. Ben
Neel, Harvard University, Cambridge, Mass.) for 2-16 hours at room
temperature; washed 3.times.10 minutes with TBST; and then
incubated with an appropriate horseradish peroxidase (HRP)
conjugate IgG (1:10,000) (Amersham Biosciences, Piscataway, N.J.)
for 30 minutes at room temperature. Binding was detected with the
ECL chemiluminescent reagent used according to the vendor's
instructions (Amersham Biosciences, Piscataway, N.J.). As shown in
FIG. 1, the levels of expression of endogenous PTP1B were decreased
only in C57B16 cells transfected with the murine PTP1B sequence
specific siRNA polynucleotides.
[0124] The effect of RNAi on endogenous expression of murine PTP1B
in a second murine cell line was examined. Mouse PTP1B:3T31R
fibroblasts were transfected with 20 nM mPTP1B1.1 (SEQ ID NO: __);
mPTP1B1.6 (SEQ ID NO: __); and mPTP1B1.8 (SEQ ID NO: __) according
to the method described above. The level of murine PTP1B expression
in the cells transfected with mPTP1B1.1 decreased approximately 80%
compared with cells transfected with a non-specific siRNA
(hPTP1B1.3 (SEQ ID NO: __); cells transfected with mPTP1B1.6
decreased approximately 40%; and cells transfected with mPTP1B1.8
decreased approximately 60%.
[0125] Interference with Murine PTP1B Expression by siRNA in
Co-Transfection Assays
[0126] A recombinant expression construct was prepared that encodes
wild-type murine PTP1B (mPTP1B) (GenBank Acc. No. NM.sub.--011201,
SEQ ID NOs: __ and __). The following oligonucleotide primers were
used for the wild-type construct. The sequences of the BamHI and
EcoRI restriction sites are underlined.
1 mPTP1B-sense (mPTP1B 5' BamHI)
5'-GGGGGGGATCCATGGAGATGGAGAAGGAGTT- CGAGG-3' (SEQ ID NO:_) mPTP1B
anti sense (mPTP1B 3' EcoRI)
5'-GGGGGAATTCTCAGTGAAAACACACCCGGTAGCAC-3' (SEQ ID NO:_)
[0127] Vector pCMVTag2B (Stratagene, La Jolla, Calif.) was digested
with restriction endonuclease BamHI (New England Biolabs, Beverly,
Mass.) for 3 hours at 37.degree. C. The digested vector was then
incubated with Klenow polymerase (New England Biolabs) for 15
minutes at 25.degree. C. to fill in the recessed 3' termini,
followed by an incubation of 30 minutes at 37.degree. C. with calf
intestinal phosphatase (New England Biolabs). The GATEWAY.TM.
Reading Frame Cassette B (Invitrogen Life Technologies, Carlsbad,
Calif.) was inserted into the pCMVTag2B vector by ligation with T4
DNA ligase (Invitrogen Life Technologies) overnight at 16.degree.
C. according to the supplier's instructions. DB3.1.TM. competent E.
coli cells were transformed with the ligated vector (GWpCMVTag2)
and DNA was isolated by standard molecular biology methods.
[0128] Vectors for expression of mPTP1B wild type were prepared as
follows. The mPTP1B construct was subcloned into a GATEWAY.TM.
entry vector pENTR3C.TM. (Invitrogen Life Technologies) by
digesting 20 .mu.l of the mPTP1B cDNA or 20 .mu.l of the
pENTR3C.TM. vector with 1 .mu.l of BamHI (New England Biolabs); 1
.mu.l of EcoRI (New England Biolabs); 5 .mu.l 10.times. EcoRi
buffer (New England Biolabs); 5 .mu.l 10.times.BSA (New England
Biolabs); and 18 .mu.l distilled water for 3 hours at 37.degree. C.
Digested DNA was run on a 1% agarose gel, digested bands were
excised, and the DNA was gel-purified using a QIAGEN Gel Extraction
kit (QIAGEN, Inc., Valencia, Calif.). Four microliters of the
mPTP1B cDNA was ligated into 2 .mu.l of the pENTR3C.TM. vector
overnight at 16.degree. C. with 1 .mu.l 10.times. Ligation Buffer
(Invitrogen Life Technologies), 1 .mu.l T4 DNA Ligase (4U/.mu.l)
(Invitrogen, Carlsbad, Calif.), and 2 .mu.l distilled water. The
construct was transformed into LIBRARY EFFICIENCY.RTM.
DH5.alpha..TM. cells. The FLAG.RTM. epitope-tagged mPTP1B construct
was prepared by cloning the pENTR3 C.TM. mPTP1B WT construct into
the GWpCMVTag2 vector. The pENTR3C.TM. construct containing the
mPTP1B polynucleotide was linearized by digesting the construct
with Vsp I (Promega Corp., Madison, Wis.) at 37.degree. C. for 2
hours. The DNA was purified using a QIAGEN PCR Purification kit
(QIAGEN, Inc.). Three microliters (100 ng/.mu.l) of the GWpCMVTag2
vector were combined in a GATEWAY.TM. LR reaction with 6 .mu.l
linearized pENTR3C.TM. mPTP1B WT, 3 .mu.l TE buffer, 4 .mu.l
Clonase.TM. Enzyme, and 4 .mu.l LR reaction buffer (Invitrogen Life
Technologies) for 1 hour at room temperature. After addition of
Proteinase K (Invitrogen Life Technologies) to the reaction for 10
minutes, LIBRARY EFFICIENCY.RTM. DH5.alpha..TM. cells were
transformed with the expression construct.
[0129] The murine PTP1B expression vector (0.5 .mu.g) was
co-transfected with 20 nM murine PTP1B sequence-specific siRNA
polynucleotides into PTP1B knockout mouse fibroblasts (PTP1B KO
mouse embryonic fibroblasts were prepared from 13-day embryos from
PTP1B knock out mice to establish the cell line, which was then
transfected with human insulin receptor (1BKO+HIR) (HIR, Julie
Moyers, Eli Lilly and Company, Indianapolis, Ind.)). Cells were
transfected with siRNAs or annealing buffer alone. Each siRNA was
diluted in 250 .mu.l O.sub.PTIMEM.RTM. low serum medium (Gibco,
Inc.) to a final concentration of 20 nM. In a separate tube, 10
.mu.l of Lipofectamine.TM. 2000 (Invitrogen Life Technologies,
Carlsbad, Calif.) was combined with 250 .mu.l O.sub.PTIMEM.RTM..
Each solution was incubated for 7 minutes. The two solutions were
then mixed and incubated at room temperature for 22 minutes. The
final volume of the mixed solution was adjusted to 100 .mu.l and
then the cells were added. After incubating the transfected cells
for 18 hours at 37.degree. C., cell lysates were prepared,
separated by 4-12% SDS-PAGE, and immunoblotted using the anti-PTP1B
murine monoclonal antibody (see above). The results are summarized
in Table 1, and it is noted that each 21-mer sequence below
contains a dinucleotide "overhang" at the 3' end, and that certain
preferred embodiments of the invention described herein should be
considered to include the 19-mer polynucleotide sequences beginning
at the 5' end therein as well as the 21-mer polynucleotide shown in
the Table.
2TABLE 1 siRNA INTERFERENCE WITH MURINE PIP-1B EXPRESSION IN
CO-TRANSFECTION ASSAYS SEQ ID Decrease in Target siRNA Sequence
siRNA Name NO: Expression Murine PTP1B 5'-gaagcccagaggagcuauatt-3'
mPTP1B1.1 95% 5'-cuacaccacauggccugactt-3' mPTP1B1.2 Not analyzed
5'-gacugccgaccagcugcgctt-3' mPTP1B1.3 Not analyzed
5'-gguaccgagaugucagccctt-3' mPTP1B1.4 25%
5'-ugacuauaucaaugccagctt-3' mPTP1B1.5 Not analyzed
5'-agaagaaaaggagaugguctt-3' mPTP1B1.6 80%
5'-cgggaagugcaaggagcuctt-3' mPTP1B1.7 Not analyzed
5'-ggaucaguggaaggagcuctc-3' mPTP1B1.8 80%
[0130] Interference with Rat PTP1B Expression by siRNA in
Co-Transfection Assays
[0131] A co-transfection assay was performed as described above in
which 1BKO+HIR mouse fibroblasts were co-transfected with an
expression vector containing the sequence encoding a rat PTP1B
polypeptide (SEQ ID NO: __) (GenBank Accession No. NM.sub.--012637)
and a sequence specific siRNA, rPTP1B1.1
(5'-agaagaaaaagagaugguctt-3' (SEQ ID NO: __) (20 nM). Additional
rat PTP1B specific siRNA polynucleotides examined in the
co-transfection assay included rPTP1B1.2
(5'-cggaugguggguggagguctt-3' (SEQ ID NO: __); rPTP1B1.3
(5'-uggcaagugcaaggagcuctt-3' (SEQ ID NO: __); and rPTP]B1.4
(5'-cuacaccaccuggccugactt-3' (SEQ ID NO: __). The level of
expression of the rat PTP1B polypeptide was determined by
immunoblotting cell lysates with an anti-human PTP1B antibody that
also specifically binds to rat PTP1B (PHO2, Oncogene Research
Products.TM. Inc. San Diego, Calif.). Expression of rat PTP1B
decreased approximately 50% in cells transfected with
rPTP1B1.1.
[0132] Interference with Human PTP-1B Expression by siRNA in
Co-Transfection Assays
[0133] Human PTP1B encoding sequence was cloned into a Pmt vector
according to standard molecular biology procedures (see Flint et
al., EMBO J. 12:1937-46 (1993)). 1BKO+HIR cells were co-transfected
with the human PTP-1B expression vector and siRNA polynucleotides
(20 nM) specific for human PTP-1B sequences overnight using
Lipofectamine 2000. Cells were lysed as described above, and the
lysates were separated by 4-12% SDS-PAGE and transferred onto a
PDVF membrane. The level of expression of human PTP-1 B was
determined by immunoblotting with an anti-human PTP-1B antibody
(PHO2, Oncogene Research Products.TM., Inc. San Diego, Calif.).
Interference with expression of human PTP-1B was observed with four
siRNA polynucleotides as indicated in Table 2, and it is noted that
each 21-mer sequence below contains a dinucleotide "overhang" at
the 3' end, and that the invention herein should be considered to
include the 19-mer polynucleotide sequences beginning at the 5' end
therein as well as the 21-mer polynucleotide shown in the
Table.
3TABLE 2 sIRNA INTERFERENCE WITH HUMAN PTP-1B EXPRESSION IN
CO-TRANSFECTION ASSAYS siRNA SEQ ID Decrease in Target siRNA
Sequence Name NO: Expression Human PTP1B
5'-cuauaccacauggccugactt-3' hPTP1B1.1 Not analyzed
5'-gcccaaaggaguuacauuctt-3' hPTP1B1.2 >95%
5'-ggaagaaaaaggaagcccctt-3' hPTP1B1.3 >95%
5'-caaugggaaaugcagggagtt-3' hPTP1B1.4 >95%
5'-ggaucaguggaaggagcuutc-3' hPTP1B1.5 >95%
[0134] Interference with Endogenous Expression of Human PTP1B by
siRNA
[0135] The effect of sequence specific siRNA on endogenous
expression of human PTP1B was examined in two different cell lines.
HeLa cells were transfected as described above with hPTP1B1.1,
hPTP1B1.2, hPTP1B1.3, hPTP1B1.4, and hPTP1B1.5 at 20 nM using
Lipofectamine 2000, and after three days, the level of expression
of PTP1B was analyzed by immunoblot. No significant decrease in
expression of human PTP1B was observed in HeLa cells transfected
with the siRNA hPTP1B1.1. In HeLa cells transfected with hPTP1B1.2
and hPTP1B1.4, the level of expression of human PTP1B decreased
80%, and in cells transfected with hPTP1B1.3, the level of
expression decreased 90%. Endogenous expression of human PTP1B in
the second cell line, 293-HEK-HIR, (gift from Julie Moyers, Eli
Lilly and Company) transfected with sequence specific siRNAs
hPTP1B1.2, hPTPB1.3, hPTP1B1.4, hPTP1B1.5 (20 nM) was reduced by
90%.
[0136] Transient Transfection of Human PTP1B and Sequence Specific
Hairpin Vectors
[0137] Effectiveness of a human PTP1B sequence-specific siRNA in
the form of a hairpin insert was examined in a transient
co-transfection assay. Cells (1BKO+HIR mouse fibroblasts) were
transfected with a human PTP1B expression vector (see above) and
co-transfected with hPTP1B hairpin vectors (1, 0.5, and 0.25 .mu.g)
according to the transfection method described above. The human
PTP1B specific sequences were inserted in frame with a human U6
small nuclear RNA promoter into a vector, which was a gift from
David Engelke (University of Michigan, Ann Arbor, Mich.) (see also
Paul et al., Nat. Biotechnol. 20:446-48 (2002)). The sequences of
each strand inserted into the hairpin vectors are as follows.
4 hPTP1B H1.2-HP4 5'-tttGCCCAAAGGAGTTACATTCGTAAGAATGTAACTC-
CTTTGGGCttttt-3' (SEQ ID NO:_) 3'-GGGTTTCCTCAATGTAAGCATTCT-
TACATTGAGGAAACCCGaaaaagatc-5' (SEQ ID NO:_) hPTP1B H1.2-HP9
5'-tttGCCCAAAGGAGTTACATTCCCTGGGTAAGAATGTAACTCCTTTGGGCtttt- t-3'
(SEQ ID NO:_) 3'-GGGTTTCCTCAATGTAAGGGACCCATTCTTACATTG-
AGGAAACCCGaaaaagatc-5' (SEQ ID NO:_)
[0138] Twenty-four hours after the cells were transfected, cell
lysates were prepared and expression of human PTP1B was determined
by immunoblotting with an anti-human PTP1B antibody (see above).
Cell lysates were also immunoblotted with an antibody specific for
human insulin receptor beta chain (IR.beta.) (C-19, Cat. No.
SC-711, Santa Cruz Biotechnology). The results are presented in
FIG. 2.
[0139] Hairpin vectors are also prepared that contain sequences
specific for murine PTP1B. The following sequences of each strand
are inserted into a hairpin vector.
5 mPTP1B M1.1-HP4 5'-tttGAAGCCCAGAGGAGCTATAAGAATATAGCTCCTC-
TGGGCTTCttttt-3' (SEQ ID NO:_) 3'-TTCGGGTCTCCTCGATA1TCTTAT-
ATCGAGGAGACCCGAAGaaaaagatc-5' (SEQ ID NO:_) mPTP1B M1.1-HP9
5'-tttGAAGCCCAGAGGAGCTATAGGGTGAGAATATAGCTCCTCTGGGCUCttttt- -3' (SEQ
ID NO:_) 3'-TTCGGGTCTCCTCGATATCCCACTCTTATATCGAGGA-
GACCCGAAGaaaaagatc-5' (SEQ ID NO:_)
EXAMPLE 2
[0140] Effect of siRNAs Specific for PTP1B on Insulin Receptor
Tyrosine Phosphorylation
[0141] This example illustrates the effect of RNAi on the function
of components in a cell signaling pathway. The role of PTP1B in the
down regulation of insulin signaling has been illustrated by data
derived from a variety of approaches (Cheng et al., Eur. J.
Biochem. 269:1050-59 (2002)), including the phenotype of the PTP1B
knockout mouse (Elchebly et al., Science 283:1544-48 (1999); Klaman
et al., Mol. Cell Biol. 20:5479-89 (2000); see also U.S. patent
application Ser. No. 10/366,547).
[0142] The effect of human PTP1B siRNA on the level of
phosphorylation of IR-.beta. was evaluated by ELISA. 292-HEK HIR
cells were transfected with 0, 1, 5, and 10 nM hPTP1B1.3 (SEQ ID
NO: __) or mPTP1B1.1 (SEQ ID NO: __). Seventy-two hours after
transfection, cells were exposed to insulin for 7 minutes at
concentrations of 0, 20, 50, and 100 nM. Cell lysates were prepared
as described in Example 1, and total cell protein was quantified by
the Bio-Rad Protein Assay performed according to the manufacturer's
instructions (Bio-Rad, Hercules, Calif.). An ELISA was performed as
follows. Dynex Immulon HB4.times. plates were coated with
anti-insulin receptor antibody Ab-1 (1 mg/ml; NeoMarkers, Inc.,
Fremont, Calif.) that was diluted 1:1000 in CMF (calcium magnesium
free)-PBS containing 5 .mu.g/ml fatty acid free BSA (faf-BSA). The
plates were incubated at 4.degree. C. for at least four hours. The
antibody solution was removed by aspiration, followed by the
addition of 300 .mu.l of 3% faf-BSA+CMF-PBS. The plates were
incubated for 1 hr with agitation on a vortex platform shaker
(setting #5) at room temperature. After aspirating the 3%
faf-BSA+CMF-PBS solution, approximately 10-20 .mu.g of lysate were
added to the wells and incubated at room temperature for one hour.
Plates were washed three times with TBST (20 mM Tris, -HCl, pH 7.5
150 mM NaCl; 0.05% Tween 20). An anti-insulin receptor
phosphotyrosine specific antibody (pTyr 1162/63, Biosource
International, Camarillo, Calif., Catalog #44-804) was diluted
1:2000 in TBST and added to the plates for one hour at room
temperature. The plates were washed three times with TBST.
HRP-conjugated anti-rabbit antibody (Amersham Biosciences, catalog
#NA934V) (1:2000 in TBST) was then added to the wells and incubated
at room temperature for one hour. The plates were washed three
times with TBST and once with deionized, sterile water. TMB
solution (Sigma Aldrich) (100 .mu.l per well) was added and
developed until a modest color change (10-30 minutes depending on
cell type and insulin response). The reaction was stopped with 100
.mu.l of 1.8 N H.sub.2S0.sub.4 and then mixed. The optical density
of each well was measured at 450 nM in a Spectramax plate reader
(Molecular Devices Corp., Sunnyvale, Calif.). The data are
presented in FIG. 3. The level of expression of PTP1B in each cell
lysates was determined by immunoblot as described above. PTP1B
polypeptide was detected using an anti-human PTP-1B antibody (PHO2,
Oncogene Research Products.TM., Inc.). The amount of PTP1B
expressed in cells transfected with varying concentrations of
either siRNA was quantified by densitometric analysis of the
immunoblot. The level of expression of human PTP1B is presented as
a percent of the level of expression in cells that were not
transfected with hPTP1B1.3 siRNA (i.e., the level of expression in
untransfected cells equals 100%) (see tables in FIG. 3).
[0143] In a second experiment, 292-HEK HIR cells were transfected
with 0, 0.5, 3, or 10 nM siRNAs. The siRNA polynucleotides
transfected into the cells included hPTP1B1.2 (SEQ ID NO: __),
hPTP1B1.3 (SEQ ID NO: __), mPTP1B1.1 (SEQ ID NO: __), and rPTP1B1.2
(SEQ ID NO: __). Seventy-two hours after transfection, cells were
exposed to insulin for 7 minutes at concentrations of 0, 1, 5, 10,
50, and 100 nM. Cell lysates were prepared and total cell protein
was quantified as described above. An ELISA was performed as
described above. Cell lysates were coated onto 96-well plates,
blocked, and probed with an anti-pYpY.sup.1162/1163-IR-.beta.
antibody. Binding was detected using an enzyme conjugated secondary
reagent. As shown in FIG. 4, increased phosphorylation of the
insulin receptor was observed in cells transfected with
hPTP1B1.3.
[0144] The percent decrease in the level of PTP1B expression was
compared with the level of phosphorylation of the insulin receptor.
In three separate experements, 292-HEK HIR cells were transfected
with 0, 0.5, 3, or 10 nM hPTP1B1.3 siRNA and then exposed to
insulin for 7 minutes at concentrations of 0, 5, 10, 20, 50, and
100 nM. An ELISA and immunoblot of cell lysates were performed as
described above. The effect of hPTP1B1.3 siRNA on the
phosphorylation state of the insulin receptor is summarized in FIG.
5. Each data point represents the average optical density measured
in duplicate wells.
EXAMPLE 3
[0145] Human and Mouse PTP1B Specific siRNA Polynucleotides
[0146] The level of expression of human PTP1B in cells that are
capable of expressing human PTP1B and that are transfected with any
one of the following siRNA polynucleotides is determined according
to methods and procedures described in Example 1. The effect of the
siRNA specific for human PTP1B on insulin receptor tyrosine
phosphorylation is determined according to the method described in
Example 2. The siRNA sequences that are incorporated into a vector
from which a hairpin vector is transcribed and/or that are
transfected via liposomes according to methods described in Example
1 are presented in Tables 3-5. The human PTP1B target sequences
were derived from the human PTP1B nucleotide sequence set forth in
GenBank Accession No. NM.sub.--002827 (SEQ ID NO: __). Table 3
presents 19-base pair human PTP1B target sequences that are
preceded by a AA dinucleotide leader sequence. Table 4 presents
19-base pair human PTP1B target sequences that are preceded by a CA
dinucleotide leader sequence. The leader sequence refers to the two
nucleotides that are 5' to the 19 base pair target mRNA sequence
and the "ending sequence" refers to the two nucleotides that are
just 3' to the mRNA sequence. The position number connotes the
nucleotide position at or about the first nucleotide of the
19-nucleotide target sequence. The regions of the mRNA are referred
to as the coding region (CR) or open reading frame/coding region
(ORF/CF) and untranslated region (UTR). The stop codon (UAG)
present in any sequence is underlined and bolded. Table 5 presents
human PTP1B siRNA polynucleotide sequences that were selected using
the Dharmacon siDESIGN system. These sequences were generated using
the following parameters: (1) leader sequences included
dinucleotides AA, CA, TA, and GA; (2) 5' UTR, coding region, and 3'
UTR were scanned; (4) the G+C content varied from approximately
31-63%; (5) overlaps of sequences within different 19 nucleotide
sequences were permitted. These sequences were then compared to
known human genome sequences using the BLAST program. Potential
target sequences were eliminated if 16 or more consecutive
nucleotides within the 19-nucleotide target sequence were
identified in another human polynucleotide sequence. The remaining
19-nucleotide siRNA sequences are presented in Table 5. The
sequences in shaded rows were identified by other methods as
well.
[0147] Similarly, the level of expression of mouse or rat PTP1B in
cells that are transfected with sequence specific siRNA
polynucleotides is determined according to the methods and
procedures described in Example 1. The effect of the siRNA specific
for mouse or rat PTP1B on insulin receptor tyrosine phosphorylation
is determined according to the method described in Example 2.
Tables 6 and 7 present 19-nucleotide siRNA sequences specific to
mouse PTP1B (GenBank Accession No. NM.sub.--011201, SEQ ID NO: __)
that have a AA dinucleotide leader sequence and a CA dinucleotide
leader sequence, respectively. Table 8 presents 1 9-nucleotide
siRNA sequences that were selected using Dharmacon siDESIGN and the
BLAST program as described above except that the sequences were
compared with known mouse genome sequences. Table 9 presents
19-nucleotide siRNA sequences that were selected using Dharmacon
siDESIGN and the BLAST program as described above except that the
sequences were compared with known rat genome sequences.
[0148] Each siRNA sequence represented in Tables 3-9 lists the
sequence of the sense strand of the siRNA and its corresponding
sequence identifier. An siRNA polynucleotide as described herein is
understood to be composed of the 19 nucleotide sense strand and its
complementary (or antisense) strand. In addition, a siRNA
polynucleotide of the present invention typically has a
dinucleotide overhang at the 3' end of each strand, which may be
any two nucleotides.
6TABLE 3 HUMAN PTP1B sIRNA POLYNUCLEOTIDE SEQUENCES Ending
Identified Leader 19 nucleotide target (mRNA) sequence Sequence
Sequence (SEQ ID NO) (mRNA) Position Region Name AA
AAGGAGUUCGAGCAGAUCG (_) AC 187 CR AA AGGAGUUCGAGCAGAUCGA CA 188 CR
AA GGAGUUCGAGCAGAUCGAC AA 189 CR AA GCCAGUGACUUCCCAUGUA GA 253 CR
AA CCGAAAUAGGUACAGAGAC GU 300 CR AA AUAGGUACAGAGACGUCAG UC 305 CR
AA UAGGUACAGAGACGUCAGU CC 306 CR AA AAUGGAAGAAGCCCAAAGG AG 393 CR
AA AUGGAAGAAGCCCAAAGGA GU 394 CR AA UGGAAGAAGCCCAAAGGAG UU 395 CR
AA GAAGCCCAAAGGAGUUACA UU 400 CR AA GCCCAAAGGAGUUACAUUC UU 403 CR
hPTP1B 1.2 AA CACAUGCGGUCACUUUUGG GA 444 CR AA CAGAGUGAUGGAGAAAGGU
UC 507 CR AA GGUUCGUUAAAAUGCGCAC AA 527 CR AA AAUGCGCACAAUACUGGCC
AC 533 CR AA AUGCGCACAAUACUGGCCA CA 534 CR AA UGCGCACAAUACUGGCCAC
AA 535 CR AA CCCAAGAAACUCGAGAGAU CU 668 CR AA UCACCAGCCUCAUUCUUGA
AC 773 CR AA CCUUCUGUCUGGCUGAUAC CU 845 CR AA GAGGAAAGACCCUUCUUCC
GU 885 CR AA AGACCCUUCUUCCGUUGAU AU 891 CR AA GACCCUUCUUCCGUUGAUA
UC 892 CR AA AUGAGGAAGUUUCGGAUGG CC 931 CR AA GCUGCCAAAUUCAUCAUGC
GG 1003 CR AA GGAGCUUUCCCACGAGGAC CU 1050 CR AA ACGAAUCCUGGAGCCACAC
AA 1116 CR AA CGAAUCCUGGAGCCACACA AU 1117 CR AA UCCUGGAGCCACACAAUCC
GA 1121 CR AA UGGGAAAUGCAGGGAGUUC UU 1137 CR AA GCAACAGACCCAGCAGGAU
AA 1179 CR AA GAGACCCACCAGGAUAAAG AC 1183 CR AA AGACUGCCCCAUCAAGGAA
GA 1200 CR AA CACUGCCCCAUCAAGGAAG AA 1201 CR AA GGAAGAAAAAGGAAGCCCC
UU 1215 CR hPTP1B 1.3 AA AAGCAAGCCCCUUAAAUGC CG 1223 CR AA
AGGAAGCCCCUUAAAUGCC CC 1224 CR AA GGAACCCCCUUAAAUGCCG CA 1225 CR AA
GCCCCUUAAAUGCCGCACC CU 1229 CR AA AUGCCCCACCCUACCGCAU CG 1238 CR AA
AGCAUGAGUCAAGACACUG AA 1261 CR AA CCAUCAGUCAACACACUGA AG 1262 CR AA
GGACGAGGACCAUGCACUG AG 1365 CR AA GCCCUUCCUGGUCAACAUG UC 1395 CR AA
CAUGUGCGUCCCUACGGUC CU 1410 CR AA CACCAACACAUAGCCUGAC CC 1470 CR
& 3'UTR AA CACAUAGCCUGACCCUCCU CC 1476 CR & 3'UTR AA
AACCCAUCUUCCCCGGAUG UC 1627 3'UTR AA ACCCAUCUUCCCCGGAUGU CU 1628
3'UTR AA CCCAUCUUCCCCGGAUGUG UC 1629 3'UTR AA ACAGAGUACCAUGCUGGCC
CC 1729 3'UTR AA GAGAGUACCAUGCUGGCGG CC 1730 3'UTR AA
CAGCCCCCCCCUUGAAUCU CC 1835 3'UTR AA AGGCAUCCAUACUGCACUA CC 1904
3'UTR AA GGCAUCCAUAGUGCACUAG CA 1905 3'UTR AA GGAGGACGGUUGUAAGCAG
UU 2072 3'UTR AA UCACUGCUCCCCCGUGUGU AU 2241 3'UTR AA
GGUCUUCUUGUGUCCUGAU GA 2276 3'UTR AA UGUGCCCCAUGUCCAAGUC CA 2341
3'UTR AA GUCCAACCUGCCUGUGCAU GA 2357 3'UTR AA CCUGCCUGUGCAUGACCUG
AU 2363 3'UTR AA GCCUGUUGCUGAAGUCAUU GU 2407 3'UTR AA
GUCAUUGUCGCUCAGCAAU AG 2420 3'UTR AA UUCCUGGCAUGACACUCUA GU 2474
3'UTR AA GCCAUAUUCACACCUCACG CU 2571 3'UTR AA GUCAACACUCUUCUUGAGC
AG 2662 3'UTR AA CACUCUUCUUGAGCAGACC GU 2667 3'UTR AA
GAGAGGCACCUGCUGGAAA CC 2697 3'UTR AA CCACACUUCUUGAAACAGC CU 2716
3'UTR AA GACCUCCACAUUAAGUGGC UU 2870 3'UTR AA CAUGAAAAACACGGCAGCU
GU 2896 3'UTR AA AAACACGGCAGCUGUAGCU CC 2902 3'UTR AA
AACACGGCAGCUGUAGCUC CC 2903 3'UTR AA ACACGGCAGCUGUAGCUCC CG 2904
3'UTR AA CAUUCGAGGUGUCACCCUG CA 3003 3'UTR AA GGCUUAGGUGCCAGGCUGU
AA 3047 3'UTR AA UGGACGUACUGGUUUAACC UC 3151 3'UTR AA
CCUCCUAUCCUUGGAGAGC AG 3168 3'UTR
[0149]
7TABLE 4 HUMAN PTP1B siRNA POLYNUCLEOTIDE SEQUENCES (CA LEADER)
Ending Identified Leader 19 nucleotide target (mRNA) sequence
Sequence Sequence (SEQ ID NO) (mRNA) Position Region Name CA
UGAAGAAGCAGCAGCGGCU AG 31 5'UTR CA GGAUAUCCGACAUGAAGCC AG 237 CR CA
UGAAGCCAGUGACUUCCCA UG 249 CR CA GUGACUUCCCAUGUAGAGU GG 257 CR CA
UGUAGUGUGGCCAAGCUUC CU 268 CR CA GAGACGUCAGUCCCUUUGA CC 314 CR CA
GUCCCUUUGACCAUAGUCG GA 323 CR CA UGCUCAACAGAGUGAUGGA GA 500 CR CA
ACAGAGUGAUGGAGAAAGG UU 506 CR CA GAGUGAUGGAGAAAGGUUC GU 509 CR CA
GUGCGACAGCUAGAAUUGG AA 637 CR CA ACCCAAGAAACUCGAGAGA UC 667 CR CA
CUAUACCACAUGGCCUGAC UU 699 CR hPTPLB 1.1 CA CAUGGCCUGACUUUGGAGU CC
707 CR CA UGGCCUGACUUUGGAGUCC CU 709 CR CA CCAGCCUCAUUCUUGAACU UU
736 CR CA GGCAUCGGCAGGUCUGGAA CC 826 CR CA UCGGCAGGUCUGGAACCUU CU
830 CR CA GGUCUGGAACCUUCUGUCU GG 836 CR CA AGAGGAAAGACCCUUCUUC CG
884 CR CA GCUGCGCUUCUCCUACCUG GC 972 CR CA GGAUCAGUGGAAGGAGCUU UC
1038 CR hPTP1B 1.5 CA GUGGAAGGAGCUUUCCCAC GA 1044 CR CA
CCCAAACGAAUCCUGGAGC CA 1111 CR CA AACGAAUCCUGGAGCCACA CA 1115 CR CA
CACAAUGGGAAAUGCAGGG AG 1132 CR CA CAAUGGGAAAUGCAGGGAG UU 1134 CR
hPTP1B 1.4 CA AUGGGAAAUGCAGGGAGUU CU 1136 CR CA GGAGGAUAAAGACUGCCCC
AU 1191 CR CA AGGAAGAAAAAGGAAGCCC CU 1214 CR CA CCCUACGGCAUCGAAAGCA
UG 1246 CR CA UGCACUGAGUUACUGGAAG CC 1377 CR CA CUGAGUUACUGGAAGCCCU
UC 1381 CR CA ACAUGUGCGUGGCUACGGU CC 1409 CR CA UGUGCGUGGCUACGGUCCU
CA 1412 CR CA GGUUCCUGUUCAACAGCAA CA 1457 CR CA ACAGCAACACAUAGCCUGA
CC 1469 CR & 3'UTR CA GCAACACAUAGCCUGACCC UC 1472 CR &
3'UTR CA ACACAUAGCCUGACCCUCC UC 1475 CR & 3'UTR CA
CAUAGCCUGACCCUCCUCC AC 1478 CR & 3'UTR CA UAGCCUGACCCUCCUCCAC
UC 1480 CR & 3'UTR CA CUCCACCUCCACCCACUGU CC 1498 3'UTR CA
GGCAUGCCGCGGUAGGUAA GG 1552 3'UTR CA CUAAAACCCAUCUUCCCCG GA 1623
3'UTR CA UCUUCCCCGGAUGUGUGUC UC 1633 3'UTR CA ACAGCCCCCCCCUUGAAUC
UG 1834 3'UTR CA AAGGCAUCCAUAGUGCACU AG 1903 3'UTR CA
AUCACUGCUCCCCCGUGUG UA 2240 3'UTR CA CUGCUCCCCCGUGUGUAUU UG 2244
3'UTR CA UGUCCAAGUCCAACCUGCC UG 2350 3'UTR CA AGUCCAACCUGCCUGUGCA
UG 2356 3'UTR CA ACCUGCCUGUGCAUGACCU GA 2362 3'UTR CA
UUACAUGGCUGUGGUUCCU AA 2386 3'UTR CA UGGCUGUGGUUCCUAAGCC UG 2391
3'UTR CA UGACACUCUAGUGACUUCC UG 2483 3'UTR CA CUCUAGUGACUUCCUGGUG
AG 2488 3'UTR CA GCCUGUCCUGGUACAGCAG GG 2514 3'UTR CA
UAUUCACACCUCACGCUCU GG 2575 3'UTR CA CACCUCACGCUCUGGACAU GA 2581
3'UTR CA CCUCACGCUCUGGACAUGA UU 2583 3'UTR CA CGCUCUGGAGAUGAUUUAG
GG 2588 3'UTR CA GCCUCCGCCAUUCCAAGUC AA 2646 3'UTR CA
ACACUCUUCUUGAGCAGAC CG 2666 3'UTR CA CUCUUCUUGAGCAGACCGU GA 2669
3'UTR CA GACCGUGAUUUGGAAGAGA GG 2682 3'UTR CA CCUGCUGGAAACCACACUU
CU 2705 3'UTR CA CACUUCUUGAAACAGCCUG GG 2719 3'UTR CA
UGAAAAACACGGCAGCUGU AG 2898 3'UTR CA GCUGUAGCUCCCGAGCUAC UC 2912
3'UTR CA CAUUUUGCCUUUCUCGUGG UA 2950 3'UTR CA UUCGAGGUGUCACCCUGCA
GA 3005 3'UTR CA CCCUGCAGAGCUAUGGUGA GG 3017 3'UTR CA
GAGCUAUGGUGAGGUGUGG AU 3024 3'UTR CA GGCUGUAAGCAUUCUGAGC UG 3060
3'UTR CA GCUGGCUCUCCACCUUGUU AC 3188 3'UTR
[0150]
8TABLE 6 MOUSE PTP1B siRNA POLYNUCLEOTIDE SEQUENCES (AA LEADER)
Ending Identified Leader 19 nucleotide target (mRNA) sequence
Sequence Sequence (SEQ ID NO) (mRNA) Position Region Name AA
CCAAACGGACAACCCAUAG UA 79 5'UTR AA ACGGACAACCCAUAGUACC CG 83 5'UTR
AA CGGACAACCCAUAGUACCC GA 84 5'UTR AA CCCAUAGUACCCGAAGACA GG 91
5'UTR AA CCAGACAAUCGUAAGCUUG AU 117 5'UTR AA GCUUGAUGGUGUUUUCCCU GA
131 5'UTR AA GCAUCUCAUGAAUGUCAGC CA 165 5'UTR AA
UGUCAGCCAAAUUCCGUAC AG 177 5'UTR AA AUUCCGUACAGUUCGGUGC GG 187
5'UTR AA UUCCGUACAGUUCGGUGCG GA 188 5'UTR AA CGAAACACCUCCUGUACCA GG
215 5'UTR AA ACACCUCCUGUACCAGGUU CC 219 5'UTR AA
CACCUCCUGUACCAGGUUC CC 220 5'UTR AA CUUCAGAAUCAUCCAGGCU UC 354
5'UTR AA UCAUCCAGGCUUCAUCAUG UU 362 5'UTR AA GGUGAGAGCCACCACAGAG GA
423 5'UTR AA CUGGUAGGCUGAACCCAUG CU 492 5'UTR AA
CCCAUGCUGAAGCUCCACC CG 505 5'UTR AA CAUGCAGAAGCCGCUGCUG GG 583
5'UTR AA GGAGUUCGAGGAGAUCGAC AA 724 5'UTR AA GCCAGCGACUUCCCAUGCA AA
788 CR AA AGUCGCGAAGCUUCCUAAG AA 808 CR AA GUCGCGAAGCUUCCUAAGA AC
809 CR AA GAACAAAAACCGGAACAGG UA 826 CR AA CAAAAACCGGAACAGGUAC CG
829 CR AA AAACCGGAACAGGUACCGA GA 832 CR AA AACCGGAACAGGUACCGAG AU
833 CR AA ACCGGAACAGGUACCGAGA UG 834 CR AA CCGGAACAGGUACCGAGAU GU
835 CR AA CAGGUACCGAGAUGUCAGC CC 841 CR AA AAAUGGAAGAAGCCCAGAG GA
927 CR AA AAUGGAAGAAGCCCAGAGG AG 928 CR AA AUGGAAGAAGCCCAGAGGA GC
929 CR AA UGGAAGAAGCCCAGAGGAG CU 930 CR AA GAAGCCCAGAGGAGCUAUA UU
935 CR mPTPIB 1.1 AA GCCCAGAGGAGCUAUAUUC UC 938 CR AA
CCGCAUCAUGGAGAAAGGC UC 1042 CR AA AGGCUCGUUAAAAUGUGCC CA 1057 CR AA
GGCUCGUUAAAAUGUGCCC AG 1058 CR AA AAUGUGCCCAGUAUUGGCC AC 1068 CR AA
AUGUGCCCAGUAUUGGCCA CA 1069 CR AA UGUGCCCAGUAUUGGCCAC AG 1070 CR AA
GGAGAUGGUCUUUGAUGAC AC 1102 CR AA AACCUGACUACCAAGGAGA CU 1193 CR AA
ACCUGACUACCAAGGAGAC UC 1194 CR AA CCUGACUACCAAGGAGACU CG 1195 CR AA
GGAGACUCGAGAGAUCCUG CA 1207 CR AA AGUCCGAGAGUCAGGCUCA CU 1300 CR AA
GUCCGAGAGUCAGGCUCAC UC 1301 CR AA GAGGAAAGACCCAUCUUCC GU 1420 CR AA
AGACCCAUCUUCCGUGGAC AU 1426 CR AA GACCCAUCUUCCGUGGACA UC 1427 CR AA
GAAAGUACUGCUGGAGAUG CG 1450 CR AA AGUACUGCUGGAGAUGCGC AG 1453 CR AA
GUACUGCUGGAGAUGCGCA GG 1454 CR AA ACGCACACUGGAGCCUCAC AA 1651 CR AA
CGCACACUGGAGCCUCACA AC 1652 CR AA GUGCAAGGAGCUCUUCUCC AG 1678 CR AA
GGAGCUCUUCUCCAGCCAC CA 1684 CR AA GGCAGAGCCCAGUCAAGUG CC 1757 CR AA
GUGCCAUGCACAGCGUGAG CA 1773 CR AA GUUAGGAGACGGAUGGUGG GU 1814 CR AA
AGUGCUCAGGCGUCUGUCC CC 1847 CR AA GAGCUGUCCUCCACUGAGG AG 1877 CR AA
CACAAGGCACAUUGGCCAA GU 1901 CR AA GGCACAUUGGCCAAGUCAC UG 1906 CR AA
GUCACUGGAAGCCCUUCCU GG 1920 CR AA GCCCUUCCUGGUCAAUGUG UG 1930 CR AA
UGUGUGCAUGGCCACGCUC CU 1945 CR AA CAACAACUCGCAAGCCUGC UC 2079 3'UTR
AA CAACUCGCAAGCCUGCUCU GG 2082 3'UTR AA CUCGCAAGCCUGCUCUGGA AC 2085
3'UTR AA GCCUGCUCUGGAACUGGAA GG 2092 3'UTR AA CCUGUUCAGGAGAAGUAGA
GG 2195 3'UTR AA UACUCUUCUUGCUCUCACC UC 2226 3'UTR AA
CAUUUAUAAAGGCAGGCCC GA 2322 3'UTR AA CGGGAAGUGCAAGGAGCUC UU 1674 CR
mPTPIB 1.7 AA UGACUAUAUCAAUGCCAGC UU 903 CR mPTPIB 1.5
[0151]
9TABLE 7 MOUSE PTP1B siRNA POLYNUCLEOTIDE SEQUENCES (CA LEADER)
Ending Identified Leader 19 nucleotide target (mRNA) sequence
Sequence Sequence (SEQ ID NO) (mRNA) Position Region Name CA
CAUUCCUAGUUAGCAGUGC (_) AU 21 5'UTR CA GUGCAUACUCAUCAGACUG (_) GA
36 5'UTR CA AACGGACAACCCAUAGUAC (_) CC 82 5'UTR CA
ACCCAUAGUACCCGAAGAC AG 90 5'UTR CA GCCAAAUUCCGUACAGUUC GG 182 5'UTR
CA AAUUCCGUACAGUUCGGUG CG 186 5'UTR CA GUUCGGUGCGGAUCCGAAC GA 197
5'UTR CA CCUCCUGUACCAGGUUCCC GU 222 5'UTR CA GGUUCCCGUGUCGCUCUCA AU
234 5'UTR CA GAAUCAUCCAGGCUUCAUC AU 359 5'UTR CA
GGCUUCAUCAUGUUUUCCC AC 369 5'UTR CA UCAUGUUUUCCCACCUCCA GC 376
5'UTR CA UGUUUUCCCACCUCCAGCA AG 379 5'UTR CA CCUCCAGCAAGAACCGAGG GC
389 5'UTR CA UGAAGGUGAGAGCCACCAC AG 419 5'UTR CA
CCACAGAGGAGACGCAUGG GA 434 5'UTR CA CAGACGAUGACGAAGACGC GC 460
5'UTR CA GACGAUGACGAAGACGCGC CA 462 5'UTR CA CGUGUGGAACUGGUAGGCU GA
483 5'UTR CA UGCUGAAGCUCCACCCGUA GU 509 5'UTR CA
GGCAUGGCGGAGGCUAGAU GC 546 5'UTR CA UCCAGAACAUGCAGAAGCC GC 576
5'UTR CA GAACAUGCAGAAGCCGCUG CU 580 5'UTR CA UGGAGAUGGAGAAGGAGUU CG
711 CR CA GGACAUUCGACAUGAAGCC AG 772 CR CA UUCGACAUGAAGCCAGCGA CU
777 CR CA UGAAGCCAGCGACUUCCCA UG 784 CR CA GCGACUUCCCAUGCAAAGU CG
792 CR CA UGCAAAGUCGCGAAGCUUC CU 803 CR CA AAGUCGCGAAGCUUCCUAA GA
807 CR CA AAAACCGGAACAGGUACCG AG 831 CR CA GGUACCGAGAUGUCAGCCC UU
843 CR CA GCCCUUUUGACCACAGUCG GA 858 CR CA GAGGAGCUAUAUUCUCACC CA
943 CR CA UGCUCAACCGCAUCAUGGA GA 1035 CR CA ACCGCAUCAUGGAGAAAGG CU
1041 CR CA UCAUGGAGAAAGGCUCGUU AA 1047 CR CA GUAUUGGCCACAGCAAGAA GA
1078 CR CA CAGUACGACAGUUGGAGUU GG 1170 CR CA GUACGACAGUUGGAGUUGG AA
1172 CR CA GUUGGAGUUGGAAAACCUG AC 1180 CR CA AGGAGACUCGAGAGAUCCU GC
1206 CR CA UUUCCACUACACCACAUGG CC 1228 CR CA CUACACCACAUGGCCUGAC UU
1234 CR mPTPIB 1.2 CA CCACAUGGCCUGACUUUGG AG 1239 CR CA
CAUGGCCUGACUUUGGAGU CC 1242 CR CA UGGCCUGACUUUGGAGUCC CC 1244 CR CA
CCGGCUUCUUUCCUCAAUU UC 1271 CR CA AAGUCCGAGAGUCAGGCUC AC 1299 CR CA
UGGCCCCAUUGUGGUCCAC UG 1333 CR CA UUGUGGUCCACUGCAGCGC CG 1341 CR CA
CCUGCCUCUUACUGAUGGA CA 1398 CR CA AGAGGAAAGACCCAUCUUC CG 1419 CR CA
UCUUCCGUGGACAUCAAGA AA 1433 CR CA UCCAGACUGCCGACCAGCU GC 1491 CR CA
GCUGCGCUUCUCCUACCUG GC 1507 CR CA GUGCAGGAUCAGUGGAAGG AG 1568 CR CA
GGAUCAGUGGAAGGAGCUC UC 1573 CR mPTPIB 1.8 CA CCCAAACGCACACUGGAGC CU
1646 CR CA AACGCACACUGGAGCCUCA CA 1650 CR CA CACUGGAGCCUCACAACGG GA
1656 CR CA AGGAGCUCUUCUCCAGCCA CC 1683 CR CA GAGAGGAAGGCAGAGCCCA GU
1749 CR CA GAGCCCAGUCAAGUGCCAU GC 1761 CR CA GUCAAGUGCCAUGCACAGC GU
1768 CR CA AGUGCCAUGCACAGCGUGA GC 1772 CR CA UGCACAGCGUGAGCAGCAU GA
1779 CR CA CAGCGUGAGCAGCAUGAGU CC 1783 CR CA GCGUGAGCAGCAUGAGUCC AG
1785 CR CA GCAUGAGUCCAGACACUGA AG 1794 CR CA UGAGUCCAGACACUGAAGU UA
1797 CR CA GACACUGAAGUUAGGAGAC GG 1805 CR CA CUGAAGUUAGGAGACGGAU GG
1809 CR CA AAGUGCUCAGGCGUCUGUC CC 1846 CR CA CCGAGGAAGAGCUGUCCUC CA
1869 CR CA CUGAGGAGGAACACAAGGC AC 1890 CR CA CAAGGCACAUUGGCCAAGU CA
1903 CR CA AGGCACAUUGGCCAAGUCA CU 1905 CR CA CAUUGGCCAAGUCACUGGA AG
1910 CR CA UUGGCCAAGUCACUGGAAG CC 1912 CR CA AGUCACUGGAAGCCCUUCC UG
1919 CR CA CUGGAAGCCCUUCCUGGUC AA 1924 CR CA AUGUGUGCAUGGCCACGCU CC
1944 CR CA CCGGCGCGUACUUGUGCUA CC 1971 CR CA CUGCCACUGCCCAGCUUAG GA
2023 3'UTR CA CUGCCCAGCUUAGGAUGCG GU 2029 3'UTR CA
GCUUAGGAUGCGGUCUGCG GC 2036 3'UTR CA ACAACUCGCAAGCCUGCUC UG 2081
3'UTR CA ACUCGCAAGCCUGCUCUGG AA 2084 3'UTR CA AGCCUGCUCUGGAACUGGA
AG 2091 3'UTR CA GGAGAAGUAGAGGAAAUGC CA 2203 3'UTR CA
CCUCACUCCUCCCCUUUCU CU 2243 3'UTR CA CUCCUCCCCUUUCUCUGAU UC 2248
3'UTR CA UUUAUAAAGGCAGGCCCGA AU 2324 3'UTR CA GGUACCGAGAUGUCAGCCC
UU 846 CR mPTPIB 1.4 CA AGAAGAAAAGGAGAUGGUC UU 1095 CR mPTPIB 1.6
CA GACUGCCGACCAGCUGCGC UU 1497 CR mPTP1B 1.3
[0152]
10TABLE 9 RAT PTP1B sIRNA POLYNUCLEOTIDE SEQUENCES (POST- BLAST) 19
nucleotide target (mRNA) Position (SEQ ID NO) Region Number
UCGAUAAGGCUGGGAACUG ORF/CR 148 GAAUAGCGAAACUUCCUAA ORF/CR 217
AUAGCGAAACUUCCUAAGA ORF/CR 219 CCACAGUCGGAUUAAAUUG ORF/CR 278
CAGUCGGAUUAAAUUGCAU ORF/CR 281 GUCGGAUUAAAUUGCAUCA ORF/CR 283
UUGCAUCAGGAAGAUAAUG ORF/CR 294 GCCCAGAGGAGCUAUAUCC ORF/CR 348
UCCUCACCCAGGGCCCUUU ORF/CR 364 ACCGCAUCAUGGAGAAAGG ORF/CR 451
UCAUGGAGAAAGGCUCGUU ORF/CR 457 UGGAGAAAGGCUCGUUAAA ORF/CR 460
GUAUUGGCCACAGAAAGAA ORF/CR 488 AGAGAUGGUCUUCGAUGAC ORF/CR 512
CACCAAUUUGAAGCUGACA ORF/CR 530 CCAAUUUGAAGCUGACACU ORF/CR 532
UUACACAGUACGGCAGUUG ORF/CR 575 CAGUACGGCAGUUGGAGUU ORF/CR 580
GGCUCGAGAGAUCCUGCAU ORF/CR 620 CUGAUGGACAAGAGGAAAG ORF/CR 819
CAUCAAGAAAGUGCUGUUG ORF/CR 854 GGGUGCAAAGUUCAUCAUG ORF/CR 947
UCAUGGGCGACUCGUCAGU ORF/CR 961 CUCGUCAGUGCAGGAUCAG ORF/CR 971
CGCACAUUGGAGCCUCACA ORF/CR 1062 AGUGCAAGGAGCUCUUCUC ORF/CR 1087
GCAUGAGCAGUAUGAGUCA ORF/CR 1195 GUAUGAGUCAAGACACUGA ORF/CR 1204
GUCAAGACACUGAAGUUAG ORF/CR 1210 UGGUGGGUGGAGGUCUUCA ORF/CR 1237
AAGGCACACAGGCCAGUUC ORF/CR 1314 AGGCACACAGGCCAGUUCA ORF/CR 1315
GGCACACAGGCCAGUUCAC ORF/CR 1316 AGCCCUUCCUGGUCAACGU ORF/CR 1339
UUUGGUCUGCGGCGUCUAA 3'UTR 1472 GAAGAAACAACAGCUUACA 3'UTR 1500
AGAAACAACAGCUUACAAG 3'UTR 1502 GUCUAAUCUCAGGGCCUUA 3'UTR 1604
AUGCCAAAUACUCUUCUUG 3'UTR 1647 UCAGAUUCACGAUUUACGU 3'UTR 1838
GCCACUCCACUGAGGUGUA 3'UTR 2197 CUCCACUGAGGUGUAAAGC 3'UTR 2201
GCCUUGGUGUCAUGGAAGU 3'UTR 2238 ACAACCUCUGAAACACUCA 3'UTR 2279
GUCUGGACUCAUGAAACAC 3'UTR 2381 AACACCGCCGAGCGCUUAC 3'UTR 2395
ACACCGCCGAGCGCUUACU 3'UTR 2396 GCCGCUCCACUGUUAUUUA 3'UTR 2764
UUCACUUUGCCCACAGACA 3'UTR 2783 CAGACAACAGUGGUGACAU 3'UTR 2975
GACAACAGUGGUGACAUGU 3'UTR 2977 ACAGUGGUGACAUGUAAAG 3'UTR 2981
CUGAUGACAUGUGUAGGAU 3'UTR 3012 CUCCCGGCAGGACUCUUCA 3'UTR 3231
CCUCAUUCCCUGGACACUU 3'UTR 3304 CCAGUCACCUUGCUCAGAA 3'UTR 3488
GUCACCUUGCUCAGAAGUG 3'UTR 3491 UAAGCGAAGGCAGCUGGAA 3'UTR 3602
AAGCUGCUGCAUGCCUUAA 3'UTR 3837 AGCUGCUGCAUGCCUUAAG 3'UTR 3838
CAACAAAGUGCUCUGGAAU 3'UTR 3977 CUGUCCGACUGCACCGUUU 3'UTR 4049
CUGCACCGUUUCCAACUUG 3'UTR 4057 ACUUGUGUCUCACUAAUGG 3'UTR 4071
ADDITIONAL REFERENCES
[0153] Agami et al., Cell 102:55-66 (2000)
[0154] Bass, Brenda L., Cell 101:235:238 (2000)
[0155] Brummelkamp et al., Science 296:550-53 (2002)
[0156] Carthew, Richard W., Current Opinion in Cell Biology
13:244-248 (2001)
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(2000)
[0158] Elbashir et al., Genes & Development 15:188-200
(2001)
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[0160] Fire et al., Nature 391:806-11 (1993)
[0161] Flint et al., Proc. Natl. Acad. Sci. USA 94:1680-1685
(1997)
[0162] Fukada et al., J. Biol. Chem. 276:25512-25519 (2001)
[0163] Harborth et al., J. Cell Sci. 114:4557-4565 (2001)
[0164] Hutvagner et al., Curr. Opin. Gen. & Dev. 12:225-232
(2002)
[0165] Kisielow et al., Biochem. J. 363:1-5 (2002)
[0166] Paddison et al., Genes & Development 16:948-958
(2002)
[0167] Salmeen et al., Moleular Cell 6:1401-1412 (2000)
[0168] Scadden et al., EMBO Reports 2:1107-1111 (2001)
[0169] Sharp, Phillip A., Genes & Development 13:139-141
(1999)
[0170] Sharp, Phillip A., Genes & Development 15:485-490
(2001)
[0171] Shen et al., Proc. Natl. Acad. Sci. USA 24:13613-13618
(2001)
[0172] Sui et al., Proc. Natl. Acad Sci. USA 99:5515-5520
(2002)
[0173] Tonks et al, Curr. Opin. Cell Biol. 13:182-195 (2001)
[0174] Tuschl, Thomas, Chembiochem. 2:239-245 (2001)
[0175] Ui-Tei et al., FEBS Letters 479:79-82 (2000)
[0176] Wen et al., Proc. Natl. Acad Sci. 98:4622-4627 (2001)
[0177] Zamore et al., Cell 101:25-33 (2000)
[0178] EPI 152 056
[0179] U.S. Pat. No. 2001/0029617
[0180] U.S. Pat. No. 2002/0007051
[0181] U.S. Pat. No. 6,326,193
[0182] U.S. Pat. No. 6,342,595
[0183] U.S. Pat. No. 6,506,559
[0184] WO 01/29058
[0185] WO 01/34815
[0186] WO 01/42443
[0187] WO 01/68836
[0188] WO 01/75164
[0189] WO 01/92513
[0190] WO 01/96584
[0191] WO 99/32619
[0192] From the foregoing, it will be appreciated that, although
specific embodiments of the invention have been described herein
for the purpose of illustration, various modifications may be made
without deviating from the spirit and scope of the invention.
Accordingly, the present invention is not limited except as by the
appended claims.
Sequence CWU 1
1
599 1 7 PRT Unknown Unique signature sequence motif contained
within the conserved domain of the PTP family of enzymes. 1 Cys Xaa
Xaa Xaa Xaa Xaa Arg 1 5 2 11 PRT Unknown An 11 amino acid conserved
sequence containing the signature sequence motif in a majority of
PTPs. 2 Xaa His Cys Xaa Ala Gly Xaa Xaa Arg Xaa Gly 1 5 10 3 3247
DNA Homo sapiens 3 gggcgggcct cggggctaag agcgcgacgc ctagagcggc
agacggcgca gtgggccgag 60 aaggaggcgc agcagccgcc ctggcccgtc
atggagatgg aaaaggagtt cgagcagatc 120 gacaagtccg ggagctgggc
ggccatttac caggatatcc gacatgaagc cagtgacttc 180 ccatgtagag
tggccaagct tcctaagaac aaaaaccgaa ataggtacag agacgtcagt 240
ccctttgacc atagtcggat taaactacat caagaagata atgactatat caacgctagt
300 ttgataaaaa tggaagaagc ccaaaggagt tacattctta cccagggccc
tttgcctaac 360 acatgcggtc acttttggga gatggtgtgg gagcagaaaa
gcaggggtgt cgtcatgctc 420 aacagagtga tggagaaagg ttcgttaaaa
tgcgcacaat actggccaca aaaagaagaa 480 aaagagatga tctttgaaga
cacaaatttg aaattaacat tgatctctga agatatcaag 540 tcatattata
cagtgcgaca gctagaattg gaaaacctta caacccaaga aactcgagag 600
atcttacatt tccactatac cacatggcct gactttggag tccctgaatc accagcctca
660 ttcttgaact ttcttttcaa agtccgagag tcagggtcac tcagcccgga
gcacgggccc 720 gttgtggtgc actgcagtgc aggcatcggc aggtctggaa
ccttctgtct ggctgatacc 780 tgcctcctgc tgatggacaa gaggaaagac
ccttcttccg ttgatatcaa gaaagtgctg 840 ttagaaatga ggaagtttcg
gatggggttg atccagacag ccgaccagct gcgcttctcc 900 tacctggctg
tgatcgaagg tgccaaattc atcatggggg actcttccgt gcaggatcag 960
tggaaggagc tttcccacga ggacctggag cccccacccg agcatatccc cccacctccc
1020 cggccaccca aacgaatcct ggagccacac aatgggaaat gcagggagtt
cttcccaaat 1080 caccagtggg tgaaggaaga gacccaggag gataaagact
gccccatcaa ggaagaaaaa 1140 ggaagcccct taaatgccgc accctacggc
atcgaaagca tgagtcaaga cactgaagtt 1200 agaagtcggg tcgtgggggg
aagtcttcga ggtgcccagg ctgcctcccc agccaaaggg 1260 gagccgtcac
tgcccgagaa ggacgaggac catgcactga gttactggaa gcccttcctg 1320
gtcaacatgt gcgtggctac ggtcctcacg gccggcgctt acctctgcta caggttcctg
1380 ttcaacagca acacatagcc tgaccctcct ccactccacc tccacccact
gtccgcctct 1440 gcccgcagag cccacgcccg actagcaggc atgccgcggt
aggtaagggc cgccggaccg 1500 cgtagagagc cgggccccgg acggacgttg
gttctgcact aaaacccatc ttccccggat 1560 gtgtgtctca cccctcatcc
ttttactttt tgccccttcc actttgagta ccaaatccac 1620 aagccatttt
ttgaggagag tgaaagagag taccatgctg gcggcgcaga gggaaggggc 1680
ctacacccgt cttggggctc gccccaccca gggctccctc ctggagcatc ccaggcggcg
1740 cacgccaaca gcccccccct tgaatctgca gggagcaact ctccactcca
tatttattta 1800 aacaattttt tccccaaagg catccatagt gcactagcat
tttcttgaac caataatgta 1860 ttaaaatttt ttgatgtcag ccttgcatca
agggctttat caaaaagtac aataataaat 1920 cctcaggtag tactgggaat
ggaaggcttt gccatgggcc tgctgcgtca gaccagtact 1980 gggaaggagg
acggttgtaa gcagttgtta tttagtgata ttgtgggtaa cgtgagaaga 2040
tagaacaatg ctataatata taatgaacac gtgggtattt aataagaaac atgatgtgag
2100 attactttgt cccgcttatt ctcctccctg ttatctgcta gatctagttc
tcaatcactg 2160 ctcccccgtg tgtattagaa tgcatgtaag gtcttcttgt
gtcctgatga aaaatatgtg 2220 cttgaaatga gaaactttga tctctgctta
ctaatgtgcc ccatgtccaa gtccaacctg 2280 cctgtgcatg acctgatcat
tacatggctg tggttcctaa gcctgttgct gaagtcattg 2340 tcgctcagca
atagggtgca gttttccagg aataggcatt tgctaattcc tggcatgaca 2400
ctctagtgac ttcctggtga ggcccagcct gtcctggtac agcagggtct tgctgtaact
2460 cagacattcc aagggtatgg gaagccatat tcacacctca cgctctggac
atgatttagg 2520 gaagcaggga caccccccgc cccccacctt tgggatcagc
ctccgccatt ccaagtcaac 2580 actcttcttg agcagaccgt gatttggaag
agaggcacct gctggaaacc acacttcttg 2640 aaacagcctg ggtgacggtc
ctttaggcag cctgccgccg tctctgtccc ggttcacctt 2700 gccgagagag
gcgcgtctgc cccaccctca aaccctgtgg ggcctgatgg tgctcacgac 2760
tcttcctgca aagggaactg aagacctcca cattaagtgg ctttttaaca tgaaaaacac
2820 ggcagctgta gctcccgagc tactctcttg ccagcatttt cacattttgc
ctttctcgtg 2880 gtagaagcca gtacagagaa attctgtggt gggaacattc
gaggtgtcac cctgcagagc 2940 tatggtgagg tgtggataag gcttaggtgc
caggctgtaa gcattctgag ctggcttgtt 3000 gtttttaagt cctgtatatg
tatgtagtag tttgggtgtg tatatatagt agcatttcaa 3060 aatggacgta
ctggtttaac ctcctatcct tggagagcag ctggctctcc accttgttac 3120
acattatgtt agagaggtag cgagctgctc tgctatatgc cttaagccaa tatttactca
3180 tcaggtcatt attttttaca atggccatgg aataaaccat ttttacaaaa
ataaaaacaa 3240 aaaaagc 3247 4 435 PRT Homo sapiens 4 Met Glu Met
Glu Lys Glu Phe Glu Gln Ile Asp Lys Ser Gly Ser Trp 1 5 10 15 Ala
Ala Ile Tyr Gln Asp Ile Arg His Glu Ala Ser Asp Phe Pro Cys 20 25
30 Arg Val Ala Lys Leu Pro Lys Asn Lys Asn Arg Asn Arg Tyr Arg Asp
35 40 45 Val Ser Pro Phe Asp His Ser Arg Ile Lys Leu His Gln Glu
Asp Asn 50 55 60 Asp Tyr Ile Asn Ala Ser Leu Ile Lys Met Glu Glu
Ala Gln Arg Ser 65 70 75 80 Tyr Ile Leu Thr Gln Gly Pro Leu Pro Asn
Thr Cys Gly His Phe Trp 85 90 95 Glu Met Val Trp Glu Gln Lys Ser
Arg Gly Val Val Met Leu Asn Arg 100 105 110 Val Met Glu Lys Gly Ser
Leu Lys Cys Ala Gln Tyr Trp Pro Gln Lys 115 120 125 Glu Glu Lys Glu
Met Ile Phe Glu Asp Thr Asn Leu Lys Leu Thr Leu 130 135 140 Ile Ser
Glu Asp Ile Lys Ser Tyr Tyr Thr Val Arg Gln Leu Glu Leu 145 150 155
160 Glu Asn Leu Thr Thr Gln Glu Thr Arg Glu Ile Leu His Phe His Tyr
165 170 175 Thr Thr Trp Pro Asp Phe Gly Val Pro Glu Ser Pro Ala Ser
Phe Leu 180 185 190 Asn Phe Leu Phe Lys Val Arg Glu Ser Gly Ser Leu
Ser Pro Glu His 195 200 205 Gly Pro Val Val Val His Cys Ser Ala Gly
Ile Gly Arg Ser Gly Thr 210 215 220 Phe Cys Leu Ala Asp Thr Cys Leu
Leu Leu Met Asp Lys Arg Lys Asp 225 230 235 240 Pro Ser Ser Val Asp
Ile Lys Lys Val Leu Leu Glu Met Arg Lys Phe 245 250 255 Arg Met Gly
Leu Ile Gln Thr Ala Asp Gln Leu Arg Phe Ser Tyr Leu 260 265 270 Ala
Val Ile Glu Gly Ala Lys Phe Ile Met Gly Asp Ser Ser Val Gln 275 280
285 Asp Gln Trp Lys Glu Leu Ser His Glu Asp Leu Glu Pro Pro Pro Glu
290 295 300 His Ile Pro Pro Pro Pro Arg Pro Pro Lys Arg Ile Leu Glu
Pro His 305 310 315 320 Asn Gly Lys Cys Arg Glu Phe Phe Pro Asn His
Gln Trp Val Lys Glu 325 330 335 Glu Thr Gln Glu Asp Lys Asp Cys Pro
Ile Lys Glu Glu Lys Gly Ser 340 345 350 Pro Leu Asn Ala Ala Pro Tyr
Gly Ile Glu Ser Met Ser Gln Asp Thr 355 360 365 Glu Val Arg Ser Arg
Val Val Gly Gly Ser Leu Arg Gly Ala Gln Ala 370 375 380 Ala Ser Pro
Ala Lys Gly Glu Pro Ser Leu Pro Glu Lys Asp Glu Asp 385 390 395 400
His Ala Leu Ser Tyr Trp Lys Pro Phe Leu Val Asn Met Cys Val Ala 405
410 415 Thr Val Leu Thr Ala Gly Ala Tyr Leu Cys Tyr Arg Phe Leu Phe
Asn 420 425 430 Ser Asn Thr 435 5 3318 DNA Homo sapiens 5
gtgatgcgta gttccggctg ccggttgaca tgaagaagca gcagcggcta gggcggcggt
60 agctgcaggg gtcggggatt gcagcgggcc tcggggctaa gagcgcgacg
cggcctagag 120 cggcagacgg cgcagtgggc cgagaaggag gcgcagcagc
cgccctggcc cgtcatggag 180 atggaaaagg agttcgagca gatcgacaag
tccgggagct gggcggccat ttaccaggat 240 atccgacatg aagccagtga
cttcccatgt agagtggcca agcttcctaa gaacaaaaac 300 cgaaataggt
acagagacgt cagtcccttt gaccatagtc ggattaaact acatcaagaa 360
gataatgact atatcaacgc tagtttgata aaaatggaag aagcccaaag gagttacatt
420 cttacccagg gccctttgcc taacacatgc ggtcactttt gggagatggt
gtgggagcag 480 aaaagcaggg gtgtcgtcat gctcaacaga gtgatggaga
aaggttcgtt aaaatgcgca 540 caatactggc cacaaaaaga agaaaaagag
atgatctttg aagacacaaa tttgaaatta 600 acattgatct ctgaagatat
caagtcatat tatacagtgc gacagctaga attggaaaac 660 cttacaaccc
aagaaactcg agagatctta catttccact ataccacatg gcctgacttt 720
ggagtccctg aatcaccagc ctcattcttg aactttcttt tcaaagtccg agagtcaggg
780 tcactcagcc cggagcacgg gcccgttgtg gtgcactgca gtgcaggcat
cggcaggtct 840 ggaaccttct gtctggctga tacctgcctc ttgctgatgg
acaagaggaa agacccttct 900 tccgttgata tcaagaaagt gctgttagaa
atgaggaagt ttcggatggg gctgatccag 960 acagccgacc agctgcgctt
ctcctacctg gctgtgatcg aaggtgccaa attcatcatg 1020 ggggactctt
ccgtgcagga tcagtggaag gagctttccc acgaggacct ggagccccca 1080
cccgagcata tccccccacc tccccggcca cccaaacgaa tcctggagcc acacaatggg
1140 aaatgcaggg agttcttccc aaatcaccag tgggtgaagg aagagaccca
ggaggataaa 1200 gactgcccca tcaaggaaga aaaaggaagc cccttaaatg
ccgcacccta cggcatcgaa 1260 agcatgagtc aagacactga agttagaagt
cgggtcgtgg ggggaagtct tcgaggtgcc 1320 caggctgcct ccccagccaa
aggggagccg tcactgcccg agaaggacga ggaccatgca 1380 ctgagttact
ggaagccctt cctggtcaac atgtgcgtgg ctacggtcct cacggccggc 1440
gcttacctct gctacaggtt cctgttcaac agcaacacat agcctgaccc tcctccactc
1500 cacctccacc cactgtccgc ctctgcccgc agagcccacg cccgactagc
aggcatgccg 1560 cggtaggtaa gggccgccgg accgcgtaga gagccgggcc
ccggacggac gttggttctg 1620 cactaaaacc catcttcccc ggatgtgtgt
ctcacccctc atccttttac tttttgcccc 1680 ttccactttg agtaccaaat
ccacaagcca ttttttgagg agagtgaaag agagtaccat 1740 gctggcggcg
cagagggaag gggcctacac ccgtcttggg gctcgcccca cccagggctc 1800
cctcctggag catcccaggc gggcggcacg ccaacagccc cccccttgaa tctgcaggga
1860 gcaactctcc actccatatt tatttaaaca attttttccc caaaggcatc
catagtgcac 1920 tagcattttc ttgaaccaat aatgtattaa aattttttga
tgtcagcctt gcatcaaggg 1980 ctttatcaaa aagtacaata ataaatcctc
aggtagtact gggaatggaa ggctttgcca 2040 tgggcctgct gcgtcagacc
agtactggga aggaggacgg ttgtaagcag ttgttattta 2100 gtgatattgt
gggtaacgtg agaagataga acaatgctat aatatataat gaacacgtgg 2160
gtatttaata agaaacatga tgtgagatta ctttgtcccg cttattctcc tccctgttat
2220 ctgctagatc tagttctcaa tcactgctcc cccgtgtgta ttagaatgca
tgtaaggtct 2280 tcttgtgtcc tgatgaaaaa tatgtgcttg aaatgagaaa
ctttgatctc tgcttactaa 2340 tgtgccccat gtccaagtcc aacctgcctg
tgcatgacct gatcattaca tggctgtggt 2400 tcctaagcct gttgctgaag
tcattgtcgc tcagcaatag ggtgcagttt tccaggaata 2460 ggcatttgcc
taattcctgg catgacactc tagtgacttc ctggtgaggc ccagcctgtc 2520
ctggtacagc agggtcttgc tgtaactcag acattccaag ggtatgggaa gccatattca
2580 cacctcacgc tctggacatg atttagggaa gcagggacac cccccgcccc
ccacctttgg 2640 gatcagcctc cgccattcca agtcaacact cttcttgagc
agaccgtgat ttggaagaga 2700 ggcacctgct ggaaaccaca cttcttgaaa
cagcctgggt gacggtcctt taggcagcct 2760 gccgccgtct ctgtcccggt
tcaccttgcc gagagaggcg cgtctgcccc accctcaaac 2820 cctgtggggc
ctgatggtgc tcacgactct tcctgcaaag ggaactgaag acctccacat 2880
taagtggctt tttaacatga aaaacacggc agctgtagct cccgagctac tctcttgcca
2940 gcattttcac attttgcctt tctcgtggta gaagccagta cagagaaatt
ctgtggtggg 3000 aacattcgag gtgtcaccct gcagagctat ggtgaggtgt
ggataaggct taggtgccag 3060 gctgtaagca ttctgagctg ggcttgttgt
ttttaagtcc tgtatatgta tgtagtagtt 3120 tgggtgtgta tatatagtag
catttcaaaa tggacgtact ggtttaacct cctatccttg 3180 gagagcagct
ggctctccac cttgttacac attatgttag agaggtagcg agctgctctg 3240
ctatatgcct taagccaata tttactcatc aggtcattat tttttacaat ggccatggaa
3300 taaaccattt ttacaaaa 3318 6 435 PRT Homo sapiens 6 Met Glu Met
Glu Lys Glu Phe Glu Gln Ile Asp Lys Ser Gly Ser Trp 1 5 10 15 Ala
Ala Ile Tyr Gln Asp Ile Arg His Glu Ala Ser Asp Phe Pro Cys 20 25
30 Arg Val Ala Lys Leu Pro Lys Asn Lys Asn Arg Asn Arg Tyr Arg Asp
35 40 45 Val Ser Pro Phe Asp His Ser Arg Ile Lys Leu His Gln Glu
Asp Asn 50 55 60 Asp Tyr Ile Asn Ala Ser Leu Ile Lys Met Glu Glu
Ala Gln Arg Ser 65 70 75 80 Tyr Ile Leu Thr Gln Gly Pro Leu Pro Asn
Thr Cys Gly His Phe Trp 85 90 95 Glu Met Val Trp Glu Gln Lys Ser
Arg Gly Val Val Met Leu Asn Arg 100 105 110 Val Met Glu Lys Gly Ser
Leu Lys Cys Ala Gln Tyr Trp Pro Gln Lys 115 120 125 Glu Glu Lys Glu
Met Ile Phe Glu Asp Thr Asn Leu Lys Leu Thr Leu 130 135 140 Ile Ser
Glu Asp Ile Lys Ser Tyr Tyr Thr Val Arg Gln Leu Glu Leu 145 150 155
160 Glu Asn Leu Thr Thr Gln Glu Thr Arg Glu Ile Leu His Phe His Tyr
165 170 175 Thr Thr Trp Pro Asp Phe Gly Val Pro Glu Ser Pro Ala Ser
Phe Leu 180 185 190 Asn Phe Leu Phe Lys Val Arg Glu Ser Gly Ser Leu
Ser Pro Glu His 195 200 205 Gly Pro Val Val Val His Cys Ser Ala Gly
Ile Gly Arg Ser Gly Thr 210 215 220 Phe Cys Leu Ala Asp Thr Cys Leu
Leu Leu Met Asp Lys Arg Lys Asp 225 230 235 240 Pro Ser Ser Val Asp
Ile Lys Lys Val Leu Leu Glu Met Arg Lys Phe 245 250 255 Arg Met Gly
Leu Ile Gln Thr Ala Asp Gln Leu Arg Phe Ser Tyr Leu 260 265 270 Ala
Val Ile Glu Gly Ala Lys Phe Ile Met Gly Asp Ser Ser Val Gln 275 280
285 Asp Gln Trp Lys Glu Leu Ser His Glu Asp Leu Glu Pro Pro Pro Glu
290 295 300 His Ile Pro Pro Pro Pro Arg Pro Pro Lys Arg Ile Leu Glu
Pro His 305 310 315 320 Asn Gly Lys Cys Arg Glu Phe Phe Pro Asn His
Gln Trp Val Lys Glu 325 330 335 Glu Thr Gln Glu Asp Lys Asp Cys Pro
Ile Lys Glu Glu Lys Gly Ser 340 345 350 Pro Leu Asn Ala Ala Pro Tyr
Gly Ile Glu Ser Met Ser Gln Asp Thr 355 360 365 Glu Val Arg Ser Arg
Val Val Gly Gly Ser Leu Arg Gly Ala Gln Ala 370 375 380 Ala Ser Pro
Ala Lys Gly Glu Pro Ser Leu Pro Glu Lys Asp Glu Asp 385 390 395 400
His Ala Leu Ser Tyr Trp Lys Pro Phe Leu Val Asn Met Cys Val Ala 405
410 415 Thr Val Leu Thr Ala Gly Ala Tyr Leu Cys Tyr Arg Phe Leu Phe
Asn 420 425 430 Ser Asn Thr 435 7 2346 DNA Mus musculus 7
gaattcggga tccttttgca cattcctagt tagcagtgca tactcatcag actggagatg
60 tttaatgaca tcagggaacc aaacggacaa cccatagtac ccgaagacag
ggtgaaccag 120 acaatcgtaa gcttgatggt gttttccctg actgggtagt
tgaagcatct catgaatgtc 180 agccaaattc cgtacagttc ggtgcggatc
cgaacgaaac acctcctgta ccaggttccc 240 gtgtcgctct caatttcaat
cagctcatct atttgtttgg gagtcttgat tttatttacc 300 gtgaagacct
tctctggctg gccccgggct ctcatgttgg tgtcatgaat taacttcaga 360
atcatccagg cttcatcatg ttttcccacc tccagcaaga accgagggct ttctggcatg
420 aaggtgagag ccaccacaga ggagacgcat gggagcgcac agacgatgac
gaagacgcgc 480 cacgtgtgga actggtaggc tgaacccatg ctgaagctcc
acccgtagtg gggaatgatg 540 gcccaggcat ggcggaggct agatgccgcc
aatcatccag aacatgcaga agccgctgct 600 ggggagcttg gggctgcggt
ggtggcgggt gacgggcttc gggacgcgga gcgacgcggc 660 ctagcgcggc
ggacggccgt gggaactcgg gcagccgacc cgtcccgcca tggagatgga 720
gaaggagttc gaggagatcg acaaggctgg gaactgggcg gctatttacc aggacattcg
780 acatgaagcc agcgacttcc catgcaaagt cgcgaagctt cctaagaaca
aaaaccggaa 840 caggtaccga gatgtcagcc cttttgacca cagtcggatt
aaattgcacc aggaagataa 900 tgactatatc aatgccagct tgataaaaat
ggaagaagcc cagaggagct atattctcac 960 ccagggccct ttaccaaaca
catgtgggca cttctgggag atggtgtggg agcagaagag 1020 caggggcgtg
gtcatgctca accgcatcat ggagaaaggc tcgttaaaat gtgcccagta 1080
ttggccacag caagaagaaa aggagatggt ctttgatgac acaggtttga agttgacact
1140 aatctctgaa gatgtcaagt catattacac agtacgacag ttggagttgg
aaaacctgac 1200 taccaaggag actcgagaga tcctgcattt ccactacacc
acatggcctg actttggagt 1260 ccccgagtca ccggcttctt tcctcaattt
ccttttcaaa gtccgagagt caggctcact 1320 cagcctggag catggcccca
ttgtggtcca ctgcagcgcc ggcatcggga ggtcagggac 1380 cttctgtctg
gctgacacct gcctcttact gatggacaag aggaaagacc catcttccgt 1440
ggacatcaag aaagtactgc tggagatgcg caggttccgc atggggctca tccagactgc
1500 cgaccagctg cgcttctcct acctggctgt catcgagggc gccaagttca
tcatgggcga 1560 ctcgtcagtg caggatcagt ggaaggagct ctcccgggag
gatctagacc ttccacccga 1620 gcacgtgccc ccacctcccc ggccacccaa
acgcacactg gagcctcaca acgggaagtg 1680 caaggagctc ttctccagcc
accagtgggt gagcgaggag acctgtgggg atgaagacag 1740 cctggccaga
gaggaaggca gagcccagtc aagtgccatg cacagcgtga gcagcatgag 1800
tccagacact gaagttagga gacggatggt gggtggaggt cttcaaagtg ctcaggcgtc
1860 tgtccccacc gaggaagagc tgtcctccac tgaggaggaa cacaaggcac
attggccaag 1920 tcactggaag cccttcctgg tcaatgtgtg catggccacg
ctcctggcca ccggcgcgta 1980 cttgtgctac cgggtgtgtt ttcactgaca
gactgggagg cactgccact gcccagctta 2040 ggatgcggtc tgcggcgtct
gacctggtgt agagggaaca acaactcgca agcctgctct 2100 ggaactggaa
gggcctgccc caggagggta ttagtgcact gggctttgaa ggagcccctg 2160
gtcccacgaa cagagtctaa tctcagggcc ttaacctgtt caggagaagt agaggaaatg
2220 ccaaatactc ttcttgctct cacctcactc ctcccctttc tctgattcat
ttgtttttgg 2280 aaaaaaaaaa aaaaagaatt acaacacatt gttgttttta
acatttataa aggcaggccc 2340 gaattc 2346 8 432 PRT Mus musculus 8 Met
Glu Met Glu Lys Glu Phe Glu Glu Ile Asp Lys Ala Gly Asn Trp 1 5 10
15 Ala Ala Ile Tyr Gln Asp Ile Arg His Glu Ala Ser Asp Phe Pro Cys
20 25
30 Lys Val Ala Lys Leu Pro Lys Asn Lys Asn Arg Asn Arg Tyr Arg Asp
35 40 45 Val Ser Pro Phe Asp His Ser Arg Ile Lys Leu His Gln Glu
Asp Asn 50 55 60 Asp Tyr Ile Asn Ala Ser Leu Ile Lys Met Glu Glu
Ala Gln Arg Ser 65 70 75 80 Tyr Ile Leu Thr Gln Gly Pro Leu Pro Asn
Thr Cys Gly His Phe Trp 85 90 95 Glu Met Val Trp Glu Gln Lys Ser
Arg Gly Val Val Met Leu Asn Arg 100 105 110 Ile Met Glu Lys Gly Ser
Leu Lys Cys Ala Gln Tyr Trp Pro Gln Gln 115 120 125 Glu Glu Lys Glu
Met Val Phe Asp Asp Thr Gly Leu Lys Leu Thr Leu 130 135 140 Ile Ser
Glu Asp Val Lys Ser Tyr Tyr Thr Val Arg Gln Leu Glu Leu 145 150 155
160 Glu Asn Leu Thr Thr Lys Glu Thr Arg Glu Ile Leu His Phe His Tyr
165 170 175 Thr Thr Trp Pro Asp Phe Gly Val Pro Glu Ser Pro Ala Ser
Phe Leu 180 185 190 Asn Phe Leu Phe Lys Val Arg Glu Ser Gly Ser Leu
Ser Leu Glu His 195 200 205 Gly Pro Ile Val Val His Cys Ser Ala Gly
Ile Gly Arg Ser Gly Thr 210 215 220 Phe Cys Leu Ala Asp Thr Cys Leu
Leu Leu Met Asp Lys Arg Lys Asp 225 230 235 240 Pro Ser Ser Val Asp
Ile Lys Lys Val Leu Leu Glu Met Arg Arg Phe 245 250 255 Arg Met Gly
Leu Ile Gln Thr Ala Asp Gln Leu Arg Phe Ser Tyr Leu 260 265 270 Ala
Val Ile Glu Gly Ala Lys Phe Ile Met Gly Asp Ser Ser Val Gln 275 280
285 Asp Gln Trp Lys Glu Leu Ser Arg Glu Asp Leu Asp Leu Pro Pro Glu
290 295 300 His Val Pro Pro Pro Pro Arg Pro Pro Lys Arg Thr Leu Glu
Pro His 305 310 315 320 Asn Gly Lys Cys Lys Glu Leu Phe Ser Ser His
Gln Trp Val Ser Glu 325 330 335 Glu Thr Cys Gly Asp Glu Asp Ser Leu
Ala Arg Glu Glu Gly Arg Ala 340 345 350 Gln Ser Ser Ala Met His Ser
Val Ser Ser Met Ser Pro Asp Thr Glu 355 360 365 Val Arg Arg Arg Met
Val Gly Gly Gly Leu Gln Ser Ala Gln Ala Ser 370 375 380 Val Pro Thr
Glu Glu Glu Leu Ser Ser Thr Glu Glu Glu His Lys Ala 385 390 395 400
His Trp Pro Ser His Trp Lys Pro Phe Leu Val Asn Val Cys Met Ala 405
410 415 Thr Leu Leu Ala Thr Gly Ala Tyr Leu Cys Tyr Arg Val Cys Phe
His 420 425 430 9 3215 DNA Homo sapiens 9 gcgcgacgcg gcctagagcg
gcagacggcg cagtgggccg agaaggaggc gcagcagccg 60 ccctggcccg
tcatggagat ggaaaaggag ttcgagcaga tcgacaagtc cgggagctgg 120
gcggccattt accaggatat ccgacatgaa gccagtgact tcccatgtag agtggccaag
180 cttcctaaga acaaaaaccg aaataggtac agagacgtca gtccctttga
ccatagtcgg 240 attaaactac atcaagaaga taatgactat atcaacgcta
gtttgataaa aatggaagaa 300 gcccaaagga gttacattct tacccagggc
cctttgccta acacatgcgg tcacttttgg 360 gagatggtgt gggagcagaa
aagcaggggt gtcgtcatgc tcaacagagt gatggagaaa 420 ggttcgttaa
aatgcgcaca atactggcca caaaaagaag aaaaagagat gatctttgaa 480
gacacaaatt tgaaattaac attgatctct gaagatatca agtcatatta tacagtgcga
540 cagctagaat tggaaaacct tacaacccaa gaaactcgag agatcttaca
tttccactat 600 accacatggc ctgactttgg agtccctgaa tcaccagcct
cattcttgaa ctttcttttc 660 aaagtccgag agtcagggtc actcagcccg
gagcacgggc ccgttgtggt gcactgcagt 720 gcaggcatcg gcaggtctgg
aaccttctgt ctggctgata cctgcctctt gctgatggac 780 aagaggaaag
acccttcttc cgttgatatc aagaaagtgc tgttagaaat gaggaagttt 840
cggatggggc tgatccagac agccgaccag ctgcgcttct cctacctggc tgtgatcgaa
900 ggtgccaaat tcatcatggg ggactcttcc gtgcaggatc agtggaagga
gctttcccac 960 gaggacctgg agcccccacc cgagcatatc cccccacctc
cccggccacc caaacgaatc 1020 ctggagccac acaatgggaa atgcagggag
ttcttcccaa atcaccagtg ggtgaaggaa 1080 gagacccagg aggataaaga
ctgccccatc aaggaagaaa aaggaagccc cttaaatgcc 1140 gcaccctacg
gcatcgaaag catgagtcaa gacactgaag ttagaagtcg ggtcgtgggg 1200
ggaagtcttc gaggtgccca ggctgcctcc ccagccaaag gggagccgtc actgcccgag
1260 aaggacgagg accatgcact gagttactgg aagcccttcc tggtcaacat
gtgcgtggct 1320 acggtcctca cggccggcgc ttacctctgc tacaggttcc
tgttcaacag caacacatag 1380 cctgaccctc ctccactcca cctccaccca
ctgtccgcct ctgcccgcag agcccacgcc 1440 cgactagcag gcatgccgcg
gtaggtaagg gccgccggac cgcgtagaga gccgggcccc 1500 ggacggacgt
tggttctgca ctaaaaccca tcttccccgg atgtgtgtct cacccctcat 1560
ccttttactt tttgcccctt ccactttgag taccaaatcc acaagccatt ttttgaggag
1620 agtgaaagag agtaccatgc tggcggcgca gagggaaggg gcctacaccc
gtcttggggc 1680 tcgccccacc cagggctccc tcctggagca tcccaggcgg
gcggcacgcc agacagcccc 1740 ccccttgaat ctgcagggag caactctcca
ctccatattt atttaaacaa ttttttcccc 1800 aaaggcatcc atagtgcact
agcattttct tgaaccaata atgtattaaa attttttgat 1860 gtcagccttg
catcaagggc tttatcaaaa agtacaataa taaatcctca ggtagtactg 1920
ggaatggaag gctttgccat gggcctgctg cgtcagacca gtactgggaa ggaggacggt
1980 tgtaagcagt tgttatttag tgatattgtg ggtaacgtga gaagatagaa
caatgctata 2040 atatataatg aacacgtggg tatttaataa gaaacatgat
gtgagattac tttgtcccgc 2100 ttattctgct ccctgttatc tgctagatct
agttctcaat cactgctccc ccgtgtgtat 2160 tagaatgcat gtaaggtctt
cttgtgtcct gatgaaaaat atgtgcttga aatgagaaac 2220 tttgatctct
gcttactaat gtgccccatg tccaagtcca acctgcctgt gcatgacctg 2280
atcattacat ggctgtggtt cctaagcctg ttgctgaagt cattgtcgct cagcaatagg
2340 gtgcagtttt ccaggaatag gcatttgcct aattcctggc atgacactct
agtgacttcc 2400 tggtgaggcc cagcctgtcc tggtacagca gggtcttgct
gtaactcaga cattccaagg 2460 gtatgggaag ccatattcac acctcacgct
ctggacatga tttagggaag cagggacacc 2520 ccccgccccc cacctttggg
atcagcctcc gccattccaa gtcgacactc ttcttgagca 2580 gaccgtgatt
tggaagagag gcacctgctg gaaaccacac ttcttgaaac agcctgggtg 2640
acggtccttt aggcagcctg ccgccgtctc tgtcccggtt caccttgccg agagaggcgc
2700 gtctgcccca ccctcaaacc ctgtggggcc tgatggtgct cacgactctt
cctgcaaagg 2760 gaactgaaga cctccacatt aagtggcttt ttaacatgaa
aaacacggca gctgtagctc 2820 ccgagctact ctcttgccag cattttcaca
ttttgccttt ctcgtggtag aagccagtac 2880 agagaaattc tgtggtggga
acattcgagg tgtcaccctg cagagctatg gtgaggtgtg 2940 gataaggctt
aggtgccagg ctgtaagcat tctgagctgg cttgttgttt ttaagtcctg 3000
tatatgtatg tagtagtttg ggtgtgtata tatagtagca tttcaaaatg gacgtactgg
3060 tttaacctcc tatccttgga gagcagctgg ctctccacct tgttacacat
tatgttagag 3120 aggtagcgag ctgctctgct atgtccttaa gccaatattt
actcatcagg tcattatttt 3180 ttacaatggc catggaataa accattttta caaaa
3215 10 435 PRT Homo sapiens 10 Met Glu Met Glu Lys Glu Phe Glu Gln
Ile Asp Lys Ser Gly Ser Trp 1 5 10 15 Ala Ala Ile Tyr Gln Asp Ile
Arg His Glu Ala Ser Asp Phe Pro Cys 20 25 30 Arg Val Ala Lys Leu
Pro Lys Asn Lys Asn Arg Asn Arg Tyr Arg Asp 35 40 45 Val Ser Pro
Phe Asp His Ser Arg Ile Lys Leu His Gln Glu Asp Asn 50 55 60 Asp
Tyr Ile Asn Ala Ser Leu Ile Lys Met Glu Glu Ala Gln Arg Ser 65 70
75 80 Tyr Ile Leu Thr Gln Gly Pro Leu Pro Asn Thr Cys Gly His Phe
Trp 85 90 95 Glu Met Val Trp Glu Gln Lys Ser Arg Gly Val Val Met
Leu Asn Arg 100 105 110 Val Met Glu Lys Gly Ser Leu Lys Cys Ala Gln
Tyr Trp Pro Gln Lys 115 120 125 Glu Glu Lys Glu Met Ile Phe Glu Asp
Thr Asn Leu Lys Leu Thr Leu 130 135 140 Ile Ser Glu Asp Ile Lys Ser
Tyr Tyr Thr Val Arg Gln Leu Glu Leu 145 150 155 160 Glu Asn Leu Thr
Thr Gln Glu Thr Arg Glu Ile Leu His Phe His Tyr 165 170 175 Thr Thr
Trp Pro Asp Phe Gly Val Pro Glu Ser Pro Ala Ser Phe Leu 180 185 190
Asn Phe Leu Phe Lys Val Arg Glu Ser Gly Ser Leu Ser Pro Glu His 195
200 205 Gly Pro Val Val Val His Cys Ser Ala Gly Ile Gly Arg Ser Gly
Thr 210 215 220 Phe Cys Leu Ala Asp Thr Cys Leu Leu Leu Met Asp Lys
Arg Lys Asp 225 230 235 240 Pro Ser Ser Val Asp Ile Lys Lys Val Leu
Leu Glu Met Arg Lys Phe 245 250 255 Arg Met Gly Leu Ile Gln Thr Ala
Asp Gln Leu Arg Phe Ser Tyr Leu 260 265 270 Ala Val Ile Glu Gly Ala
Lys Phe Ile Met Gly Asp Ser Ser Val Gln 275 280 285 Asp Gln Trp Lys
Glu Leu Ser His Glu Asp Leu Glu Pro Pro Pro Glu 290 295 300 His Ile
Pro Pro Pro Pro Arg Pro Pro Lys Arg Ile Leu Glu Pro His 305 310 315
320 Asn Gly Lys Cys Arg Glu Phe Phe Pro Asn His Gln Trp Val Lys Glu
325 330 335 Glu Thr Gln Glu Asp Lys Asp Cys Pro Ile Lys Glu Glu Lys
Gly Ser 340 345 350 Pro Leu Asn Ala Ala Pro Tyr Gly Ile Glu Ser Met
Ser Gln Asp Thr 355 360 365 Glu Val Arg Ser Arg Val Val Gly Gly Ser
Leu Arg Gly Ala Gln Ala 370 375 380 Ala Ser Pro Ala Lys Gly Glu Pro
Ser Leu Pro Glu Lys Asp Glu Asp 385 390 395 400 His Ala Leu Ser Tyr
Trp Lys Pro Phe Leu Val Asn Met Cys Val Ala 405 410 415 Thr Val Leu
Thr Ala Gly Ala Tyr Leu Cys Tyr Arg Phe Leu Phe Asn 420 425 430 Ser
Asn Thr 435 11 4127 DNA Rattus norvegicus 11 agccgctgct ggggaggttg
gggctgaggt ggtggcgggc gacgggcctc gagacgcgga 60 gcgacgcggc
ctagcgcggc ggacggccga gggaactcgg gcagtcgtcc cgtcccgcca 120
tggaaatgga gaaggaattc gagcagatcg ataaggctgg gaactgggcg gctatttacc
180 aggatattcg acatgaagcc agtgacttcc catgcagaat agcgaaactt
cctaagaaca 240 aaaaccggaa caggtaccga gatgtcagcc cttttgacca
cagtcggatt aaattgcatc 300 aggaagataa tgactatatc aatgccagct
tgataaaaat ggaggaagcc cagaggagct 360 atatcctcac ccagggccct
ttaccaaaca cgtgcgggca cttctgggag atggtgtggg 420 agcagaagag
caggggcgtg gtcatgctca accgcatcat ggagaaaggc tcgttaaaat 480
gtgcccagta ttggccacag aaagaagaaa aagagatggt cttcgatgac accaatttga
540 agctgacact gatctctgaa gatgtcaagt catattacac agtacggcag
ttggagttgg 600 agaacctggc tacccaggag gctcgagaga tcctgcattt
ccactacacc acctggcctg 660 actttggagt ccctgagtca cctgcctctt
tcctcaattt cctattcaaa gtccgagagt 720 caggctcact cagcccagag
cacggcccca ttgtggtcca ctgcagtgct ggcattggca 780 ggtcagggac
cttctgcctg gctgacacct gcctcttact gatggacaag aggaaagacc 840
cgtcctctgt ggacatcaag aaagtgctgt tggagatgcg caggttccgc atggggctca
900 tccagacggc cgaccaactg cgcttctcct acctggctgt gatcgagggt
gcaaagttca 960 tcatgggcga ctcgtcagtg caggatcagt ggaaggagct
ttcccatgaa gacctggagc 1020 ctccccctga gcacgtgccc ccacctcccc
ggccacccaa acgcacattg gagcctcaca 1080 atggcaagtg caaggagctc
ttctccaacc accagtgggt gagcgaggag agctgtgagg 1140 atgaggacat
cctggccaga gaggaaagca gagccccctc aattgctgtg cacagcatga 1200
gcagtatgag tcaagacact gaagttagga aacggatggt gggtggaggt cttcaaagtg
1260 ctcaggcatc tgtccccact gaggaagagc tgtccccaac cgaggaggaa
caaaaggcac 1320 acaggccagt tcactggaag cccttcctgg tcaacgtgtg
catggccacg gccctggcga 1380 ctggcgcgta cctctgttac cgggtatgtt
ttcactgaca gactgctgtg aggcatgagc 1440 gtggtgggcg ctgccactgc
ccaggttagg atttggtctg cggcgtctaa cctggtgtag 1500 aagaaacaac
agcttacaag cctgtggtgg aactggaagg gccagcccca ggaggggcat 1560
ctgtgcactg ggctttgaag gagcccctgg tcccaagaac agagtctaat ctcagggcct
1620 taacctgttc aggagaagta gaggaaatgc caaatactct tcttgctctc
acctcactcc 1680 tcccctttct ctggttcgtt tgtttttgga aaaaaaaaaa
aaagaattac aacacattgt 1740 tgtttttaac atttataaag gcaggttttt
gttattttta gagaaaacaa aagatgctag 1800 gcactggtga gattctcttg
tgccctttgg catgtgatca gattcacgat ttacgtttat 1860 ttccggggga
gggtcccacc tgtcaggact gtaaagttcc tgctggcttg gtcagccccc 1920
ccaccccccc accccgagct tgcaggtgcc ctgctgtgag gagagcagca gcagaggctg
1980 cccctggaca gaagcccagc tctgcttccc tcaggtgtcc ctgcgtttcc
atcctccttc 2040 tttgtgaccg ccatcttgca gatgacccag tcctcagcac
cccacccctg cagatgggtt 2100 tctccgaggg cctgcctcag ggtcatcaga
ggttggctgc cagcttagag ctggggcttc 2160 catttgattg gaaagtcatt
actattctat gtagaagcca ctccactgag gtgtaaagca 2220 agactcataa
aggaggagcc ttggtgtcat ggaagtcact ccgcgcgcag gacctgtaac 2280
aacctctgaa acactcagtc ctgctgcagt gacgtccttg aaggcatcag acagatgatt
2340 tgcagactgc caagacttgt cctgagccgt gatttttaga gtctggactc
atgaaacacc 2400 gccgagcgct tactgtgcag cctctgatgc tggttggctg
aggctgcggg gaggtggaca 2460 ctgtgggtgc atccagtgca gttgcttttg
tgcagttggg tccagcagca cagcccgcac 2520 tccagcctca gctgcaggcc
acagtggcca tggaggccgc cagagcgagc tggggtggat 2580 gcttgttcac
ttggagcagc cttcccagga cgtgcagctc ccttcctgct ttgtccttct 2640
gcttccttcc ctggagtagc aagcccacga gcaatcgtga ggggtgtgag ggagctgcag
2700 aggcatcaga gtggcctgca gcggcgtgag gccccttccc ctccgacacc
cccctccaga 2760 ggagccgctc cactgttatt tattcacttt gcccacagac
acccctgagt gagcacaccc 2820 tgaaactgac cgtgtaaggt gtcagcctgc
acccaggacc gtcaggtgca gcaccgggtc 2880 agtcctaggg ttgaggtagg
actgacacag ccactgtgtg gctggtgctg gggcaggggc 2940 aggagctgag
ggtcttagaa gcaatcttca ggaacagaca acagtggtga catgtaaagt 3000
ccctgtggct actgatgaca tgtgtaggat gaaggctggc ctttctccca tgactttcta
3060 gatcccgttc cccgtctgct ttccctgtga gttagaaaac acacaggctc
ctgtcctggt 3120 ggtgccgtgt gcttgacatg ggaaacttag atgcctgctc
actggcgggc acctcggcat 3180 cgccaccact cagagtgaga gcagtgctgt
ccagtgccga ggccgcctga ctcccggcag 3240 gactcttcag gctctggcct
gccccagcac accccgctgg atctcagaca ttccacaccc 3300 acacctcatt
ccctggacac ttgggcaagc aggcccgccc ttccacctct ggggtcagcc 3360
cctccattcc gagttcacac tgctctggag caggccagga ccggaagcaa ggcagctggt
3420 gaggagcacc ctcctgggaa cagtgtaggt gacagtcctg agagtcagct
tgctagcgct 3480 gctggcacca gtcaccttgc tcagaagtgt gtggctcttg
aggctgaaga gactgatgat 3540 ggtgctcatg actcttctgt gaggggaact
tgaccttcac attgggtggc tttttttaaa 3600 ataagcgaag gcagctggaa
ctccagtctg cctcttgcca gcacttcaca ttttgccttt 3660 cacccagaga
agccagcaca gagccactgg ggaaggcgat ggccttgcct gcacaggctg 3720
aggagatggc tcagccggcg tccaggctgt gtctggagca gggggtgcac agcagcctca
3780 caggtggggg cctcagagca ggcgctgccc tgtcccctgc cccgctggag
gcagcaaagc 3840 tgctgcatgc cttaagtcaa tacttactca gcagggcgct
ctcgttctct ctctctctct 3900 ctctctctct ctctctctct ctctctctct
ctctaaatgg ccatagaata aaccatttta 3960 caaaaataaa agccaacaac
aaagtgctct ggaatagcac ctttgcagga gcggggggtg 4020 tctcagggtc
ttctgtgacc tcaccgaact gtccgactgc accgtttcca acttgtgtct 4080
cactaatggg tctgcattag ttgcaacaat aaatgttttt aaagaac 4127 12 432 PRT
Rattus norvegicus 12 Met Glu Met Glu Lys Glu Phe Glu Gln Ile Asp
Lys Ala Gly Asn Trp 1 5 10 15 Ala Ala Ile Tyr Gln Asp Ile Arg His
Glu Ala Ser Asp Phe Pro Cys 20 25 30 Arg Ile Ala Lys Leu Pro Lys
Asn Lys Asn Arg Asn Arg Tyr Arg Asp 35 40 45 Val Ser Pro Phe Asp
His Ser Arg Ile Lys Leu His Gln Glu Asp Asn 50 55 60 Asp Tyr Ile
Asn Ala Ser Leu Ile Lys Met Glu Glu Ala Gln Arg Ser 65 70 75 80 Tyr
Ile Leu Thr Gln Gly Pro Leu Pro Asn Thr Cys Gly His Phe Trp 85 90
95 Glu Met Val Trp Glu Gln Lys Ser Arg Gly Val Val Met Leu Asn Arg
100 105 110 Ile Met Glu Lys Gly Ser Leu Lys Cys Ala Gln Tyr Trp Pro
Gln Lys 115 120 125 Glu Glu Lys Glu Met Val Phe Asp Asp Thr Asn Leu
Lys Leu Thr Leu 130 135 140 Ile Ser Glu Asp Val Lys Ser Tyr Tyr Thr
Val Arg Gln Leu Glu Leu 145 150 155 160 Glu Asn Leu Ala Thr Gln Glu
Ala Arg Glu Ile Leu His Phe His Tyr 165 170 175 Thr Thr Trp Pro Asp
Phe Gly Val Pro Glu Ser Pro Ala Ser Phe Leu 180 185 190 Asn Phe Leu
Phe Lys Val Arg Glu Ser Gly Ser Leu Ser Pro Glu His 195 200 205 Gly
Pro Ile Val Val His Cys Ser Ala Gly Ile Gly Arg Ser Gly Thr 210 215
220 Phe Cys Leu Ala Asp Thr Cys Leu Leu Leu Met Asp Lys Arg Lys Asp
225 230 235 240 Pro Ser Ser Val Asp Ile Lys Lys Val Leu Leu Glu Met
Arg Arg Phe 245 250 255 Arg Met Gly Leu Ile Gln Thr Ala Asp Gln Leu
Arg Phe Ser Tyr Leu 260 265 270 Ala Val Ile Glu Gly Ala Lys Phe Ile
Met Gly Asp Ser Ser Val Gln 275 280 285 Asp Gln Trp Lys Glu Leu Ser
His Glu Asp Leu Glu Pro Pro Pro Glu 290 295 300 His Val Pro Pro Pro
Pro Arg Pro Pro Lys Arg Thr Leu Glu Pro His 305 310 315 320 Asn Gly
Lys Cys Lys Glu Leu Phe Ser Asn His Gln Trp Val Ser Glu 325 330 335
Glu Ser Cys Glu Asp Glu Asp Ile Leu Ala Arg Glu Glu Ser Arg Ala 340
345 350 Pro Ser Ile Ala Val His Ser Met Ser Ser Met Ser Gln Asp Thr
Glu 355 360 365 Val Arg Lys Arg Met Val Gly Gly Gly Leu Gln Ser Ala
Gln Ala Ser 370 375 380 Val Pro Thr Glu Glu Glu Leu Ser Pro Thr Glu
Glu Glu Gln Lys Ala 385 390 395
400 His Arg Pro Val His Trp Lys Pro Phe Leu Val Asn Val Cys Met Ala
405 410 415 Thr Ala Leu Ala Thr Gly Ala Tyr Leu Cys Tyr Arg Val Cys
Phe His 420 425 430 13 4127 DNA Rattus norvegicus 13 agccgctgct
ggggaggttg gggctgaggt ggtggcgggc gacgggcctc gagacgcgga 60
gcgacgcggc ctagcgcggc ggacggccga gggaactcgg gcagtcgtcc cgtcccgcca
120 tggaaatgga gaaggaattc gagcagatcg ataaggctgg gaactgggcg
gctatttacc 180 aggatattcg acatgaagcc agtgacttcc catgcagaat
agcgaaactt cctaagaaca 240 aaaaccggaa caggtaccga gatgtcagcc
cttttgacca cagtcggatt aaattgcatc 300 aggaagataa tgactatatc
aatgccagct tgataaaaat ggaggaagcc cagaggagct 360 atatcctcac
ccagggccct ttaccaaaca cgtgcgggca cttctgggag atggtgtggg 420
agcagaagag caggggcgtg gtcatgctca accgcatcat ggagaaaggc tcgttaaaat
480 gtgcccagta ttggccacag aaagaagaaa aagagatggt cttcgatgac
accaatttga 540 agctgacact gatctctgaa gatgtcaagt catattacac
agtacggcag ttggagttgg 600 agaacctggc tacccaggag gctcgagaga
tcctgcattt ccactacacc acctggcctg 660 actttggagt ccctgagtca
cctgcctctt tcctcaattt cctattcaaa gtccgagagt 720 caggctcact
cagcccagag cacggcccca ttgtggtcca ctgcagtgct ggcattggca 780
ggtcagggac cttctgcctg gctgacacct gcctcttact gatggacaag aggaaagacc
840 cgtcctctgt ggacatcaag aaagtgctgt tggagatgcg caggttccgc
atggggctca 900 tccagacggc cgaccaactg cgcttctcct acctggctgt
gatcgagggt gcaaagttca 960 tcatgggcga ctcgtcagtg caggatcagt
ggaaggagct ttcccatgaa gacctggagc 1020 ctccccctga gcacgtgccc
ccacctcccc ggccacccaa acgcacattg gagcctcaca 1080 atggcaagtg
caaggagctc ttctccaacc accagtgggt gagcgaggag agctgtgagg 1140
atgaggacat cctggccaga gaggaaagca gagccccctc aattgctgtg cacagcatga
1200 gcagtatgag tcaagacact gaagttagga aacggatggt gggtggaggt
cttcaaagtg 1260 ctcaggcatc tgtccccact gaggaagagc tgtccccaac
cgaggaggaa caaaaggcac 1320 acaggccagt tcactggaag cccttcctgg
tcaacgtgtg catggccacg gccctggcga 1380 ctggcgcgta cctctgttac
cgggtatgtt ttcactgaca gactgctgtg aggcatgagc 1440 gtggtgggcg
ctgccactgc ccaggttagg atttggtctg cggcgtctaa cctggtgtag 1500
aagaaacaac agcttacaag cctgtggtgg aactggaagg gccagcccca ggaggggcat
1560 ctgtgcactg ggctttgaag gagcccctgg tcccaagaac agagtctaat
ctcagggcct 1620 taacctgttc aggagaagta gaggaaatgc caaatactct
tcttgctctc acctcactcc 1680 tcccctttct ctggttcgtt tgtttttgga
aaaaaaaaaa aaagaattac aacacattgt 1740 tgtttttaac atttataaag
gcaggttttt gttattttta gagaaaacaa aagatgctag 1800 gcactggtga
gattctcttg tgccctttgg catgtgatca gattcacgat ttacgtttat 1860
ttccggggga gggtcccacc tgtcaggact gtaaagttcc tgctggcttg gtcagccccc
1920 ccaccccccc accccgagct tgcaggtgcc ctgctgtgag gagagcagca
gcagaggctg 1980 cccctggaca gaagcccagc tctgcttccc tcaggtgtcc
ctgcgtttcc atcctccttc 2040 tttgtgaccg ccatcttgca gatgacccag
tcctcagcac cccacccctg cagatgggtt 2100 tctccgaggg cctgcctcag
ggtcatcaga ggttggctgc cagcttagag ctggggcttc 2160 catttgattg
gaaagtcatt actattctat gtagaagcca ctccactgag gtgtaaagca 2220
agactcataa aggaggagcc ttggtgtcat ggaagtcact ccgcgcgcag gacctgtaac
2280 aacctctgaa acactcagtc ctgctgcagt gacgtccttg aaggcatcag
acagatgatt 2340 tgcagactgc caagacttgt cctgagccgt gatttttaga
gtctggactc atgaaacacc 2400 gccgagcgct tactgtgcag cctctgatgc
tggttggctg aggctgcggg gaggtggaca 2460 ctgtgggtgc atccagtgca
gttgcttttg tgcagttggg tccagcagca cagcccgcac 2520 tccagcctca
gctgcaggcc acagtggcca tggaggccgc cagagcgagc tggggtggat 2580
gcttgttcac ttggagcagc cttcccagga cgtgcagctc ccttcctgct ttgtccttct
2640 gcttccttcc ctggagtagc aagcccacga gcaatcgtga ggggtgtgag
ggagctgcag 2700 aggcatcaga gtggcctgca gcggcgtgag gccccttccc
ctccgacacc cccctccaga 2760 ggagccgctc cactgttatt tattcacttt
gcccacagac acccctgagt gagcacaccc 2820 tgaaactgac cgtgtaaggt
gtcagcctgc acccaggacc gtcaggtgca gcaccgggtc 2880 agtcctaggg
ttgaggtagg actgacacag ccactgtgtg gctggtgctg gggcaggggc 2940
aggagctgag ggtcttagaa gcaatcttca ggaacagaca acagtggtga catgtaaagt
3000 ccctgtggct actgatgaca tgtgtaggat gaaggctggc ctttctccca
tgactttcta 3060 gatcccgttc cccgtctgct ttccctgtga gttagaaaac
acacaggctc ctgtcctggt 3120 ggtgccgtgt gcttgacatg ggaaacttag
atgcctgctc actggcgggc acctcggcat 3180 cgccaccact cagagtgaga
gcagtgctgt ccagtgccga ggccgcctga ctcccggcag 3240 gactcttcag
gctctggcct gccccagcac accccgctgg atctcagaca ttccacaccc 3300
acacctcatt ccctggacac ttgggcaagc aggcccgccc ttccacctct ggggtcagcc
3360 cctccattcc gagttcacac tgctctggag caggccagga ccggaagcaa
ggcagctggt 3420 gaggagcacc ctcctgggaa cagtgtaggt gacagtcctg
agagtcagct tgctagcgct 3480 gctggcacca gtcaccttgc tcagaagtgt
gtggctcttg aggctgaaga gactgatgat 3540 ggtgctcatg actcttctgt
gaggggaact tgaccttcac attgggtggc tttttttaaa 3600 ataagcgaag
gcagctggaa ctccagtctg cctcttgcca gcacttcaca ttttgccttt 3660
cacccagaga agccagcaca gagccactgg ggaaggcgat ggccttgcct gcacaggctg
3720 aggagatggc tcagccggcg tccaggctgt gtctggagca gggggtgcac
agcagcctca 3780 caggtggggg cctcagagca ggcgctgccc tgtcccctgc
cccgctggag gcagcaaagc 3840 tgctgcatgc cttaagtcaa tacttactca
gcagggcgct ctcgttctct ctctctctct 3900 ctctctctct ctctctctct
ctctctctct ctctaaatgg ccatagaata aaccatttta 3960 caaaaataaa
agccaacaac aaagtgctct ggaatagcac ctttgcagga gcggggggtg 4020
tctcagggtc ttctgtgacc tcaccgaact gtccgactgc accgtttcca acttgtgtct
4080 cactaatggg tctgcattag ttgcaacaat aaatgttttt aaagaac 4127 14
432 PRT Rattus norvegicus 14 Met Glu Met Glu Lys Glu Phe Glu Gln
Ile Asp Lys Ala Gly Asn Trp 1 5 10 15 Ala Ala Ile Tyr Gln Asp Ile
Arg His Glu Ala Ser Asp Phe Pro Cys 20 25 30 Arg Ile Ala Lys Leu
Pro Lys Asn Lys Asn Arg Asn Arg Tyr Arg Asp 35 40 45 Val Ser Pro
Phe Asp His Ser Arg Ile Lys Leu His Gln Glu Asp Asn 50 55 60 Asp
Tyr Ile Asn Ala Ser Leu Ile Lys Met Glu Glu Ala Gln Arg Ser 65 70
75 80 Tyr Ile Leu Thr Gln Gly Pro Leu Pro Asn Thr Cys Gly His Phe
Trp 85 90 95 Glu Met Val Trp Glu Gln Lys Ser Arg Gly Val Val Met
Leu Asn Arg 100 105 110 Ile Met Glu Lys Gly Ser Leu Lys Cys Ala Gln
Tyr Trp Pro Gln Lys 115 120 125 Glu Glu Lys Glu Met Val Phe Asp Asp
Thr Asn Leu Lys Leu Thr Leu 130 135 140 Ile Ser Glu Asp Val Lys Ser
Tyr Tyr Thr Val Arg Gln Leu Glu Leu 145 150 155 160 Glu Asn Leu Ala
Thr Gln Glu Ala Arg Glu Ile Leu His Phe His Tyr 165 170 175 Thr Thr
Trp Pro Asp Phe Gly Val Pro Glu Ser Pro Ala Ser Phe Leu 180 185 190
Asn Phe Leu Phe Lys Val Arg Glu Ser Gly Ser Leu Ser Pro Glu His 195
200 205 Gly Pro Ile Val Val His Cys Ser Ala Gly Ile Gly Arg Ser Gly
Thr 210 215 220 Phe Cys Leu Ala Asp Thr Cys Leu Leu Leu Met Asp Lys
Arg Lys Asp 225 230 235 240 Pro Ser Ser Val Asp Ile Lys Lys Val Leu
Leu Glu Met Arg Arg Phe 245 250 255 Arg Met Gly Leu Ile Gln Thr Ala
Asp Gln Leu Arg Phe Ser Tyr Leu 260 265 270 Ala Val Ile Glu Gly Ala
Lys Phe Ile Met Gly Asp Ser Ser Val Gln 275 280 285 Asp Gln Trp Lys
Glu Leu Ser His Glu Asp Leu Glu Pro Pro Pro Glu 290 295 300 His Val
Pro Pro Pro Pro Arg Pro Pro Lys Arg Thr Leu Glu Pro His 305 310 315
320 Asn Gly Lys Cys Lys Glu Leu Phe Ser Asn His Gln Trp Val Ser Glu
325 330 335 Glu Ser Cys Glu Asp Glu Asp Ile Leu Ala Arg Glu Glu Ser
Arg Ala 340 345 350 Pro Ser Ile Ala Val His Ser Met Ser Ser Met Ser
Gln Asp Thr Glu 355 360 365 Val Arg Lys Arg Met Val Gly Gly Gly Leu
Gln Ser Ala Gln Ala Ser 370 375 380 Val Pro Thr Glu Glu Glu Leu Ser
Pro Thr Glu Glu Glu Gln Lys Ala 385 390 395 400 His Arg Pro Val His
Trp Lys Pro Phe Leu Val Asn Val Cys Met Ala 405 410 415 Thr Ala Leu
Ala Thr Gly Ala Tyr Leu Cys Tyr Arg Val Cys Phe His 420 425 430 15
36 DNA Artificial Sequence Oligonucleotide primer mPTP1B-sense 15
gggggggatc catggagatg gagaaggagt tcgagg 36 16 35 DNA Artificial
Sequence Oligonucleotide primer mPTP1B-anti sense 16 gggggaattc
tcagtgaaaa cacacccggt agcac 35 17 21 RNA Artificial Sequence Small
interfering RNA - mPTP1B1.1 17 gaagcccaga ggagcuauan n 21 18 19 RNA
Artificial Sequence Small interfering RNA - mPTP1B1.1 18 gaagcccaga
ggagcuaua 19 19 19 RNA Artificial Sequence Small interfering RNA -
mPTP1B1.1 19 uauagcuccu cugggcuuc 19 20 21 RNA Artificial Sequence
Small interfering RNA - mPTP1B1.1 20 gaagcccaga ggagcuauan n 21 21
21 RNA Artificial Sequence Small interfering RNA - mPTP1B1.1 21
nnuauagcuc cucugggcuu c 21 22 21 RNA Artificial Sequence Small
interfering RNA - mPTP1B1.2 22 cuacaccaca uggccugacn n 21 23 19 RNA
Artificial Sequence Small interfering RNA - mPTP1B1.2 23 cuacaccaca
uggccugac 19 24 19 RNA Artificial Sequence Small interfering RNA -
mPTP1B1.2 24 gucaggccau gugguguag 19 25 21 RNA Artificial Sequence
Small interfering RNA - mPTP1B1.2 25 cuacaccaca uggccugacn n 21 26
21 RNA Artificial Sequence Small interfering RNA - mPTP1B1.2 26
nngucaggcc auguggugua g 21 27 21 RNA Artificial Sequence Small
interfering RNA - mPTP1B1.3 27 gacugccgac cagcugcgcn n 21 28 19 RNA
Artificial Sequence Small interfering RNA - mPTP1B1.3 28 gacugccgac
cagcugcgc 19 29 19 RNA Artificial Sequence Small interfering RNA -
mPTP1B1.3 29 gcgcagcugg ucggcaguc 19 30 21 RNA Artificial Sequence
Small interfering RNA - mPTP1B1.3 30 gacugccgac cagcugcgcn n 21 31
21 RNA Artificial Sequence Small interfering RNA - mPTP1B1.3 31
nngcgcagcu ggucggcagu c 21 32 21 RNA Artificial Sequence Small
interfering RNA - mPTP1B1.4 32 gguaccgaga ugucagcccn n 21 33 19 RNA
Artificial Sequence Small interfering RNA - mPTP1B1.4 33 gguaccgaga
ugucagccc 19 34 19 RNA Artificial Sequence Small interfering RNA -
mPTP1B1.4 34 gggcugacau cucgguacc 19 35 21 RNA Artificial Sequence
Small interfering RNA - mPTP1B1.4 35 gguaccgaga ugucagcccn n 21 36
21 RNA Artificial Sequence Small interfering RNA - mPTP1B1.4 36
nngggcugac aucucgguac c 21 37 21 RNA Artificial Sequence Small
interfering RNA - mPTP1B1.5 37 ugacuauauc aaugccagcn n 21 38 19 RNA
Artificial Sequence Small interfering RNA - mPTP1B1.5 38 ugacuauauc
aaugccagc 19 39 19 RNA Artificial Sequence Small interfering RNA -
mPTP1B1.5 39 gcuggcauug auauaguca 19 40 21 RNA Artificial Sequence
Small interfering RNA - mPTP1B1.5 40 ugacuauauc aaugccagcn n 21 41
21 RNA Artificial Sequence Small interfering RNA - mPTP1B1.5 41
nngcuggcau ugauauaguc a 21 42 21 RNA Artificial Sequence Small
interfering RNA - mPTP1B1.6 42 agaagaaaag gagauggucn n 21 43 19 RNA
Artificial Sequence Small interfering RNA - mPTP1B1.6 43 agaagaaaag
gagaugguc 19 44 19 RNA Artificial Sequence Small interfering RNA -
mPTP1B1.6 44 gaccaucucc uuuucuucu 19 45 21 RNA Artificial Sequence
Small interfering RNA - mPTP1B1.6 45 agaagaaaag gagauggucn n 21 46
21 RNA Artificial Sequence Small interfering RNA - mPTP1B1.6 46
nngaccaucu ccuuuucuuc u 21 47 21 RNA Artificial Sequence Small
interfering RNA - mPTP1B1.7 47 cgggaagugc aaggagcucn n 21 48 19 RNA
Artificial Sequence Small interfering RNA - mPTP1B1.7 48 cgggaagugc
aaggagcuc 19 49 19 RNA Artificial Sequence Small interfering RNA -
mPTP1B1.7 49 gagcuccuug cacuucccg 19 50 21 RNA Artificial Sequence
Small interfering RNA - mPTP1B1.7 50 cgggaagugc aaggagcucn n 21 51
21 RNA Artificial Sequence Small interfering RNA - mPTP1B1.7 51
nngagcuccu ugcacuuccc g 21 52 21 RNA Artificial Sequence Small
interfering RNA - mPTP1B1.8 52 ggaucagugg aaggagcucn c 21 53 19 RNA
Artificial Sequence Small interfering RNA - mPTP1B1.8 53 ggaucagugg
aaggagcuc 19 54 19 RNA Artificial Sequence Small interfering RNA -
mPTP1B1.8 54 gagcuccuuc cacugaucc 19 55 21 RNA Artificial Sequence
Small interfering RNA - mPTP1B1.8 55 ggaucagugg aaggagcucn n 21 56
21 RNA Artificial Sequence Small interfering RNA - mPTP1B1.8 56
nngagcuccu uccacugauc c 21 57 21 RNA Artificial Sequence Small
interfering RNA - rPTP1B1.1 57 agaagaaaaa gagauggucn n 21 58 19 RNA
Artificial Sequence Small interfering RNA - rPTP1B1.1 58 agaagaaaaa
gagaugguc 19 59 19 RNA Artificial Sequence Small interfering RNA -
rPTP1B1.1 59 gaccaucucu uuuucuucu 19 60 21 RNA Artificial Sequence
Small interfering RNA - rPTP1B1.1 60 agaagaaaaa gagauggucn n 21 61
21 RNA Artificial Sequence Small interfering RNA - rPTP1B1.1 61
nngaccaucu cuuuuucuuc u 21 62 21 RNA Artificial Sequence Small
interfering RNA - rPTP1B1.2 62 cggauggugg guggaggucn n 21 63 19 RNA
Artificial Sequence Small interfering RNA - rPTP1B1.2 63 cggauggugg
guggagguc 19 64 19 RNA Artificial Sequence Small interfering RNA -
rPTP1B1.2 64 gaccuccacc caccauccg 19 65 21 RNA Artificial Sequence
Small interfering RNA - rPTP1B1.2 65 cggauggugg guggaggucn n 21 66
21 RNA Artificial Sequence Small interfering RNA - rPTP1B1.2 66
nngaccucca cccaccaucc g 21 67 21 RNA Artificial Sequence Small
interfering RNA - rPTP1B1.3 67 uggcaagugc aaggagcucn n 21 68 19 RNA
Artificial Sequence Small interfering RNA - rPTP1B1.3 68 uggcaagugc
aaggagcuc 19 69 19 RNA Artificial Sequence Small interfering RNA -
rPTP1B1.3 69 gagcuccuug cacuugcca 19 70 21 RNA Artificial Sequence
Small interfering RNA - rPTP1B1.3 70 uggcaagugc aaggagcucn n 21 71
21 RNA Artificial Sequence Small interfering RNA - rPTP1B1.3 71
nngagcuccu ugcacuugcc a 21 72 21 RNA Artificial Sequence Small
interfering RNA - rPTP1B1.4 72 cuacaccacc uggccugacn n 21 73 19 RNA
Artificial Sequence Small interfering RNA - rPTP1B1.4 73 cuacaccacc
uggccugac 19 74 19 RNA Artificial Sequence Small interfering RNA -
rPTP1B1.4 74 gucaggccag gugguguag 19 75 21 RNA Artificial Sequence
Small interfering RNA - rPTP1B1.4 75 cuacaccacc uggccugacn n 21 76
21 RNA Artificial Sequence Small interfering RNA - rPTP1B1.4 76
nngucaggcc agguggugua g 21 77 21 RNA Artificial Sequence Small
interfering RNA - hPTP1B1.1 77 cuauaccaca uggccugacn n 21 78 19 RNA
Artificial Sequence Small interfering RNA - hPTP1B1.1 78 cuauaccaca
uggccugac 19 79 19 RNA Artificial Sequence Small interfering RNA -
hPTP1B1.1 79 gucaggccau gugguauag 19 80 21 RNA Artificial Sequence
Small interfering RNA - hPTP1B1.1 80 cuauaccaca uggccugacn n 21 81
21 RNA Artificial Sequence Small interfering RNA - hPTP1B1.1 81
nngucaggcc augugguaua g 21 82 21 RNA Artificial Sequence Small
interfering RNA - hPTP1B1.2 82 gcccaaagga guuacauucn n 21 83 19 RNA
Artificial Sequence Small interfering RNA - hPTP1B1.2 83 gcccaaagga
guuacauuc 19 84 19 RNA Artificial Sequence Small interfering RNA -
hPTP1B1.2 84 gaauguaacu ccuuugggc 19 85 21 RNA Artificial Sequence
Small interfering RNA - hPTP1B1.2 85 gcccaaagga guuacauucn n 21 86
21 RNA Artificial Sequence Small interfering RNA - hPTP1B1.2 86
nngaauguaa cuccuuuggg c 21 87 21 RNA Artificial Sequence Small
interfering RNA - hPTP1B1.3 87 ggaagaaaaa ggaagccccn n 21 88 19 RNA
Artificial Sequence Small interfering RNA - hPTP1B1.3 88 ggaagaaaaa
ggaagcccc 19 89 19 RNA Artificial Sequence Small interfering RNA -
hPTP1B1.3 89 ggggcuuccu uuuucuucc 19 90 21 RNA Artificial Sequence
Small interfering RNA - hPTP1B1.3 90 ggaagaaaaa ggaagccccn n 21 91
21 RNA Artificial Sequence Small interfering RNA - hPTP1B1.3 91
nnggggcuuc cuuuuucuuc c 21 92 21 RNA Artificial Sequence Small
interfering RNA - hPTP1B1.4 92 caaugggaaa ugcagggagn n 21 93 19 RNA
Artificial Sequence Small interfering RNA - hPTP1B1.4 93 caaugggaaa
ugcagggag 19 94 19 RNA Artificial Sequence Small interfering RNA -
hPTP1B1.4 94 cucccugcau uucccauug 19 95 21 RNA Artificial Sequence
Small interfering RNA - hPTP1B1.4 95 caaugggaaa ugcagggagn n 21 96
21 RNA Artificial Sequence misc_feature 1, 2 n = A,T,C,U or G 96
nncucccugc auuucccauu g 21 97 21 RNA Artificial Sequence Small
interfering RNA - hPTP1B1.5 97 ggaucagugg aaggagcuun c 21 98 19 RNA
Artificial Sequence Small interfering RNA - hPTP1B1.5 98 ggaucagugg
aaggagcuu 19 99 19 RNA Artificial Sequence Small interfering RNA -
hPTP1B1.5 99 aagcuccuuc cacugaucc 19 100 21 RNA Artificial Sequence
Small interfering RNA - hPTP1B1.5 100 ggaucagugg aaggagcuun n 21
101 21 RNA Artificial Sequence Small interfering RNA - hPTP1B1.5
101 nnaagcuccu uccacugauc c 21 102 50 DNA Artificial Sequence
Hairpin vector - hPTP1B H1.2-HP4 102 tttgcccaaa ggagttacat
tcgtaagaat gtaactcctt tgggcttttt 50 103 50 DNA Artificial Sequence
Hairpin vector - hPTP1B H1.2-HP4 103 ctagaaaaag cccaaaggag
ttacattctt acgaatgtaa ctcctttggg 50 104 50 RNA Artificial Sequence
Hairpin vector - hPTP1B H1.2-HP4 104 uuugcccaaa ggaguuacau
ucguaagaau guaacuccuu ugggcuuuuu 50 105 50 RNA Artificial Sequence
Hairpin vector - hPTP1B H1.2-HP4 105 cuagaaaaag cccaaaggag
uuacauucuu acgaauguaa cuccuuuggg 50 106 55 DNA Artificial Sequence
Hairpin vector - hPTP1B H1.2-HP9 106 tttgcccaaa ggagttacat
tccctgggta agaatgtaac tcctttgggc ttttt 55 107 55 DNA Artificial
Sequence Hairpin vector - hPTP1B H1.2-HP9 107 ctagaaaaag cccaaaggag
ttacattctt acccagggaa tgtaactcct ttggg 55 108 55 RNA Artificial
Sequence Hairpin vector - hPTP1B H1.2-HP9 108 uuugcccaaa ggaguuacau
ucccugggua agaauguaac uccuuugggc uuuuu 55 109 55 RNA Artificial
Sequence Hairpin vector - hPTP1B H1.2-HP9 109 cuagaaaaag cccaaaggag
uuacauucuu acccagggaa uguaacuccu uuggg 55 110 50 DNA Artificial
Sequence Hairpin vector - mPTP1B M1.1-HP4 110 tttgaagccc agaggagcta
taagaatata gctcctctgg gcttcttttt 50 111 50 DNA Artificial Sequence
Hairpin vector - mPTP1B M1.1-HP4 111 ctagaaaaag aagcccagag
gagctatatt cttatagctc ctctgggctt 50 112 50 RNA Artificial Sequence
Hairpin vector - mPTP1B M1.1-HP4 112 uuugaagccc agaggagcua
uaagaauaua gcuccucugg gcuucuuuuu 50 113 50 RNA Artificial Sequence
Hairpin vector - mPTP1B M1.1-HP4 113 cuagaaaaag aagcccagag
gagcuauauu cuuauagcuc cucugggcuu 50 114 55 DNA Artificial Sequence
Hairpin vector - mPTP1B M1.1-HP9 114 tttgaagccc agaggagcta
tagggtgaga atatagctcc tctgggcttc ttttt 55 115 55 DNA Artificial
Sequence Hairpin vector - mPTP1B M1.1-HP9 115 ctagaaaaag aagcccagag
gagctatatt ctcaccctat agctcctctg ggctt 55 116 55 RNA Artificial
Sequence Hairpin vector - mPTP1B M1.1-HP9 116 uuugaagccc agaggagcua
uagggugaga auauagcucc ucugggcuuc uuuuu 55 117 55 RNA Artificial
Sequence Hairpin vector - mPTP1B M1.1-HP9 117 cuagaaaaag aagcccagag
gagcuauauu cucacccuau agcuccucug ggcuu 55 118 19 RNA Artificial
Sequence Small interfering RNA 118 aaggaguucg agcagaucg 19 119 19
RNA Artificial Sequence Small interfering RNA 119 aggaguucga
gcagaucga 19 120 19 RNA Artificial Sequence Small interfering RNA
120 ggaguucgag cagaucgac 19 121 19 RNA Artificial Sequence Small
interfering RNA 121 gccagugacu ucccaugua 19 122 19 RNA Artificial
Sequence Small interfering RNA 122 ccgaaauagg uacagagac 19 123 19
RNA Artificial Sequence Small interfering RNA 123 auagguacag
agacgucag 19 124 19 RNA Artificial Sequence Small interfering RNA
124 uagguacaga gacgucagu 19 125 19 RNA Artificial Sequence Small
interfering RNA 125 aauggaagaa gcccaaagg 19 126 19 RNA Artificial
Sequence Small interfering RNA 126 auggaagaag cccaaagga 19 127 19
RNA Artificial Sequence Small interfering RNA 127 uggaagaagc
ccaaaggag 19 128 19 RNA Artificial Sequence Small interfering RNA
128 gaagcccaaa ggaguuaca 19 129 19 RNA Artificial Sequence Small
interfering RNA 129 gcccaaagga guuacauuc 19 130 19 RNA Artificial
Sequence Small interfering RNA 130 cacaugcggu cacuuuugg 19 131 19
RNA Artificial Sequence Small interfering RNA 131 cagagugaug
gagaaaggu 19 132 19 RNA Artificial Sequence Small interfering RNA
132 gguucguuaa aaugcgcac 19 133 19 RNA Artificial Sequence Small
interfering RNA 133 aaugcgcaca auacuggcc 19 134 19 RNA Artificial
Sequence Small interfering RNA 134 augcgcacaa uacuggcca 19 135 19
RNA Artificial Sequence Small interfering RNA 135 ugcgcacaau
acuggccac 19 136 19 RNA Artificial Sequence Small interfering RNA
136 cccaagaaac ucgagagau 19 137 19 RNA Artificial Sequence Small
interfering RNA 137 ucaccagccu cauucuuga 19 138 19 RNA Artificial
Sequence Small interfering RNA 138 ccuucugucu ggcugauac 19 139 19
RNA Artificial Sequence Small interfering RNA 139 gaggaaagac
ccuucuucc 19 140 19 RNA Artificial Sequence Small interfering RNA
140 agacccuucu uccguugau 19 141 19 RNA Artificial Sequence Small
interfering RNA 141 gacccuucuu ccguugaua 19 142 19 RNA Artificial
Sequence Small interfering RNA 142 augaggaagu uucggaugg 19 143 19
RNA Artificial Sequence Small interfering RNA 143 ggugccaaau
ucaucaugg 19 144 19 RNA Artificial Sequence Small interfering RNA
144 ggagcuuucc cacgaggac 19 145 19 RNA Artificial Sequence Small
interfering RNA 145 acgaauccug gagccacac 19 146 19 RNA Artificial
Sequence Small interfering RNA 146 cgaauccugg agccacaca 19 147 19
RNA Artificial Sequence Small interfering RNA 147 uccuggagcc
acacaaugg 19 148 19 RNA Artificial Sequence Small interfering RNA
148 ugggaaaugc agggaguuc 19 149 19 RNA Artificial Sequence Small
interfering RNA 149 ggaagagacc caggaggau 19 150 19 RNA Artificial
Sequence Small interfering RNA 150 gagacccagg aggauaaag 19 151 19
RNA Artificial Sequence Small interfering RNA 151 agacugcccc
aucaaggaa 19 152 19 RNA Artificial Sequence Small interfering RNA
152 gacugcccca ucaaggaag 19 153 19 RNA Artificial Sequence Small
interfering RNA 153 ggaagaaaaa ggaagcccc 19 154 19 RNA Artificial
Sequence Small interfering RNA 154 aaggaagccc cuuaaaugc 19 155 19
RNA Artificial Sequence Small interfering RNA 155 aggaagcccc
uuaaaugcc 19 156 19 RNA Artificial Sequence Small interfering RNA
156 ggaagccccu uaaaugccg 19 157 19 RNA Artificial Sequence Small
interfering RNA 157 gccccuuaaa ugccgcacc 19 158 19 RNA Artificial
Sequence Small interfering RNA 158 augccgcacc cuacggcau 19 159 19
RNA Artificial Sequence Small interfering RNA 159 agcaugaguc
aagacacug 19 160 19 RNA Artificial Sequence Small interfering RNA
160 gcaugaguca agacacuga 19 161 19 RNA Artificial Sequence Small
interfering RNA 161 ggacgaggac caugcacug 19 162 19 RNA Artificial
Sequence Small interfering RNA 162 gcccuuccug gucaacaug 19 163 19
RNA Artificial Sequence Small interfering RNA 163 caugugcgug
gcuacgguc 19 164 19 RNA Artificial Sequence Small interfering RNA
164 cagcaacaca uagccugac 19 165 19 RNA Artificial Sequence Small
interfering RNA 165 cacauagccu gacccuccu 19 166 19 RNA Artificial
Sequence Small interfering RNA 166 aacccaucuu ccccggaug 19 167 19
RNA Artificial Sequence Small interfering RNA 167 acccaucuuc
cccggaugu 19 168 19 RNA Artificial Sequence Small interfering RNA
168 cccaucuucc ccggaugug 19 169 19 RNA Artificial Sequence Small
interfering RNA 169 agagaguacc augcuggcg 19 170 19 RNA Artificial
Sequence Small interfering RNA 170 gagaguacca ugcuggcgg 19 171 19
RNA Artificial Sequence Small interfering RNA 171 cagccccccc
cuugaaucu 19 172 19 RNA Artificial Sequence Small interfering RNA
172 aggcauccau agugcacua 19 173 19 RNA Artificial Sequence Small
interfering RNA 173 ggcauccaua gugcacuag 19 174 19 RNA Artificial
Sequence Small interfering RNA 174 ggaggacggu uguaagcag 19 175 19
RNA Artificial Sequence Small interfering RNA 175 ucacugcucc
cccgugugu 19 176 19 RNA Artificial Sequence Small interfering RNA
176 ggucuucuug uguccugau 19 177 19 RNA Artificial Sequence Small
interfering RNA 177 ugugccccau guccaaguc 19 178 19 RNA Artificial
Sequence Small interfering RNA 178 guccaaccug ccugugcau 19 179 19
RNA Artificial Sequence Small interfering RNA 179 ccugccugug
caugaccug 19 180 19 RNA Artificial Sequence Small interfering RNA
180 gccuguugcu gaagucauu 19 181 19 RNA Artificial Sequence Small
interfering RNA 181 gucauugucg cucagcaau 19 182 19 RNA Artificial
Sequence Small interfering RNA 182 uuccuggcau gacacucua 19 183 19
RNA Artificial Sequence Small interfering RNA 183 gccauauuca
caccucacg 19 184 19 RNA Artificial Sequence Small interfering RNA
184 gucaacacuc uucuugagc 19 185 19 RNA Artificial Sequence Small
interfering RNA 185 cacucuucuu gagcagacc 19 186 19 RNA Artificial
Sequence Small interfering RNA 186 gagaggcacc ugcuggaaa 19 187 19
RNA Artificial Sequence Small interfering RNA 187 ccacacuucu
ugaaacagc 19 188 19 RNA Artificial Sequence Small interfering RNA
188 gaccuccaca uuaaguggc 19 189 19 RNA Artificial Sequence Small
interfering RNA 189 caugaaaaac acggcagcu 19 190 19 RNA Artificial
Sequence Small interfering RNA 190 aaacacggca gcuguagcu 19 191 19
RNA Artificial Sequence Small interfering RNA 191 aacacggcag
cuguagcuc 19 192 19 RNA Artificial Sequence Small interfering RNA
192 acacggcagc uguagcucc 19 193 19 RNA Artificial Sequence Small
interfering RNA 193 cauucgaggu gucacccug 19 194 19 RNA Artificial
Sequence Small interfering RNA 194 ggcuuaggug ccaggcugu 19 195 19
RNA Artificial Sequence Small interfering RNA 195 uggacguacu
gguuuaacc 19 196 19 RNA Artificial Sequence Small interfering RNA
196 ccuccuaucc uuggagagc 19 197 19 RNA Artificial Sequence Small
interfering RNA 197 ugaagaagca gcagcggcu 19 198 19 RNA Artificial
Sequence Small interfering RNA 198 ggauauccga caugaagcc 19 199 19
RNA Artificial Sequence Small interfering RNA 199 ugaagccagu
gacuuccca 19 200 19 RNA Artificial Sequence Small interfering RNA
200 gugacuuccc auguagagu 19 201 19 RNA Artificial Sequence Small
interfering RNA 201 uguagugugg ccaagcuuc 19 202 19 RNA Artificial
Sequence Small interfering RNA 202 gagacgucag ucccuuuga 19 203 19
RNA Artificial Sequence Small interfering RNA 203 gucccuuuga
ccauagucg 19 204 19 RNA Artificial Sequence Small interfering RNA
204 ugcucaacag agugaugga 19 205 19 RNA Artificial Sequence Small
interfering RNA 205 acagagugau ggagaaagg 19 206 19 RNA Artificial
Sequence Small interfering RNA 206 gagugaugga gaaagguuc 19 207 19
RNA Artificial Sequence Small interfering RNA 207 gugcgacagc
uagaauugg 19 208 19 RNA Artificial Sequence Small interfering RNA
208 acccaagaaa cucgagaga 19 209 19 RNA Artificial Sequence Small
interfering RNA 209 cuauaccaca uggccugac 19 210 19 RNA Artificial
Sequence Small interfering RNA 210 cauggccuga cuuuggagu 19 211 19
RNA Artificial Sequence Small interfering RNA 211 uggccugacu
uuggagucc 19 212 19 RNA Artificial Sequence Small interfering RNA
212 ccagccucau ucuugaacu 19 213 19 RNA Artificial Sequence Small
interfering RNA 213 ggcaucggca ggucuggaa 19 214 19 RNA Artificial
Sequence Small interfering RNA 214 ucggcagguc uggaaccuu 19 215 19
RNA Artificial Sequence Small interfering RNA 215 ggucuggaac
cuucugucu 19 216 19 RNA Artificial Sequence Small interfering RNA
216 agaggaaaga cccuucuuc
19 217 19 RNA Artificial Sequence Small interfering RNA 217
gcugcgcuuc uccuaccug 19 218 19 RNA Artificial Sequence Small
interfering RNA 218 ggaucagugg aaggagcuu 19 219 19 RNA Artificial
Sequence Small interfering RNA 219 guggaaggag cuuucccac 19 220 19
RNA Artificial Sequence Small interfering RNA 220 cccaaacgaa
uccuggagc 19 221 19 RNA Artificial Sequence Small interfering RNA
221 aacgaauccu ggagccaca 19 222 19 RNA Artificial Sequence Small
interfering RNA 222 cacaauggga aaugcaggg 19 223 19 RNA Artificial
Sequence Small interfering RNA 223 caaugggaaa ugcagggag 19 224 19
RNA Artificial Sequence Small interfering RNA 224 augggaaaug
cagggaguu 19 225 19 RNA Artificial Sequence Small interfering RNA
225 ggaggauaaa gacugcccc 19 226 19 RNA Artificial Sequence Small
interfering RNA 226 aggaagaaaa aggaagccc 19 227 19 RNA Artificial
Sequence Small interfering RNA 227 cccuacggca ucgaaagca 19 228 19
RNA Artificial Sequence Small interfering RNA 228 ugcacugagu
uacuggaag 19 229 19 RNA Artificial Sequence Small interfering RNA
229 cugaguuacu ggaagcccu 19 230 19 RNA Artificial Sequence Small
interfering RNA 230 acaugugcgu ggcuacggu 19 231 19 RNA Artificial
Sequence Small interfering RNA 231 ugugcguggc uacgguccu 19 232 19
RNA Artificial Sequence Small interfering RNA 232 gguuccuguu
caacagcaa 19 233 19 RNA Artificial Sequence Small interfering RNA
233 acagcaacac auagccuga 19 234 19 RNA Artificial Sequence Small
interfering RNA 234 gcaacacaua gccugaccc 19 235 19 RNA Artificial
Sequence Small interfering RNA 235 acacauagcc ugacccucc 19 236 19
RNA Artificial Sequence Small interfering RNA 236 cauagccuga
cccuccucc 19 237 19 RNA Artificial Sequence Small interfering RNA
237 uagccugacc cuccuccac 19 238 19 RNA Artificial Sequence Small
interfering RNA 238 cuccaccucc acccacugu 19 239 19 RNA Artificial
Sequence Small interfering RNA 239 ggcaugccgc gguagguaa 19 240 19
RNA Artificial Sequence Small interfering RNA 240 cuaaaaccca
ucuuccccg 19 241 19 RNA Artificial Sequence Small interfering RNA
241 ucuuccccgg auguguguc 19 242 19 RNA Artificial Sequence Small
interfering RNA 242 acagcccccc ccuugaauc 19 243 19 RNA Artificial
Sequence Small interfering RNA 243 aaggcaucca uagugcacu 19 244 19
RNA Artificial Sequence Small interfering RNA 244 aucacugcuc
ccccgugug 19 245 19 RNA Artificial Sequence Small interfering RNA
245 cugcuccccc guguguauu 19 246 19 RNA Artificial Sequence Small
interfering RNA 246 uguccaaguc caaccugcc 19 247 19 RNA Artificial
Sequence Small interfering RNA 247 aguccaaccu gccugugca 19 248 19
RNA Artificial Sequence Small interfering RNA 248 accugccugu
gcaugaccu 19 249 19 RNA Artificial Sequence Small interfering RNA
249 uuacauggcu gugguuccu 19 250 19 RNA Artificial Sequence Small
interfering RNA 250 uggcuguggu uccuaagcc 19 251 19 RNA Artificial
Sequence Small interfering RNA 251 ugacacucua gugacuucc 19 252 19
RNA Artificial Sequence Small interfering RNA 252 cucuagugac
uuccuggug 19 253 19 RNA Artificial Sequence Small interfering RNA
253 gccuguccug guacagcag 19 254 19 RNA Artificial Sequence Small
interfering RNA 254 uauucacacc ucacgcucu 19 255 19 RNA Artificial
Sequence Small interfering RNA 255 caccucacgc ucuggacau 19 256 19
RNA Artificial Sequence Small interfering RNA 256 ccucacgcuc
uggacauga 19 257 19 RNA Artificial Sequence Small interfering RNA
257 cgcucuggag augauuuag 19 258 19 RNA Artificial Sequence Small
interfering RNA 258 gccuccgcca uuccaaguc 19 259 19 RNA Artificial
Sequence Small interfering RNA 259 acacucuucu ugagcagac 19 260 19
RNA Artificial Sequence Small interfering RNA 260 cucuucuuga
gcagaccgu 19 261 19 RNA Artificial Sequence Small interfering RNA
261 gaccgugauu uggaagaga 19 262 19 RNA Artificial Sequence Small
interfering RNA 262 ccugcuggaa accacacuu 19 263 19 RNA Artificial
Sequence Small interfering RNA 263 cacuucuuga aacagccug 19 264 19
RNA Artificial Sequence Small interfering RNA 264 ugaaaaacac
ggcagcugu 19 265 19 RNA Artificial Sequence Small interfering RNA
265 gcuguagcuc ccgagcuac 19 266 19 RNA Artificial Sequence Small
interfering RNA 266 cauuuugccu uucucgugg 19 267 19 RNA Artificial
Sequence Small interfering RNA 267 uucgaggugu cacccugca 19 268 19
RNA Artificial Sequence Small interfering RNA 268 cccugcagag
cuaugguga 19 269 19 RNA Artificial Sequence Small interfering RNA
269 gagcuauggu gaggugugg 19 270 19 RNA Artificial Sequence Small
interfering RNA 270 ggcuguaagc auucugagc 19 271 19 RNA Artificial
Sequence Small interfering RNA 271 gcuggcucuc caccuuguu 19 272 19
RNA Artificial Sequence Small interfering RNA 272 ccaggauauc
cgacaugaa 19 273 19 RNA Artificial Sequence Small interfering RNA
273 uccgacauga agccaguga 19 274 19 RNA Artificial Sequence Small
interfering RNA 274 aaaccgaaau agguacaga 19 275 19 RNA Artificial
Sequence Small interfering RNA 275 gagacgucag ucccuuuga 19 276 19
RNA Artificial Sequence Small interfering RNA 276 uuaacauuga
ucucugaag 19 277 19 RNA Artificial Sequence Small interfering RNA
277 uuauacagug cgacagcua 19 278 19 RNA Artificial Sequence Small
interfering RNA 278 gugcgacagc uagaauugg 19 279 19 RNA Artificial
Sequence Small interfering RNA 279 agaaacucga gagaucuua 19 280 19
RNA Artificial Sequence Small interfering RNA 280 gaaacucgag
agaucuuac 19 281 19 RNA Artificial Sequence Small interfering RNA
281 aacucgagag aucuuacau 19 282 19 RNA Artificial Sequence Small
interfering RNA 282 acucgagaga ucuuacauu 19 283 19 RNA Artificial
Sequence Small interfering RNA 283 cucgagagau cuuacauuu 19 284 19
RNA Artificial Sequence Small interfering RNA 284 ucuuacauuu
ccacuauac 19 285 19 RNA Artificial Sequence Small interfering RNA
285 ggcaucggca ggucuggaa 19 286 19 RNA Artificial Sequence Small
interfering RNA 286 agacccuucu uccguugau 19 287 19 RNA Artificial
Sequence Small interfering RNA 287 gacccuucuu ccguugaua 19 288 19
RNA Artificial Sequence Small interfering RNA 288 cccuucuucc
guugauauc 19 289 19 RNA Artificial Sequence Small interfering RNA
289 agaaagugcu guuagaaau 19 290 19 RNA Artificial Sequence Small
interfering RNA 290 gaaagugcug uuagaaaug 19 291 19 RNA Artificial
Sequence Small interfering RNA 291 cgaauccugg agccacaca 19 292 19
RNA Artificial Sequence Small interfering RNA 292 ugagucaaga
cacugaagu 19 293 19 RNA Artificial Sequence Small interfering RNA
293 gaaggacgag gaccaugca 19 294 19 RNA Artificial Sequence Small
interfering RNA 294 auaaauccuc agguaguac 19 295 19 RNA Artificial
Sequence Small interfering RNA 295 uaaauccuca gguaguacu 19 296 19
RNA Artificial Sequence Small interfering RNA 296 gguaguacug
ggaauggaa 19 297 19 RNA Artificial Sequence Small interfering RNA
297 aggaggacgg uuguaagca 19 298 19 RNA Artificial Sequence Small
interfering RNA 298 ggacgguugu aagcaguug 19 299 19 RNA Artificial
Sequence Small interfering RNA 299 uauugugggu aacgugaga 19 300 19
RNA Artificial Sequence Small interfering RNA 300 augaacacgu
ggguauuua 19 301 19 RNA Artificial Sequence Small interfering RNA
301 caugauguga gauuacuuu 19 302 19 RNA Artificial Sequence Small
interfering RNA 302 gauuacuuug ucccgcuua 19 303 19 RNA Artificial
Sequence Small interfering RNA 303 uuacuuuguc ccgcuuauu 19 304 19
RNA Artificial Sequence Small interfering RNA 304 gaucuaguuc
ucaaucacu 19 305 19 RNA Artificial Sequence Small interfering RNA
305 gaaugcaugu aaggucuuc 19 306 19 RNA Artificial Sequence Small
interfering RNA 306 ugcauguaag gucuucuug 19 307 18 RNA Artificial
Sequence Small interfering RNA 307 ucauuacaug gcuguggu 18 308 19
RNA Artificial Sequence Small interfering RNA 308 uuccuggcau
gacacucua 19 309 19 RNA Artificial Sequence Small interfering RNA
309 cgcucuggac augauuuag 19 310 19 RNA Artificial Sequence Small
interfering RNA 310 ucagccuccg ccauuccaa 19 311 19 RNA Artificial
Sequence Small interfering RNA 311 gccuccgcca uuccaaguc 19 312 19
RNA Artificial Sequence Small interfering RNA 312 gaccgugauu
uggaagaga 19 313 19 RNA Artificial Sequence Small interfering RNA
313 acugaagacc uccacauua 19 314 19 RNA Artificial Sequence Small
interfering RNA 314 gcuacucucu ugccagcau 19 315 19 RNA Artificial
Sequence Small interfering RNA 315 uggugaggug uggauaagg 19 316 19
RNA Artificial Sequence Small interfering RNA 316 ggugccaggc
uguaagcau 19 317 19 RNA Artificial Sequence Small interfering RNA
317 uaugccuuaa gccaauauu 19 318 19 RNA Artificial Sequence Small
interfering RNA 318 uuuacucauc aggucauua 19 319 19 RNA Artificial
Sequence Small interfering RNA 319 ccaaacggac aacccauag 19 320 19
RNA Artificial Sequence Small interfering RNA 320 acggacaacc
cauaguacc 19 321 19 RNA Artificial Sequence Small interfering RNA
321 cggacaaccc auaguaccc 19 322 19 RNA Artificial Sequence Small
interfering RNA 322 cccauaguac ccgaagaca 19 323 19 RNA Artificial
Sequence Small interfering RNA 323 ccagacaauc guaagcuug 19 324 19
RNA Artificial Sequence Small interfering RNA 324 gcuugauggu
guuuucccu 19 325 19 RNA Artificial Sequence Small interfering RNA
325 gcaucucaug aaugucagc 19 326 19 RNA Artificial Sequence Small
interfering RNA 326 ugucagccaa auuccguac 19 327 19 RNA Artificial
Sequence Small interfering RNA 327 auuccguaca guucggugc 19 328 19
RNA Artificial Sequence Small interfering RNA 328 uuccguacag
uucggugcg 19 329 19 RNA Artificial Sequence Small interfering RNA
329 cgaaacaccu ccuguacca 19 330 19 RNA Artificial Sequence Small
interfering RNA 330 acaccuccug uaccagguu 19 331 19 RNA Artificial
Sequence Small interfering RNA 331 caccuccugu accagguuc 19 332 19
RNA Artificial Sequence Small interfering RNA 332 cuucagaauc
auccaggcu 19 333 19 RNA Artificial Sequence Small interfering RNA
333 ucauccaggc uucaucaug 19 334 19 RNA Artificial Sequence Small
interfering RNA 334 ggugagagcc accacagag 19 335 19 RNA Artificial
Sequence Small interfering RNA 335 cugguaggcu gaacccaug 19 336 19
RNA Artificial Sequence Small interfering RNA 336 cccaugcuga
agcuccacc 19 337 19 RNA Artificial Sequence Small interfering RNA
337 caugcagaag ccgcugcug 19 338 19 RNA Artificial Sequence Small
interfering RNA 338 ggaguucgag gagaucgac 19 339 19 RNA Artificial
Sequence Small interfering RNA 339 gccagcgacu ucccaugca 19 340 19
RNA Artificial Sequence Small interfering RNA 340 agucgcgaag
cuuccuaag 19 341 19 RNA Artificial Sequence Small interfering RNA
341 gucgcgaagc uuccuaaga 19 342 19 RNA Artificial Sequence Small
interfering RNA 342 gaacaaaaac cggaacagg 19 343 19 RNA Artificial
Sequence Small interfering RNA 343 caaaaaccgg aacagguac 19 344 19
RNA Artificial Sequence Small interfering RNA 344 aaaccggaac
agguaccga 19 345 19 RNA Artificial Sequence Small interfering RNA
345 aaccggaaca gguaccgag 19 346 19 RNA Artificial Sequence Small
interfering RNA 346 accggaacag guaccgaga 19 347 19 RNA Artificial
Sequence Small interfering RNA 347 ccggaacagg uaccgagau 19 348 19
RNA Artificial Sequence Small interfering RNA 348 cagguaccga
gaugucagc 19 349 19 RNA Artificial Sequence Small interfering RNA
349 aaauggaaga agcccagag 19 350 19 RNA Artificial Sequence Small
interfering RNA 350 aauggaagaa gcccagagg 19 351 19 RNA Artificial
Sequence Small interfering RNA 351 auggaagaag cccagagga 19 352 19
RNA Artificial Sequence Small interfering RNA 352 uggaagaagc
ccagaggag 19 353 19 RNA Artificial Sequence Small interfering RNA
353 gaagcccaga ggagcuaua 19 354 19 RNA Artificial Sequence Small
interfering RNA 354 gcccagagga gcuauauuc 19 355 19 RNA Artificial
Sequence Small interfering RNA 355 ccgcaucaug gagaaaggc 19 356 19
RNA Artificial Sequence Small
interfering RNA 356 aggcucguua aaaugugcc 19 357 19 RNA Artificial
Sequence Small interfering RNA 357 ggcucguuaa aaugugccc 19 358 19
RNA Artificial Sequence Small interfering RNA 358 aaugugccca
guauuggcc 19 359 19 RNA Artificial Sequence Small interfering RNA
359 augugcccag uauuggcca 19 360 19 RNA Artificial Sequence Small
interfering RNA 360 ugugcccagu auuggccac 19 361 19 RNA Artificial
Sequence Small interfering RNA 361 ggagaugguc uuugaugac 19 362 19
RNA Artificial Sequence Small interfering RNA 362 aaccugacua
ccaaggaga 19 363 19 RNA Artificial Sequence Small interfering RNA
363 accugacuac caaggagac 19 364 19 RNA Artificial Sequence Small
interfering RNA 364 ccugacuacc aaggagacu 19 365 19 RNA Artificial
Sequence Small interfering RNA 365 ggagacucga gagauccug 19 366 19
RNA Artificial Sequence Small interfering RNA 366 aguccgagag
ucaggcuca 19 367 19 RNA Artificial Sequence Small interfering RNA
367 guccgagagu caggcucac 19 368 19 RNA Artificial Sequence Small
interfering RNA 368 gaggaaagac ccaucuucc 19 369 19 RNA Artificial
Sequence Small interfering RNA 369 agacccaucu uccguggac 19 370 19
RNA Artificial Sequence Small interfering RNA 370 gacccaucuu
ccguggaca 19 371 19 RNA Artificial Sequence Small interfering RNA
371 gaaaguacug cuggagaug 19 372 19 RNA Artificial Sequence Small
interfering RNA 372 aguacugcug gagaugcgc 19 373 19 RNA Artificial
Sequence Small interfering RNA 373 guacugcugg agaugcgca 19 374 19
RNA Artificial Sequence Small interfering RNA 374 acgcacacug
gagccucac 19 375 19 RNA Artificial Sequence Small interfering RNA
375 cgcacacugg agccucaca 19 376 19 RNA Artificial Sequence Small
interfering RNA 376 gugcaaggag cucuucucc 19 377 19 RNA Artificial
Sequence Small interfering RNA 377 ggagcucuuc uccagccac 19 378 19
RNA Artificial Sequence Small interfering RNA 378 ggcagagccc
agucaagug 19 379 19 RNA Artificial Sequence Small interfering RNA
379 gugccaugca cagcgugag 19 380 19 RNA Artificial Sequence Small
interfering RNA 380 guuaggagac ggauggugg 19 381 19 RNA Artificial
Sequence Small interfering RNA 381 agugcucagg cgucugucc 19 382 19
RNA Artificial Sequence Small interfering RNA 382 gagcuguccu
ccacugagg 19 383 19 RNA Artificial Sequence Small interfering RNA
383 cacaaggcac auuggccaa 19 384 19 RNA Artificial Sequence Small
interfering RNA 384 ggcacauugg ccaagucac 19 385 19 RNA Artificial
Sequence Small interfering RNA 385 gucacuggaa gcccuuccu 19 386 19
RNA Artificial Sequence Small interfering RNA 386 gcccuuccug
gucaaugug 19 387 19 RNA Artificial Sequence Small interfering RNA
387 ugugugcaug gccacgcuc 19 388 19 RNA Artificial Sequence Small
interfering RNA 388 caacaacucg caagccugc 19 389 19 RNA Artificial
Sequence Small interfering RNA 389 caacucgcaa gccugcucu 19 390 19
RNA Artificial Sequence Small interfering RNA 390 cucgcaagcc
ugcucugga 19 391 19 RNA Artificial Sequence Small interfering RNA
391 gccugcucug gaacuggaa 19 392 19 RNA Artificial Sequence Small
interfering RNA 392 ccuguucagg agaaguaga 19 393 19 RNA Artificial
Sequence Small interfering RNA 393 uacucuucuu gcucucacc 19 394 19
RNA Artificial Sequence Small interfering RNA 394 cauuuauaaa
ggcaggccc 19 395 19 RNA Artificial Sequence Small interfering RNA
395 cgggaagugc aaggagcuc 19 396 19 RNA Artificial Sequence Small
interfering RNA 396 ugacuauauc aaugccagc 19 397 19 RNA Artificial
Sequence Small interfering RNA 397 cauuccuagu uagcagugc 19 398 19
RNA Artificial Sequence Small interfering RNA 398 gugcauacuc
aucagacug 19 399 19 RNA Artificial Sequence Small interfering RNA
399 aacggacaac ccauaguac 19 400 19 RNA Artificial Sequence Small
interfering RNA 400 acccauagua cccgaagac 19 401 19 RNA Artificial
Sequence Small interfering RNA 401 gccaaauucc guacaguuc 19 402 19
RNA Artificial Sequence Small interfering RNA 402 aauuccguac
aguucggug 19 403 19 RNA Artificial Sequence Small interfering RNA
403 guucggugcg gauccgaac 19 404 19 RNA Artificial Sequence Small
interfering RNA 404 ccuccuguac cagguuccc 19 405 19 RNA Artificial
Sequence Small interfering RNA 405 gguucccgug ucgcucuca 19 406 19
RNA Artificial Sequence Small interfering RNA 406 gaaucaucca
ggcuucauc 19 407 19 RNA Artificial Sequence Small interfering RNA
407 ggcuucauca uguuuuccc 19 408 19 RNA Artificial Sequence Small
interfering RNA 408 ucauguuuuc ccaccucca 19 409 19 RNA Artificial
Sequence Small interfering RNA 409 uguuuuccca ccuccagca 19 410 19
RNA Artificial Sequence Small interfering RNA 410 ccuccagcaa
gaaccgagg 19 411 19 RNA Artificial Sequence Small interfering RNA
411 ugaaggugag agccaccac 19 412 19 RNA Artificial Sequence Small
interfering RNA 412 ccacagagga gacgcaugg 19 413 19 RNA Artificial
Sequence Small interfering RNA 413 cagacgauga cgaagacgc 19 414 19
RNA Artificial Sequence Small interfering RNA 414 gacgaugacg
aagacgcgc 19 415 19 RNA Artificial Sequence Small interfering RNA
415 cguguggaac ugguaggcu 19 416 19 RNA Artificial Sequence Small
interfering RNA 416 ugcugaagcu ccacccgua 19 417 19 RNA Artificial
Sequence Small interfering RNA 417 ggcauggcgg aggcuagau 19 418 19
RNA Artificial Sequence Small interfering RNA 418 uccagaacau
gcagaagcc 19 419 19 RNA Artificial Sequence Small interfering RNA
419 gaacaugcag aagccgcug 19 420 19 RNA Artificial Sequence Small
interfering RNA 420 uggagaugga gaaggaguu 19 421 19 RNA Artificial
Sequence Small interfering RNA 421 ggacauucga caugaagcc 19 422 19
RNA Artificial Sequence Small interfering RNA 422 uucgacauga
agccagcga 19 423 19 RNA Artificial Sequence Small interfering RNA
423 ugaagccagc gacuuccca 19 424 19 RNA Artificial Sequence Small
interfering RNA 424 gcgacuuccc augcaaagu 19 425 19 RNA Artificial
Sequence Small interfering RNA 425 ugcaaagucg cgaagcuuc 19 426 19
RNA Artificial Sequence Small interfering RNA 426 aagucgcgaa
gcuuccuaa 19 427 19 RNA Artificial Sequence Small interfering RNA
427 aaaaccggaa cagguaccg 19 428 19 RNA Artificial Sequence Small
interfering RNA 428 gguaccgaga ugucagccc 19 429 19 RNA Artificial
Sequence Small interfering RNA 429 gcccuuuuga ccacagucg 19 430 19
RNA Artificial Sequence Small interfering RNA 430 gaggagcuau
auucucacc 19 431 19 RNA Artificial Sequence Small interfering RNA
431 ugcucaaccg caucaugga 19 432 19 RNA Artificial Sequence Small
interfering RNA 432 accgcaucau ggagaaagg 19 433 19 RNA Artificial
Sequence Small interfering RNA 433 ucauggagaa aggcucguu 19 434 19
RNA Artificial Sequence Small interfering RNA 434 guauuggcca
cagcaagaa 19 435 19 RNA Artificial Sequence Small interfering RNA
435 caguacgaca guuggaguu 19 436 19 RNA Artificial Sequence Small
interfering RNA 436 guacgacagu uggaguugg 19 437 19 RNA Artificial
Sequence Small interfering RNA 437 guuggaguug gaaaaccug 19 438 19
RNA Artificial Sequence Small interfering RNA 438 aggagacucg
agagauccu 19 439 19 RNA Artificial Sequence Small interfering RNA
439 uuuccacuac accacaugg 19 440 19 RNA Artificial Sequence Small
interfering RNA 440 cuacaccaca uggccugac 19 441 19 RNA Artificial
Sequence Small interfering RNA 441 ccacauggcc ugacuuugg 19 442 19
RNA Artificial Sequence Small interfering RNA 442 cauggccuga
cuuuggagu 19 443 19 RNA Artificial Sequence Small interfering RNA
443 uggccugacu uuggagucc 19 444 19 RNA Artificial Sequence Small
interfering RNA 444 ccggcuucuu uccucaauu 19 445 19 RNA Artificial
Sequence Small interfering RNA 445 aaguccgaga gucaggcuc 19 446 19
RNA Artificial Sequence Small interfering RNA 446 uggccccauu
gugguccac 19 447 19 RNA Artificial Sequence Small interfering RNA
447 uuguggucca cugcagcgc 19 448 19 RNA Artificial Sequence Small
interfering RNA 448 ccugccucuu acugaugga 19 449 19 RNA Artificial
Sequence Small interfering RNA 449 agaggaaaga cccaucuuc 19 450 19
RNA Artificial Sequence Small interfering RNA 450 ucuuccgugg
acaucaaga 19 451 19 RNA Artificial Sequence Small interfering RNA
451 uccagacugc cgaccagcu 19 452 19 RNA Artificial Sequence Small
interfering RNA 452 gcugcgcuuc uccuaccug 19 453 19 RNA Artificial
Sequence Small interfering RNA 453 gugcaggauc aguggaagg 19 454 19
RNA Artificial Sequence Small interfering RNA 454 ggaucagugg
aaggagcuc 19 455 19 RNA Artificial Sequence Small interfering RNA
455 cccaaacgca cacuggagc 19 456 19 RNA Artificial Sequence Small
interfering RNA 456 aacgcacacu ggagccuca 19 457 19 RNA Artificial
Sequence Small interfering RNA 457 cacuggagcc ucacaacgg 19 458 19
RNA Artificial Sequence Small interfering RNA 458 aggagcucuu
cuccagcca 19 459 19 RNA Artificial Sequence Small interfering RNA
459 gagaggaagg cagagccca 19 460 19 RNA Artificial Sequence Small
interfering RNA 460 gagcccaguc aagugccau 19 461 19 RNA Artificial
Sequence Small interfering RNA 461 gucaagugcc augcacagc 19 462 19
RNA Artificial Sequence Small interfering RNA 462 agugccaugc
acagcguga 19 463 19 RNA Artificial Sequence Small interfering RNA
463 ugcacagcgu gagcagcau 19 464 19 RNA Artificial Sequence Small
interfering RNA 464 cagcgugagc agcaugagu 19 465 19 RNA Artificial
Sequence Small interfering RNA 465 gcgugagcag caugagucc 19 466 19
RNA Artificial Sequence Small interfering RNA 466 gcaugagucc
agacacuga 19 467 19 RNA Artificial Sequence Small interfering RNA
467 ugaguccaga cacugaagu 19 468 19 RNA Artificial Sequence Small
interfering RNA 468 gacacugaag uuaggagac 19 469 19 RNA Artificial
Sequence Small interfering RNA 469 cugaaguuag gagacggau 19 470 19
RNA Artificial Sequence Small interfering RNA 470 aagugcucag
gcgucuguc 19 471 19 RNA Artificial Sequence Small interfering RNA
471 ccgaggaaga gcuguccuc 19 472 19 RNA Artificial Sequence Small
interfering RNA 472 cugaggagga acacaaggc 19 473 19 RNA Artificial
Sequence Small interfering RNA 473 caaggcacau uggccaagu 19 474 19
RNA Artificial Sequence Small interfering RNA 474 aggcacauug
gccaaguca 19 475 19 RNA Artificial Sequence Small interfering RNA
475 cauuggccaa gucacugga 19 476 19 RNA Artificial Sequence Small
interfering RNA 476 uuggccaagu cacuggaag 19 477 19 RNA Artificial
Sequence Small interfering RNA 477 agucacugga agcccuucc 19 478 19
RNA Artificial Sequence Small interfering RNA 478 cuggaagccc
uuccugguc 19 479 19 RNA Artificial Sequence Small interfering RNA
479 augugugcau ggccacgcu 19 480 19 RNA Artificial Sequence Small
interfering RNA 480 ccggcgcgua cuugugcua 19 481 19 RNA Artificial
Sequence Small interfering RNA 481 cugccacugc ccagcuuag 19 482 19
RNA Artificial Sequence Small interfering RNA 482 cugcccagcu
uaggaugcg 19 483 19 RNA Artificial Sequence Small interfering RNA
483 gcuuaggaug cggucugcg 19 484 19 RNA Artificial Sequence Small
interfering RNA 484 acaacucgca agccugcuc 19 485 19 RNA Artificial
Sequence Small interfering RNA 485 acucgcaagc cugcucugg 19 486 19
RNA Artificial Sequence Small interfering RNA 486 agccugcucu
ggaacugga 19 487 19 RNA Artificial Sequence Small interfering RNA
487 ggagaaguag aggaaaugc 19 488 19 RNA Artificial Sequence Small
interfering RNA 488 ccucacuccu ccccuuucu 19 489 19 RNA Artificial
Sequence Small interfering RNA 489 cuccuccccu uucucugau 19 490 19
RNA Artificial Sequence Small interfering RNA 490 uuuauaaagg
caggcccga 19 491 19 RNA Artificial Sequence Small interfering RNA
491 gguaccgaga ugucagccc 19 492 19 RNA Artificial Sequence Small
interfering RNA 492 agaagaaaag gagaugguc 19 493 19 RNA Artificial
Sequence Small interfering RNA 493 gacugccgac cagcugcgc 19 494 19
RNA Artificial Sequence Small interfering RNA 494 accaaacgga
caacccaua 19 495 19 RNA Artificial Sequence Small interfering RNA
495 ccaaacggac aacccauag
19 496 19 RNA Artificial Sequence Small interfering RNA 496
accagacaau cguaagcuu 19 497 19 RNA Artificial Sequence Small
interfering RNA 497 gacaaucgua agcuugaug 19 498 19 RNA Artificial
Sequence Small interfering RNA 498 caaucguaag cuugauggu 19 499 19
RNA Artificial Sequence Small interfering RNA 499 ucguaagcuu
gaugguguu 19 500 19 RNA Artificial Sequence Small interfering RNA
500 guugaagcau cucaugaau 19 501 19 RNA Artificial Sequence Small
interfering RNA 501 gccaaauucc guacaguuc 19 502 19 RNA Artificial
Sequence Small interfering RNA 502 gguucccgug ucgcucuca 19 503 19
RNA Artificial Sequence Small interfering RNA 503 ggcugaaccc
augcugaag 19 504 19 RNA Artificial Sequence Small interfering RNA
504 ggcauggcgg aggcuagau 19 505 19 RNA Artificial Sequence Small
interfering RNA 505 ggcuagaugc cgccaauca 19 506 19 RNA Artificial
Sequence Small interfering RNA 506 ucgacaaggc ugggaacug 19 507 19
RNA Artificial Sequence Small interfering RNA 507 aagucgcgaa
gcuuccuaa 19 508 19 RNA Artificial Sequence Small interfering RNA
508 agucgcgaag cuuccuaag 19 509 19 RNA Artificial Sequence Small
interfering RNA 509 gucgcgaagc uuccuaaga 19 510 19 RNA Artificial
Sequence Small interfering RNA 510 ccggaacagg uaccgagau 19 511 19
RNA Artificial Sequence Small interfering RNA 511 ccacagucgg
auuaaauug 19 512 19 RNA Artificial Sequence Small interfering RNA
512 aauugcacca ggaagauaa 19 513 19 RNA Artificial Sequence Small
interfering RNA 513 auugcaccag gaagauaau 19 514 19 RNA Artificial
Sequence Small interfering RNA 514 gaagcccaga ggagcuaua 19 515 19
RNA Artificial Sequence Small interfering RNA 515 agcccagagg
agcuauauu 19 516 19 RNA Artificial Sequence Small interfering RNA
516 ugcucaaccg caucaugga 19 517 19 RNA Artificial Sequence Small
interfering RNA 517 accgcaucau ggagaaagg 19 518 19 RNA Artificial
Sequence Small interfering RNA 518 ucauggagaa aggcucguu 19 519 19
RNA Artificial Sequence Small interfering RNA 519 uggagaaagg
cucguuaaa 19 520 19 RNA Artificial Sequence Small interfering RNA
520 gauggucuuu gaugacaca 19 521 19 RNA Artificial Sequence Small
interfering RNA 521 gguuugaagu ugacacuaa 19 522 19 RNA Artificial
Sequence Small interfering RNA 522 ucucugaaga ugucaaguc 19 523 19
RNA Artificial Sequence Small interfering RNA 523 gucauauuac
acaguacga 19 524 19 RNA Artificial Sequence Small interfering RNA
524 caguacgaca guuggaguu 19 525 19 RNA Artificial Sequence Small
interfering RNA 525 ccugacuacc aaggagacu 19 526 19 RNA Artificial
Sequence Small interfering RNA 526 gacucgagag auccugcau 19 527 19
RNA Artificial Sequence Small interfering RNA 527 uccugcauuu
ccacuacac 19 528 19 RNA Artificial Sequence Small interfering RNA
528 cugauggaca agaggaaag 19 529 19 RNA Artificial Sequence Small
interfering RNA 529 cccaucuucc guggacauc 19 530 19 RNA Artificial
Sequence Small interfering RNA 530 ucaugggcga cucgucagu 19 531 19
RNA Artificial Sequence Small interfering RNA 531 cucgucagug
caggaucag 19 532 19 RNA Artificial Sequence Small interfering RNA
532 cgcacacugg agccucaca 19 533 19 RNA Artificial Sequence Small
interfering RNA 533 cagcgugagc agcaugagu 19 534 19 RNA Artificial
Sequence Small interfering RNA 534 gcaugagucc agacacuga 19 535 19
RNA Artificial Sequence Small interfering RNA 535 uaaaggcagg
cccgaauuc 19 536 19 RNA Artificial Sequence Small interefering RNA
536 ucgauaaggc ugggaacug 19 537 19 RNA Artificial Sequence Small
interefering RNA 537 gaauagcgaa acuuccuaa 19 538 19 RNA Artificial
Sequence Small interefering RNA 538 auagcgaaac uuccuaaga 19 539 19
RNA Artificial Sequence Small interefering RNA 539 ccacagucgg
auuaaauug 19 540 19 RNA Artificial Sequence Small interefering RNA
540 cagucggauu aaauugcau 19 541 19 RNA Artificial Sequence Small
interefering RNA 541 gucggauuaa auugcauca 19 542 19 RNA Artificial
Sequence Small interefering RNA 542 uugcaucagg aagauaaug 19 543 19
RNA Artificial Sequence Small interefering RNA 543 gcccagagga
gcuauaucc 19 544 19 RNA Artificial Sequence Small interefering RNA
544 uccucaccca gggcccuuu 19 545 19 RNA Artificial Sequence Small
interefering RNA 545 accgcaucau ggagaaagg 19 546 19 RNA Artificial
Sequence Small interefering RNA 546 ucauggagaa aggcucguu 19 547 19
RNA Artificial Sequence Small interefering RNA 547 uggagaaagg
cucguuaaa 19 548 19 RNA Artificial Sequence Small interefering RNA
548 guauuggcca cagaaagaa 19 549 19 RNA Artificial Sequence Small
interefering RNA 549 agagaugguc uucgaugac 19 550 19 RNA Artificial
Sequence Small interefering RNA 550 caccaauuug aagcugaca 19 551 19
RNA Artificial Sequence Small interefering RNA 551 ccaauuugaa
gcugacacu 19 552 19 RNA Artificial Sequence Small interefering RNA
552 uuacacagua cggcaguug 19 553 19 RNA Artificial Sequence Small
interefering RNA 553 caguacggca guuggaguu 19 554 19 RNA Artificial
Sequence Small interefering RNA 554 ggcucgagag auccugcau 19 555 19
RNA Artificial Sequence Small interefering RNA 555 cugauggaca
agaggaaag 19 556 19 RNA Artificial Sequence Small interefering RNA
556 caucaagaaa gugcuguug 19 557 19 RNA Artificial Sequence Small
interefering RNA 557 gggugcaaag uucaucaug 19 558 19 RNA Artificial
Sequence Small interefering RNA 558 ucaugggcga cucgucagu 19 559 19
RNA Artificial Sequence Small interefering RNA 559 cucgucagug
caggaucag 19 560 19 RNA Artificial Sequence Small interefering RNA
560 cgcacauugg agccucaca 19 561 19 RNA Artificial Sequence Small
interefering RNA 561 agugcaagga gcucuucuc 19 562 19 RNA Artificial
Sequence Small interefering RNA 562 gcaugagcag uaugaguca 19 563 19
RNA Artificial Sequence Small interefering RNA 563 guaugaguca
agacacuga 19 564 19 RNA Artificial Sequence Small interefering RNA
564 gucaagacac ugaaguuag 19 565 19 RNA Artificial Sequence Small
interefering RNA 565 uggugggugg aggucuuca 19 566 19 RNA Artificial
Sequence Small interefering RNA 566 aaggcacaca ggccaguuc 19 567 19
RNA Artificial Sequence Small interefering RNA 567 aggcacacag
gccaguuca 19 568 19 RNA Artificial Sequence Small interefering RNA
568 ggcacacagg ccaguucac 19 569 19 RNA Artificial Sequence Small
interefering RNA 569 agcccuuccu ggucaacgu 19 570 19 RNA Artificial
Sequence Small interefering RNA 570 uuuggucugc ggcgucuaa 19 571 19
RNA Artificial Sequence Small interefering RNA 571 gaagaaacaa
cagcuuaca 19 572 19 RNA Artificial Sequence Small interefering RNA
572 agaaacaaca gcuuacaag 19 573 19 RNA Artificial Sequence Small
interefering RNA 573 gucuaaucuc agggccuua 19 574 19 RNA Artificial
Sequence Small interefering RNA 574 augccaaaua cucuucuug 19 575 19
RNA Artificial Sequence Small interefering RNA 575 ucagauucac
gauuuacgu 19 576 19 RNA Artificial Sequence Small interefering RNA
576 gccacuccac ugaggugua 19 577 19 RNA Artificial Sequence Small
interefering RNA 577 cuccacugag guguaaagc 19 578 19 RNA Artificial
Sequence Small interefering RNA 578 gccuuggugu cauggaagu 19 579 19
RNA Artificial Sequence Small interefering RNA 579 acaaccucug
aaacacuca 19 580 19 RNA Artificial Sequence Small interefering RNA
580 gucuggacuc augaaacac 19 581 19 RNA Artificial Sequence Small
interefering RNA 581 aacaccgccg agcgcuuac 19 582 19 RNA Artificial
Sequence Small interefering RNA 582 acaccgccga gcgcuuacu 19 583 19
RNA Artificial Sequence Small interefering RNA 583 gccgcuccac
uguuauuua 19 584 19 RNA Artificial Sequence Small interefering RNA
584 uucacuuugc ccacagaca 19 585 19 RNA Artificial Sequence Small
interefering RNA 585 cagacaacag uggugacau 19 586 19 RNA Artificial
Sequence Small interefering RNA 586 gacaacagug gugacaugu 19 587 19
RNA Artificial Sequence Small interefering RNA 587 acagugguga
cauguaaag 19 588 19 RNA Artificial Sequence Small interefering RNA
588 cugaugacau guguaggau 19 589 19 RNA Artificial Sequence Small
interefering RNA 589 cucccggcag gacucuuca 19 590 19 RNA Artificial
Sequence Small interefering RNA 590 ccucauuccc uggacacuu 19 591 19
RNA Artificial Sequence Small interefering RNA 591 ccagucaccu
ugcucagaa 19 592 19 RNA Artificial Sequence Small interefering RNA
592 gucaccuugc ucagaagug 19 593 19 RNA Artificial Sequence Small
interefering RNA 593 uaagcgaagg cagcuggaa 19 594 19 RNA Artificial
Sequence Small interefering RNA 594 aagcugcugc augccuuaa 19 595 19
RNA Artificial Sequence Small interefering RNA 595 agcugcugca
ugccuuaag 19 596 19 RNA Artificial Sequence Small interefering RNA
596 caacaaagug cucuggaau 19 597 19 RNA Artificial Sequence Small
interefering RNA 597 cuguccgacu gcaccguuu 19 598 19 RNA Artificial
Sequence Small interefering RNA 598 cugcaccguu uccaacuug 19 599 19
RNA Artificial Sequence Small interefering RNA 599 acuugugucu
cacuaaugg 19
* * * * *
References