U.S. patent application number 10/940892 was filed with the patent office on 2005-11-17 for methods and compositions for selecting sirna of improved functionality.
This patent application is currently assigned to Dharmacon, Inc.. Invention is credited to Khvorova, Anastasia, Leake, Devin, Marshall, William, Read, Steven, Reynolds, Angela, Scaringe, Stephen.
Application Number | 20050255487 10/940892 |
Document ID | / |
Family ID | 32329096 |
Filed Date | 2005-11-17 |
United States Patent
Application |
20050255487 |
Kind Code |
A1 |
Khvorova, Anastasia ; et
al. |
November 17, 2005 |
Methods and compositions for selecting siRNA of improved
functionality
Abstract
Efficient sequence specific gene silencing is possible through
the use of siRNA technology. By selecting particular siRNAs by
rational design, one can maximize the generation of an effective
gene silencing reagent, as well as methods for silencing genes.
Methods, compositions, and kits generated through rational design
of siRNAs are disclosed.
Inventors: |
Khvorova, Anastasia;
(Boulder, CO) ; Reynolds, Angela; (Conifer,
CO) ; Leake, Devin; (Boulder, CO) ; Marshall,
William; (Boulder, CO) ; Read, Steven;
(Denver, CO) ; Scaringe, Stephen; (Lafayette,
CO) |
Correspondence
Address: |
KALOW & SPRINGUT LLP
488 MADISON AVENUE
19TH FLOOR
NEW YORK
NY
10022
US
|
Assignee: |
Dharmacon, Inc.
Lafayette
CO
|
Family ID: |
32329096 |
Appl. No.: |
10/940892 |
Filed: |
September 14, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10940892 |
Sep 14, 2004 |
|
|
|
PCT/US04/14885 |
May 12, 2004 |
|
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Current U.S.
Class: |
435/6.11 ;
435/455; 435/6.16; 536/23.1; 702/20 |
Current CPC
Class: |
A61P 21/00 20180101;
C12N 2320/10 20130101; C12N 2310/14 20130101; G16B 20/00 20190201;
G16B 20/20 20190201; C12N 15/1136 20130101; A61P 35/00 20180101;
C12N 15/1138 20130101; A61P 37/02 20180101; A61P 35/02 20180101;
A61K 31/713 20130101; C12N 15/113 20130101; C12N 15/1048 20130101;
A61P 25/28 20180101; C12N 15/111 20130101; G16B 20/50 20190201;
A61P 13/12 20180101; A61P 3/10 20180101; C12N 15/1135 20130101;
C12Y 113/12007 20130101; C12N 15/1137 20130101; C12Y 502/01008
20130101; C12N 2320/11 20130101 |
Class at
Publication: |
435/006 ;
435/455; 536/023.1; 702/020 |
International
Class: |
C12Q 001/68; G06F
019/00; G01N 033/48; G01N 033/50; C07H 021/02; C12N 015/87 |
Claims
What is claimed is:
1. A kit for gene silencing, wherein said kit is comprised of a
pool of at least two siRNA duplexes, each of which is comprised of
a sequence that is complementary to a portion of the sequence of
one or more target messenger RNA, and each of which is selected
using selection criteria that are embodied in a formula comprising:
selection criteria are embodied in a formula comprising:
(-8)*A1+(-1)*A2+(12)*A3+(7)*A4+(18)*A5+-
(12)*A6+(19)*A7+(6)*A8+(-4)*A9+(-5)*A10+(-2)*A11+(-5)*A12+(17)*A13+(-3)*A1-
4+(4)*A15+(2)*A16+(8)*A17+(11)*A18+(30)*A19+(-13)*U1+(-10)*U2+(2)*U3+(-2)*-
U4+(-5)*U5+(5)*U6+(-2)*U7+(-10)*U8+(-5)*U9+(15)*U10+(-1)*U11+(0)*U12+(10)*-
U13+(-9)*U14+(-13)*U15+(-10)*U16+(3)*U17+(9)*U18+(9)*U19+(7)*C1+(3)*C2+(-2-
1)*C3+(5)*C4+(-9)*C5+(-20)*C6+(-18)*C7+(-5)*C8+(5)*C9+(1)*C10+(2)*C11+(-5)-
*C12+(-3)*C13+(-6)*C14+(-2)*C15+(-5)*C16+(-3)*C17+(-12)*C18+(-18)*C19+(14)-
*G1+(8)*G2+(7)*G3+(-10)*G4+(-4)*G5+(2)*G6+(1)*G7+(9)*G8+(5)*G9+(-11)*G10+(-
1)*G11+(9)*G12+(-24)*G13+(18)*G14+(11)*G15+(13)*G16+(-7)*G17+(-9)*G18+(-22-
)*G19+6*(number of A+U in position 15-19)-3*(number of G+C in whole
siRNA), Formula X wherein position numbering begins at the 5'-most
position of a sense strand, and A.sub.1=1 if A is the base at
position 1 of the sense strand, otherwise its value is 0; A.sub.2=1
if A is the base at position 2 of the sense strand, otherwise its
value is 0; A.sub.3=1 if A is the base at position 3 of the sense
strand, otherwise its value is 0; A.sub.4=1 if A is the base at
position 4 of the sense strand, otherwise its value is 0; A.sub.5=1
if A is the base at position 5 of the sense strand, otherwise its
value is 0; A.sub.6=1 if A is the base at position 6 of the sense
strand, otherwise its value is 0; A.sub.7=1 if A is the base at
position 7 of the sense strand, otherwise its value is 0;
A.sub.10=1 if A is the base at position 10 of the sense strand,
otherwise its value is 0; A.sub.11=1 if A is the base at position
11 of the sense strand, otherwise its value is 0; A.sub.13=1 if A
is the base at position 13 of the sense strand, otherwise its value
is 0; A.sub.19=1 if A is the base at position 19 of the sense
strand, otherwise if another base is present or the sense strand is
only 18 base pairs in length, its value is 0; C.sub.3=1 if C is the
base at position 3 of the sense strand, otherwise its value is 0;
C.sub.4=1 if C is the base at position 4 of the sense strand,
otherwise its value is 0; C.sub.5=1 if C is the base at position 5
of the sense strand, otherwise its value is 0; C.sub.6=1 if C is
the base at position 6 of the sense strand, otherwise its value is
0; C.sub.7=1 if C is the base at position 7 of the sense strand,
otherwise its value is 0; C.sub.9=1 if C is the base at position 9
of the sense strand, otherwise its value is 0; C.sub.17=1 if C is
the base at position 17 of the sense strand, otherwise its value is
0; C.sub.18=1 if C is the base at position 18 of the sense strand,
otherwise its value is 0; C.sub.19=1 if C is the base at position
19 of the sense strand, otherwise if another base is present or the
sense strand is only 18 base pairs in length, its value is 0;
G.sub.1=1 if G is the base at position 1 on the sense strand,
otherwise its value is 0; G.sub.2=1 if G is the base at position 2
of the sense strand, otherwise its value is 0; G.sub.8=1 if G is
the base at position 8 on the sense strand, otherwise its value is
0; G.sub.10=1 if G is the base at position 10 on the sense strand,
otherwise its value is 0; G.sub.13=1 if G is the base at position
13 on the sense strand, otherwise its value is 0; G.sub.19=1 if G
is the base at position 19 of the sense strand, otherwise if
another base is present or the sense strand is only 18 base pairs
in length, its value is 0; U.sub.1=1 if U is the base at position 1
on the sense strand, otherwise its value is 0; U.sub.2=1 if U is
the base at position 2 on the sense strand, otherwise its value is
0; U.sub.3=1 if U is the base at position 3 on the sense strand,
otherwise its value is 0; U.sub.4=1 if U is the base at position 4
on the sense strand, otherwise its value is 0; U.sub.7=1 if U is
the base at position 7 on the sense strand, otherwise its value is
0; U.sub.9=1 if U is the base at position 9 on the sense strand,
otherwise its value is 0; U.sub.10=1 if U is the base at position
10 on the sense strand, otherwise its value is 0; U.sub.15=1 if U
is the base at position 15 on the sense strand, otherwise its value
is 0; U.sub.16=1 if U is the base at position 16 on the sense
strand, otherwise its value is 0; U.sub.17=1 if U is the base at
position 17 on the sense strand, otherwise its value is 0;
U.sub.18=1 if U is the base at position 18 on the sense strand,
otherwise its value is 0.
2. A method for selecting an siRNA, said method comprising:
applying selection criteria to a set of potential siRNA that
comprise 18-30 base pairs; and determining the relative
functionality of the at least two siRNAs, wherein said section
criteria are non-target specific criteria, said set comprises at
least two siRNAs and each of said at least two siRNAs contains a
sequence that is at least substantially complementary to a target
gene, and said selection criteria are embodied in a formula
comprising:
(-8)*A1+(-1)*A2+(12)*A3+(7)*A4+(18)*A5+(12)*A6+(19)*A7+(6)*A8-
+(-4)*A9+(-5)*A10+(-2)*A11+(-5)*A12+(17)*A13+(-3)*A14+(4)*A15+(2)*A16+(8)*-
A17+(11)*A18+(30)*A19+(-13)*U1+(-10)*U2+(2)*U3+(-2)*U4+(-5)*U5+(5)*U6+(-2)-
*U7+(-10)*U8+(-5)*U9+(15)*U10+(-1)*U11+(0)*U12+(10)*U13+(-9)*U14+(-13)*U15-
+(-10)*U16+(3)*U17+(9)*U18+(9)*U19+(7)*C1+(3)*C2+(-21)*C3+(5)*C4+(-9)*C5+(-
-20)*C6+(-18)*C7+(-5)*C8+(5)*C9+(1)*C10+(2)*C11+(-5)*C12+(-3)*C13+(-6)*C14-
+(-2)*C15+(-5)*C16+(-3)*C17+(-12)*C18+(-18)*C19+(14)*G1+(8)*G2+(7)*G3+(-10-
)*G4+(-4)*G5+(2)*G6+(1)*G7+(9)*G8+(5)*G9+(-11)*G10+(1)*G11+(9)*G12+(-24)*G-
13+(18)*G14+(11)*G15+(13)*G16+(-7)*G17+(-9)*G18+(-22)*G19+6*(number
of A+U in position 15-19)-3*(number of G+C in whole siRNA), Formula
X wherein position numbering begins at the 5'-most position of a
sense strand, and A.sub.1=1 if A is the base at position 1 of the
sense strand, otherwise its value is 0; A.sub.2=1 if A is the base
at position 2 of the sense strand, otherwise its value is 0;
A.sub.3=1 if A is the base at position 3 of the sense strand,
otherwise its value is 0; A.sub.4=1 if A is the base at position 4
of the sense strand, otherwise its value is 0; A.sub.5=1 if A is
the base at position 5 of the sense strand, otherwise its value is
0; A.sub.6=1 if A is the base at position 6 of the sense strand,
otherwise its value is 0; A.sub.7=1 if A is the base at position 7
of the sense strand, otherwise its value is 0; A.sub.10=1 if A is
the base at position 10 of the sense strand, otherwise its value is
0; A.sub.11=1 if A is the base at position 11 of the sense strand,
otherwise its value is 0; A.sub.13=1 if A is the base at position
13 of the sense strand, otherwise its value is 0; A.sub.19=1 if A
is the base at position 19 of the sense strand, otherwise if
another base is present or the sense strand is only 18 base pairs
in length, its value is 0; C.sub.3=1 if C is the base at position 3
of the sense strand, otherwise its value is 0; C.sub.4=1 if C is
the base at position 4 of the sense strand, otherwise its value is
0; C.sub.5=1 if C is the base at position 5 of the sense strand,
otherwise its value is 0; C.sub.6=1 if C is the base at position 6
of the sense strand, otherwise its value is 0; C.sub.7=1 if C is
the base at position 7 of the sense strand, otherwise its value is
0; C.sub.9=1 if C is the base at position 9 of the sense strand,
otherwise its value is 0; C.sub.17=1 if C is the base at position
17 of the sense strand, otherwise its value is 0; C.sub.18=1 if C
is the base at position 18 of the sense strand, otherwise its value
is 0; C.sub.19=1 if C is the base at position 19 of the sense
strand, otherwise if another base is present or the sense strand is
only 18 base pairs in length, its value is 0; G.sub.1=1 if G is the
base at position 1 on the sense strand, otherwise its value is 0;
G.sub.2=1 if G is the base at position 2 of the sense strand,
otherwise its value is 0; G.sub.8=1 if G is the base at position 8
on the sense strand, otherwise its value is 0; G.sub.10=1 if G is
the base at position 10 on the sense strand, otherwise its value is
0; G.sub.13=1 if G is the base at position 13 on the sense strand,
otherwise its value is 0; G.sub.19=1 if G is the base at position
19 of the sense strand, otherwise if another base is present or the
sense strand is only 18 base pairs in length, its value is 0;
U.sub.1=1 if U is the base at position 1 on the sense strand,
otherwise its value is 0; U.sub.2=1 if U is the base at position 2
on the sense strand, otherwise its value is 0; U.sub.3=1 if U is
the base at position 3 on the sense strand, otherwise its value is
0; U.sub.4=1 if U is the base at position 4 on the sense strand,
otherwise its value is 0; U.sub.7=1 if U is the base at position 7
on the sense strand, otherwise its value is 0; U.sub.9=1 if U is
the base at position 9 on the sense strand, otherwise its value is
0; U.sub.10=1 if U is the base at position 10 on the sense strand,
otherwise its value is 0; U.sub.15=1 if U is the base at position
15 on the sense strand, otherwise its value is 0; U.sub.16=1 if U
is the base at position 16 on the sense strand, otherwise its value
is 0; U.sub.17=1 if U is the base at position 17 on the sense
strand, otherwise its value is 0; U.sub.18=1 if U is the base at
position 18 on the sense strand, otherwise its value is 0.
3. A method according to claim 1, further comprising comparing the
internal stability profiles of said at least two siRNAs.
4. A method according to claim 2, further comprising comparing the
internal stability profiles of said at least two siRNAs.
5. A method according to claim 1, further comprising selecting
either for or against sequences that contain motifs that induce
cellular stress.
6. A method according to claim 2, further comprising selecting
either for or against sequences that contain motifs that induce
cellular stress.
7. A method according to claim 1, further comprising selecting
either for or against sequences that comprise stability motifs.
8. A method according to claim 2, further comprising selecting
either for or against sequences that comprise stability motifs.
9. A method of gene silencing, comprising introducing into a cell
at least one siRNA selected according to a method of claim 1.
10. A method of gene silencing, comprising introducing into a cell
at least one siRNA selected according to a method of claim 2.
11. A method according to claim 1, wherein said introducing is by
allowing passive uptake of the at least one siRNA.
12. A method according to claim 2, wherein said introducing is by
allowing passive uptake of the at least one siRNA.
13. A method according claim 9, wherein said introducing in through
the use of a vector.
14. A method for developing an siRNA algorithm for selecting siRNA,
said method comprising: (a) selecting a set of siRNA; (b) measuring
gene silencing ability of each siRNA from said set; (c) determining
relative functionality of each siRNA; (d) determining improved
functionality based on the following variables: the presence or
absence of a particular nucleotide at a particular position, the
total number of As and Us in positions 15-19, the number of times
that the same nucleotide repeats within a given sequence, and the
total number of Gs and Cs; and (e) developing an algorithm using
the information of step (d).
15. A method of selecting an siRNA with improved functionality,
said method comprising using the algorithm of claim 14.
16. A kit, wherein said kit is comprised of at least two siRNAs,
wherein said at least two siRNAs comprise a first optimized siRNA
and a second optimized siRNA, wherein said first optimized siRNA
and said second optimized siRNA are optimized according a formula
comprising:
(-8)*A1+(-1)*A2+(12)*A3+(7)*A4+(18)*A5+(12)*A6+(19)*A7+(6)*A8+(-4)*A9+(-5-
)*A10+(-2)*A11+(-5)*A12+(17)*A13+(-3)*A14+(4)*A15+(2)*A16+(8)*A17+(11)*A18-
+(30)*A19+(-13)*U1+(-10)*U2+(2)*U3+(-2)*U4+(-5)*U5+(5)*U6+(-2)*U7+(-10)*U8-
+(-5)*U9+(15)*U10+(-1)*U11+(0)*U12+(10)*U13+(-9)*U14+(-13)*U15+(-10)*U16+(-
3)*U17+(9)*U18+(9)*U19+(7)*C1+(3)*C2+(-21)*C3+(5)*C4+(-9)*C5+(-20)*C6+(-18-
)*C7+(-5)*C8+(5)*C9+(1)*C10+(2)*C11+(-5)*C12+(-3)*C13+(-6)*C14+(-2)*C15+(--
5)*C16+(-3)*C17+(-12)*C18+(-18)*C19+(14)*G1+(8)*G2+(7)*G3+(-10)*G4+(-4)*G5-
+(2)*G6+(1)*G7+(9)*G8+(5)*G9+(-11)*G10+(1)*G11+(9)*G12+(-24)*G13+(18)*G14+-
(11)*G15+(13)*G16+(-7)*G17+(-9)*G18+(-22)*G19+6*(number of A+U in
position 15-19)-3*(number of G+C in whole siRNA), Formula X wherein
position numbering begins at the 5'-most position of a sense
strand, and A.sub.1=1 if A is the base at position 1 of the sense
strand, otherwise its value is 0; A.sub.2=1 if A is the base at
position 2 of the sense strand, otherwise its value is 0; A.sub.3=1
if A is the base at position 3 of the sense strand, otherwise its
value is 0; A.sub.4=1 if A is the base at position 4 of the sense
strand, otherwise its value is 0; A.sub.5=1 if A is the base at
position 5 of the sense strand, otherwise its value is 0; A.sub.6=1
if A is the base at position 6 of the sense strand, otherwise its
value is 0; A.sub.7=1 if A is the base at position 7 of the sense
strand, otherwise its value is 0; A.sub.10=1 if A is the base at
position 10 of the sense strand, otherwise its value is 0;
A.sub.11=1 if A is the base at position 11 of the sense strand,
otherwise its value is 0; A.sub.13=1 if A is the base at position
13 of the sense strand, otherwise its value is 0; A.sub.19=1 if A
is the base at position 19 of the sense strand, otherwise if
another base is present or the sense strand is only 18 base pairs
in length, its value is 0; C.sub.3=1 if C is the base at position 3
of the sense strand, otherwise its value is 0; C.sub.4=1 if C is
the base at position 4 of the sense strand, otherwise its value is
0; C.sub.5=1 if C is the base at position 5 of the sense strand,
otherwise its value is 0; C.sub.6=1 if C is the base at position 6
of the sense strand, otherwise its value is 0; C.sub.7=1 if C is
the base at position 7 of the sense strand, otherwise its value is
0; C.sub.9=1 if C is the base at position 9 of the sense strand,
otherwise its value is 0; C.sub.17=1 if C is the base at position
17 of the sense strand, otherwise its value is 0; C.sub.18=1 if C
is the base at position 18 of the sense strand, otherwise its value
is 0; C.sub.19=1 if C is the base at position 19 of the sense
strand, otherwise if another base is present or the sense strand is
only 18 base pairs in length, its value is 0; G.sub.1=1 if G is the
base at position 1 on the sense strand, otherwise its value is 0;
G.sub.2=1 if G is the base at position 2 of the sense strand,
otherwise its value is 0; G.sub.8=1 if G is the base at position 8
on the sense strand, otherwise its value is 0; G.sub.10=1 if G is
the base at position 10 on the sense strand, otherwise its value is
0; G.sub.13=1 if G is the base at position 13 on the sense strand,
otherwise its value is 0; G.sub.19=1 if G is the base at position
19 of the sense strand, otherwise if another base is present or the
sense strand is only 18 base pairs in length, its value is 0;
U.sub.1=1 if U is the base at position 1 on the sense strand,
otherwise its value is 0; U.sub.2=1 if U is the base at position 2
on the sense strand, otherwise its value is 0; U.sub.3=1 if U is
the base at position 3 on the sense strand, otherwise its value is
0; U.sub.4=1 if U is the base at position 4 on the sense strand,
otherwise its value is 0; U.sub.7=1 if U is the base at position 7
on the sense strand, otherwise its value is 0; U.sub.9=1 if U is
the base at position 9 on the sense strand, otherwise its value is
0; U.sub.10=1 if U is the base at position 10 on the sense strand,
otherwise its value is 0; U.sub.15=1 if U is the base at position
15 on the sense strand, otherwise its value is 0; U.sub.16=1 if U
is the base at position 16 on the sense strand, otherwise its value
is 0; U.sub.17=1 if U is the base at position 17 on the sense
strand, otherwise its value is 0; U.sub.18=1 if U is the base at
position 18 on the sense strand, otherwise its value is 0.
17. A method for identifying hyperfunctional siRNA, comprising:
applying selection criteria to a set of potential siRNA that
comprise 18-30 base pairs, wherein said selection criteria are
non-target specific criteria, and said set comprises at least two
siRNAs and each of said at least two siRNAs contains a sequence
that is at least substantially complementary to a target gene; and
determining the relative functionality of the at least two siRNAs
and assigning each of the at least two siRNAs a functionality
score; and selecting siRNAs from the at least two siRNAs that have
a functionality score that reflects greater than 80 percent
silencing at a concentration in the picomolar range, wherein said
greater than 80 percent silencing endures for greater than 120
hours.
18. A method according to claim 1, wherein said siRNA are
unimolecular.
19. A method according to claim 2, wherein said siRNA are
unimolecular.
20. A method according to claim 14, wherein said siRNA are
unimolecular.
21. A method according to claim 16, wherein said siRNA are
unimolecular.
22. A method according to claim 17, wherein said siRNA are
unimolecular.
23. A method according to claim 1, wherein said siRNA are comprised
of two separate polynucleotide strands.
24. A method according to claim 2, wherein said siRNA are comprised
of two separate polynucleotide strands.
25. A method according to claim 14, wherein said siRNA are
comprised of two separate polynucleotide strands.
26. A method according to claim 16, wherein said siRNA are
comprised of two separate polynucleotide strands.
27. A method according to claim 17, wherein said siRNA are
comprised of two separate polynucleotide strands.
28. A method according to claim 1, wherein said siRNA are expressed
from one or more vectors.
29. A method according to claim 2, wherein said siRNA are expressed
from one or more vectors.
30. A method according to claim 14, wherein said siRNA are
expressed from one or more vectors.
31. A method according to claim 16, wherein said siRNA are
expressed from one or more vectors.
32. A method according to claim 17, wherein said siRNA are
expressed from one or more vectors.
33. A method according to claim 1, wherein two or more genes are
silenced by a single administration of siRNA.
34. A method according to claim 2, wherein two or more genes are
silenced by a single administration of siRNA.
35. A method according to claim 14, wherein two or more genes are
silenced by a single administration of siRNA.
36. A method according to claim 16, wherein two or more genes are
silenced by a single administration of siRNA.
37. A method according to claim 17, wherein two or more genes are
silenced by a single administration of siRNA.
38. A kit according to claim 13, wherein one or more of said siRNA
are unimolecular.
39. A kit according to claim 13, wherein one or more of said siRNA
are comprised of two separate polynucleotide strands.
40. A kit according to claim 13, wherein one or more of said siRNA
are capable of silencing the Bcl2 gene.
41. A method for developing an siRNA algorithm for selecting
functional and hyperfunctional siRNAs for a given sequence,
comprising: (a) selecting a set of siRNAs; (b) measuring the gene
silencing ability of each siRNA from said set; (c) determining the
relative functionality of each siRNA; (d) determining the amount of
improved functionality based on the following variables: the total
GC content, melting temperature of the siRNA, GC content at
positions 15-19, the presence or absence of a particular nucleotide
at a particular position, relative thermodynamic stability at
particular positions in a duplex, and the number of times that the
same nucleotide repeats within a given sequence; and (e) developing
an algorithm using the information of step (d).
Description
REFERENCE TO TABLES SUBMITTED IN ELECTRONIC FORM
[0001] In accordance with PCT Administrative Instructions Part 8,
Applicant submits a compact disc of tables related to sequences and
hereby incorporates by reference the material submitted herewith,
on the compact disk labeled COPY 1--TABLES PART DISK 1/1, TABLES
XII and XIII (provided in triplicate, which copies are identical),
in files entitled table-xii.txt, date of creation 26 Apr. 2004,
with a size of 110,486 kb, and table-xiii.txt, date of creation 26
Apr. 2004, with a size of 23,146 kb; and in accordance with PCT
Administrative Instructions Section 801(a)(i) on the compact disk
labeled CRF (with three further copies, which copies are identical)
in a file entitled 13608PCT.txt, date of creation 26 Apr. 2004,
with a size of 556,776 kb.
FIELD OF INVENTION
[0002] The present invention relates to RNA interference
("RNAi").
BACKGROUND OF THE INVENTION
[0003] Relatively recently, researchers observed that double
stranded RNA ("dsRNA") could be used to inhibit protein expression.
This ability to silence a gene has broad potential for treating
human diseases, and many researchers and commercial entities are
currently investing considerable resources in developing therapies
based on this technology.
[0004] Double stranded RNA induced gene silencing can occur on at
least three different levels: (i) transcription inactivation, which
refers to RNA guided DNA or histone methylation; (ii) siRNA induced
mRNA degradation; and (iii) mRNA induced transcriptional
attenuation.
[0005] It is generally considered that the major mechanism of RNA
induced silencing (RNA interference, or RNAi) in mammalian cells is
mRNA degradation. Initial attempts to use RNAi in mammalian cells
focused on the use of long strands of dsRNA. However, these
attempts to induce RNAi met with limited success, due in part to
the induction of the interferon response, which results in a
general, as opposed to a target-specific, inhibition of protein
synthesis. Thus, long dsRNA is not a viable option for RNAi in
mammalian systems.
[0006] More recently it has been shown that when short (18-30 bp)
RNA duplexes are introduced into mammalian cells in culture,
sequence-specific inhibition of target mRNA can be realized without
inducing an interferon response. Certain of these short dsRNAs,
referred to as small inhibitory RNAs ("siRNAs"), can act
catalytically at sub-molar concentrations to cleave greater than
95% of the target mRNA in the cell. A description of the mechanisms
for siRNA activity, as well as some of its applications are
described in Provost et al., Ribonuclease Activity and RNA Binding
of Recombinant Human Dicer, E.M.B.O. J., 2002 Nov. 1; 21(21):
5864-5874; Tabara et al., The dsRNA Binding Protein RDE-4 Interacts
with RDE-1, DCR-1 and a DexH-box Helicase to Direct RNAi in C.
elegans, Cell 2002, Jun. 28;109(7):861-71; Ketting et al., Dicer
Functions in RNA Interference and in Synthesis of Small RNA
Involved in Developmental Timing in C. elegans; Martinez et al.,
Single-Stranded Antisense siRNAs Guide Target RNA Cleavage in RNAi,
Cell 2002, Sep. 6; 110(5):563; Hutvagner & Zamore, A micro RNA
in a multiple-turnover RNAi enzyme complex, Science 2002,
297:2056.
[0007] From a mechanistic perspective, introduction of long double
stranded RNA into plants and invertebrate cells is broken down into
siRNA by a Type III endonuclease known as Dicer. Sharp, RNA
interference--2001, Genes Dev. 2001, 15:485. Dicer, a
ribonuclease-III-like enzyme, processes the dsRNA into 19-23 base
pair short interfering RNAs with characteristic two base 3'
overhangs. Bernstein, Caudy, Hammond, & Hannon, Role for a
bidentate ribonuclease in the initiation step of RNA interference,
Nature 2001, 409:363. The siRNAs are then incorporated into an
RNA-induced silencing complex (RISC) where one or more helicases
unwind the siRNA duplex, enabling the complementary antisense
strand to guide target recognition. Nykanen, Haley, & Zamore,
ATP requirements and small interfering RNA structure in the RNA
interference pathway, Cell 2001, 107:309. Upon binding to the
appropriate target mRNA, one or more endonucleases within the RISC
cleaves the target to induce silencing. Elbashir, Lendeckel, &
Tuschl, RNA interference is mediated by 21- and 22-nucleotide RNAs,
Genes Dev 2001, 15:188, FIG. 1.
[0008] The interference effect can be long lasting and may be
detectable after many cell divisions. Moreover, RNAi exhibits
sequence specificity. Kisielow, M. et al. (2002) Isoform-specific
knockdown and expression of adaptor protein ShcA using small
interfering RNA, J. of Biochemistry 363: 1-5. Thus, the RNAi
machinery can specifically knock down one type of transcript, while
not affecting closely related mRNA. These properties make siRNA a
potentially valuable tool for inhibiting gene expression and
studying gene function and drug target validation. Moreover, siRNAs
are potentially useful as therapeutic agents against: (1) diseases
that are caused by over-expression or misexpression of genes; and
(2) diseases brought about by expression of genes that contain
mutations.
[0009] Successful siRNA-dependent gene silencing depends on a
number of factors. One of the most contentious issues in RNAi is
the question of the necessity of siRNA design, i.e., considering
the sequence of the siRNA used. Early work in C. elegans and plants
circumvented the issue of design by introducing long dsRNA (see,
for instance, Fire, A. et al. (1998) Nature 391:806-811). In this
primitive organism, long dsRNA molecules are cleaved into siRNA by
Dicer, thus generating a diverse population of duplexes that can
potentially cover the entire transcript. While some fraction of
these molecules are non-functional (i.e., induce little or no
silencing) one or more have the potential to be highly functional,
thereby silencing the gene of interest and alleviating the need for
siRNA design. Unfortunately, due to the interferon response, this
same approach is unavailable for mammalian systems. While this
effect can be circumvented by bypassing the Dicer cleavage step and
directly introducing siRNA, this tactic carries with it the risk
that the chosen siRNA sequence may be non-functional or
semi-functional.
[0010] A number of researches have expressed the view that siRNA
design is not a crucial element of RNAi. On the other hand, others
in the field have begun to explore the possibility that RNAi can be
made more efficient by paying attention to the design of the siRNA.
Unfortunately, none of the reported methods have provided a
satisfactory scheme for reliably selecting siRNA with acceptable
levels of functionality. Accordingly, there is a need to develop
rational criteria by which to select siRNA with an acceptable level
of functionality, and to identify siRNA that have this improved
level of functionality, as well as to identify siRNAs that are
hyperfunctional.
SUMMARY OF THE INVENTION
[0011] The present invention is directed to increasing the
efficiency of RNAi, particularly in mammalian systems. Accordingly,
the present invention provides kits, siRNAs and methods for
increasing siRNA efficacy.
[0012] According to a first embodiment, the present invention
provides a kit for gene silencing, wherein said kit is comprised of
a pool of at least two siRNA duplexes, each of which is comprised
of a sequence that is complementary to a portion of the sequence of
one or more target messenger RNA, and each of which is selected
using non-target specific criteria.
[0013] According to a second embodiment, the present invention
provides a method for selecting an siRNA, said method comprising
applying selection criteria to a set of potential siRNA that
comprise 18-30 base pairs, wherein said selection criteria are
non-target specific criteria, and said set comprises at least two
siRNAs and each of said at least two siRNAs contains a sequence
that is at least substantially complementary to a target gene; and
determining the relative functionality of the at least two
siRNAs.
[0014] In one embodiment, the present invention also provides a
method wherein said selection criteria are embodied in a formula
comprising:
(-14)*G.sub.13-13*A.sub.1-12*U.sub.7-11*U.sub.2-10*A.sub.11-10*U.sub.4-10*-
C.sub.3-10*C.sub.5-10*C.sub.6-9*A.sub.10-9*U.sub.9-9*C.sub.18-8*G.sub.10-7-
*U.sub.1-7*U.sub.16-7*C.sub.17-7*C.sub.19+7*U.sub.17+8*A.sub.2+8*A.sub.4+8-
*A.sub.5+8*C.sub.4+9*G.sub.8+10*A.sub.7+10*U.sub.18+11*A.sub.19+11*C.sub.9-
+15*G.sub.1+18*A.sub.3+19*U.sub.10-Tm-3*
(GC.sub.total)-6*(GC.sub.15-19)-3- 0*X; or Formula VIII
(-8)*A1+(-1)*A2+(12)*A3+(7)*A4+(18)*A5+(12)*A6+(19)*A7+(6)*A8+(-4)*A9+(-5)-
*A10+(-2)*A11+(-5)*A12+(17)*A13+(-3)*A14+(4)*A15+(2)*A16+(8)*A17+(11)*A18+-
(30)*A19+(-13)*U1+(-10)*U2+(2)*U3+(-2)*U4+(-5)*U5+(5)*U6+(-2)*U7+(-10)*U8+-
(-5)*U9+(15)*U10+(-1)*U11+(0)*U12+(10)*U13+(-9)*U14+(-13)*U15+(-10)*U16+(3-
)*U17+(9)*U18+(9)*U19+(7)*C1+(3)*C2+(-21)*C3+(5)*C4+(-9)*C5+(-20)*C6+(-18)-
*C7+(-5)*C8+(5)*C9+(1)*C10+(2)*C11+(-5)*C12+(-3)*C13+(-6)*C14+(-2)*C15+(-5-
)*C16+(-3)*C17+(-12)*C18+(-18)*C19+(14)*G1+(8)*G2+(7)*G3+(-10)*G4+(-4)*G5+-
(2)*G6+(1)*G7+(9)*G8+(5)*G9+(-11)*G10+(1)*G11+(9)*G12+(-24)*G13+(18)*G14+(-
11)*G15+(13)*G16+(-7)*G17+(-9)*G18+(-22)*G19+6*(number of A+U in
position 15-19)-3*(number of G+C in whole siRNA), Formula X
[0015] wherein position numbering begins at the 5'-most position of
a sense strand, and
[0016] A.sub.1=1 if A is the base at position 1 of the sense
strand, otherwise its value is 0;
[0017] A.sub.2=1 if A is the base at position 2 of the sense
strand, otherwise its value is 0;
[0018] A.sub.3=1 if A is the base at position 3 of the sense
strand, otherwise its value is 0;
[0019] A.sub.4=1 if A is the base at position 4 of the sense
strand, otherwise its value is 0;
[0020] A.sub.5=1 if A is the base at position 5 of the sense
strand, otherwise its value is 0;
[0021] A.sub.6=1 if A is the base at position 6 of the sense
strand, otherwise its value is 0;
[0022] A.sub.7=1 if A is the base at position 7 of the sense
strand, otherwise its value is 0;
[0023] A.sub.10=1 if A is the base at position 10 of the sense
strand, otherwise its value is 0;
[0024] A.sub.11=1 if A is the base at position 11 of the sense
strand, otherwise its value is 0;
[0025] A.sub.13=1 if A is the base at position 13 of the sense
strand, otherwise its value is 0;
[0026] A.sub.19=1 if A is the base at position 19 of the sense
strand, otherwise if another base is present or the sense strand is
only 18 base pairs in length, its value is 0;
[0027] C.sub.3=1 if C is the base at position 3 of the sense
strand, otherwise its value is 0;
[0028] C.sub.4=1 if C is the base at position 4 of the sense
strand, otherwise its value is 0;
[0029] C.sub.5=1 if C is the base at position 5 of the sense
strand, otherwise its value is 0;
[0030] C.sub.6=1 if C is the base at position 6 of the sense
strand, otherwise its value is 0;
[0031] C.sub.7=1 if C is the base at position 7 of the sense
strand, otherwise its value is 0;
[0032] C.sub.9=1 if C is the base at position 9 of the sense
strand, otherwise its value is 0;
[0033] C.sub.17=1 if C is the base at position 17 of the sense
strand, otherwise its value is 0;
[0034] C.sub.18=1 if C is the base at position 18 of the sense
strand, otherwise its value is 0;
[0035] C.sub.19=1 if C is the base at position 19 of the sense
strand, otherwise if another base is present or the sense strand is
only 18 base pairs in length, its value is 0;
[0036] G.sub.1=1 if G is the base at position 1 on the sense
strand, otherwise its value is 0;
[0037] G.sub.2=1 if G is the base at position 2 of the sense
strand, otherwise its value is 0;
[0038] G.sub.8=1 if G is the base at position 8 on the sense
strand, otherwise its value is 0;
[0039] G.sub.10=1 if G is the base at position 10 on the sense
strand, otherwise its value is 0;
[0040] G.sub.13=1 if G is the base at position 13 on the sense
strand, otherwise its value is 0;
[0041] G.sub.19=1 if G is the base at position 19 of the sense
strand, otherwise if another base is present or the sense strand is
only 18 base pairs in length, its value is 0;
[0042] U.sub.1=1 if U is the base at position 1 on the sense
strand, otherwise its value is 0;
[0043] U.sub.2=1 if U is the base at position 2 on the sense
strand, otherwise its value is 0;
[0044] U.sub.3=1 if U is the base at position 3 on the sense
strand, otherwise its value is 0;
[0045] U.sub.4=1 if U is the base at position 4 on the sense
strand, otherwise its value is 0;
[0046] U.sub.7=1 if U is the base at position 7 on the sense
strand, otherwise its value is 0;
[0047] U.sub.9=1 if U is the base at position 9 on the sense
strand, otherwise its value is 0;
[0048] U.sub.10=1 if U is the base at position 10 on the sense
strand, otherwise its value is 0;
[0049] U.sub.15=1 if U is the base at position 15 on the sense
strand, otherwise its value is 0;
[0050] U.sub.16=1 if U is the base at position 16 on the sense
strand, otherwise its value is 0;
[0051] U.sub.17=1 if U is the base at position 17 on the sense
strand, otherwise its value is 0;
[0052] U.sub.18=1 if U is the base at position 18 on the sense
strand, otherwise its value is 0.
[0053] GC.sub.15-19=the number of G and C bases within positions
15-19 of the sense strand, or within positions 15-18 if the sense
strand is only 18 base pairs in length;
[0054] GC.sub.total=the number of G and C bases in the sense
strand;
[0055] Tm=100 if the siRNA oligo has the internal repeat longer
then 4 base pairs, otherwise its value is 0; and
[0056] X=the number of times that the same nucleotide repeats four
or more times in a row.
[0057] According to a third embodiment, the invention provides a
method for developing an algorithm for selecting siRNA, said method
comprising: (a) selecting a set of siRNA; (b) measuring gene
silencing ability of each siRNA from said set; (c) determining
relative functionality of each siRNA; (d) determining improved
functionality by the presence or absence of at least one variable
selected from the group consisting of the presence or absence of a
particular nucleotide at a particular position, the total number of
As and Us in positions 15-19, the number of times that the same
nucleotide repeats within a given sequence, and the total number of
Gs and Cs; and (e) developing an algorithm using the information of
step (d).
[0058] According to a fourth embodiment, the present invention
provides a kit, wherein said kit is comprised of at least two
siRNAs, wherein said at least two siRNAs comprise a first optimized
siRNA and a second optimized siRNA, wherein said first optimized
siRNA and said second optimized siRNA are optimized according a
formula comprising Formula X.
[0059] According to a fifth embodiment, the present invention
provides a method for identifying a hyperfunctional siRNA,
comprising applying selection criteria to a set of potential siRNA
that comprise 18-30 base pairs, wherein said selection criteria are
non-target specific criteria, and said set comprises at least two
siRNAs and each of said at least two siRNAs contains a sequence
that is at least substantially complementary to a target gene;
determining the relative functionality of the at least two siRNAs
and assigning each of the at least two siRNAs a functionality
score; and selecting siRNAs from the at least two siRNAs that have
a functionality score that reflects greater than 80 percent
silencing at a concentration in the picomolar range, wherein said
greater than 80 percent silencing endures for greater than 120
hours.
[0060] According to a sixth embodiment, the present invention
provides a hyperfunctional siRNA that is capable of silencing
Bc12.
[0061] According to a seventh embodiment, the present invention
provides a method for developing an siRNA algorithm for selecting
functional and hyperfunctional siRNAs for a given sequence. The
method comprises:
[0062] (a) selecting a set of siRNAs;
[0063] (b) measuring the gene silencing ability of each siRNA from
said set;
[0064] (c) determining the relative functionality of each
siRNA;
[0065] (d) determining the amount of improved functionality by the
presence or absence of at least one variable selected from the
group consisting of the total GC content, melting temperature of
the siRNA, GC content at positions 15-19, the presence or absence
of a particular nucleotide at a particular position, relative
thermodynamic stability at particular positions in a duplex, and
the number of times that the same nucleotide repeats within a given
sequence; and
[0066] (e) developing an algorithm using the information of step
(d).
[0067] According to this embodiment, preferably the set of siRNAs
comprises at least 90 siRNAs from at least one gene, more
preferably at least 180 siRNAs from at least two different genes,
and most preferably at least 270 and 360 siRNAs from at least three
and four different genes, respectively. Additionally, in step (d)
the determination is made with preferably at least two, more
preferably at least three, even more preferably at least four, and
most preferably all of the variables. The resulting algorithm is
not target sequence specific.
[0068] In another embodiment, the present invention provides
rationally designed siRNAs identified using the formulas above.
[0069] In yet another embodiment, the present invention is directed
to hyperfunctional siRNA.
[0070] The ability to use the above algorithms, which are not
sequence or species specific, allows for the cost-effective
selection of optimized siRNAs for specific target sequences.
Accordingly, there will be both greater efficiency and reliability
in the use of siRNA technologies.
[0071] For a better understanding of the present invention together
with other and further advantages and embodiments, reference is
made to the following description taken in conjunction with the
examples, the scope of which is set forth in the appended
claims.
BRIEF DESCRIPTION OF THE FIGURES
[0072] FIG. 1 shows a model for siRNA-RISC interactions. RISC has
the ability to interact with either end of the siRNA or miRNA
molecule. Following binding, the duplex is unwound, and the
relevant target is identified, cleaved, and released.
[0073] FIG. 2 is a representation of the functionality of two
hundred and seventy siRNA duplexes that were generated to target
human cyclophilin, human diazepam-binding inhibitor (DB), and
firefly luciferase.
[0074] FIG. 3a is a representation of the silencing effect of 30
siRNAs in three different cells lines, HEK293, DU145, and Hela.
FIG. 3b shows the frequency of different functional groups (>95%
silencing (black), >80% silencing (gray), >50% silencing
(dark gray), and <50% silencing (white)) based on GC content. In
cases where a given bar is absent from a particular GC percentage,
no siRNA were identified for that particular group. FIG. 3c shows
the frequency of different functional groups based on melting
temperature (Tm).
[0075] FIG. 4 is a representation of a statistical analysis that
revealed correlations between silencing and five sequence-related
properties of siRNA: (A) an A at position 19 of the sense strand,
(B) an A at position 3 of the sense strand, (C) a U at position 10
of the sense strand, (D) a base other than G at position 13 of the
sense strand, and (E) a base other than C at position 19 of the
sense strand. All variables were correlated with siRNA silencing of
firefly luciferase and human cyclophilin. siRNAs satisfying the
criterion are grouped on the left (Selected) while those that do
not, are grouped on the right (Eliminated). Y-axis is "% Silencing
of Control." Each position on the X-axis represents a unique
siRNA.
[0076] FIGS. 5A and 5B are representations of firefly luciferase
and cyclophilin siRNA panels sorted according to functionality and
predicted values using Formula VIII. The siRNA found within the
circle represent those that have Formula VIII values
(SMARTscores.TM.) above zero. siRNA outside the indicated area have
calculated Formula VIII values that are below zero. Y-axis is
"Expression (% Control)." Each position on the X-axis represents a
unique siRNA.
[0077] FIG. 6A is a representation of the average internal
stability profile (AISP) derived from 270 siRNAs taken from three
separate genes (cyclophilin B, DBI and firefly luciferase). Graphs
represent AISP values of highly functional, functional, and
non-functional siRNA. FIG. 6B is a comparison between the AISP of
naturally derived GFP siRNA (filled squares) and the AISP of siRNA
from cyclophilin B, DBI, and luciferase having >90% silencing
properties (no fill) for the antisense strand. "DG" is the symbol
for .DELTA.G, free energy.
[0078] FIG. 7 is a histogram showing the differences in duplex
functionality upon introduction of basepair mismatches. The X-axis
shows the mismatch introduced in the siRNA and the position it is
introduced (e.g., 8C>A reveals that position 8 (which normally
has a C) has been changed to an A). The Y-axis is "% Silencing
(Normalized to Control)."
[0079] FIG. 8a is histogram that shows the effects of 5' sense and
antisense strand modification with 2'-O-methylation on
functionality. FIG. 8b is an expression profile showing a
comparison of sense strand off-target effects for IGF1R-3 and
2'-O-methyl IGF1R-3. Sense strand off-targets (lower box) are not
induced when the 5' end of the sense strand is modified with
2'-O-methyl groups (top box).
[0080] FIG. 9 shows a graph of SMARTscores.TM. versus RNAi
silencing values for more than 360 siRNA directed against 30
different genes. siRNA to the right of the vertical bar represent
those siRNA that have desirable SMARTscores.TM..
[0081] FIGS. 10A-E compare the RNAi of five different genes (SEAP,
DBI, PLK, Firefly Luciferase, and Renila Luciferase) by varying
numbers of randomly selected siRNA and four rationally designed
(SMART-selected) siRNA chosen using the algorithm described in
Formula VIII. In addition, RNAi induced by a pool of the four
SMART-selected siRNA is reported at two different concentrations
(100 and 400 nM). 10F is a comparison between a pool of randomly
selected EGFR siRNA (Pool 1) and a pool of SMART selected EGFR
siRNA (Pool 2). Pool 1, S1-S4 and Pool 2 S1-S4 represent the
individual members that made up each respective pool. Note that
numbers for random siRNAs represent the position of the 5' end of
the sense strand of the duplex. The Y-axis represents the %
expression of the control(s). The X-axis is the percent expression
of the control.
[0082] FIG. 11 shows the Western blot results from cells treated
with siRNA directed against twelve different genes involved in the
clathrin-dependent endocytosis pathway (CHC, DynII, CALM, CLCa,
CLCb, Eps15, Eps15R, Rab5a, Rab5b, Rab5c, .beta.2 subunit of AP-2
and EEA.1). siRNA were selected using Formula VIII. "Pool"
represents a mixture of duplexes 1-4. Total concentration of each
siRNA in the pool is 25 nM. Total concentration=4.times.25=100
nM.
[0083] FIG. 12 is a representation of the gene silencing
capabilities of rationally-selected siRNA directed against ten
different genes (human and mouse cyclophilin, C-myc, human lamin
A/C, QB (ubiquinol-cytochrome c reductase core protein I), MEK1 and
MEK2, ATE1 (arginyl-tRNA protein transferase), GAPDH, and Eg5). The
Y-axis is the percent expression of the control. Numbers 1, 2, 3
and 4 represent individual rationally selected siRNA. "Pool"
represents a mixture of the four individual siRNA.
[0084] FIG. 13 is the sequence of the top ten Bcl2 siRNAs as
determined by Formula VIII. Sequences are listed 5' to 3'.
[0085] FIG. 14 is the knockdown by the top ten Bcl2 siRNAs at 100
nM concentrations. The Y-axis represents the amount of expression
relative to the non-specific (ns) and transfection mixture
control.
[0086] FIG. 15 represents a functional walk where siRNA beginning
on every other base pair of a region of the luciferase gene are
tested for the ability to silence the luciferase gene. The Y-axis
represents the percent expression relative to a control. The X-axis
represents the position of each individual siRNA.
[0087] FIG. 16 is a histogram demonstrating the inhibition of
target gene expression by pools of 2 and 3 siRNAs duplexes taken
from the walk described in FIG. 15. The Y-axis represents the
percent expression relative to control. The X-axis represents the
position of the first siRNA in paired pools, or trios of siRNA. For
instance, the first paired pool contains siRNA 1 and 3. The second
paired pool contains siRNA 3 and 5. Pool 3 (of paired pools)
contains siRNA 5 and 7, and so on.
[0088] FIG. 17 is a histogram demonstrating the inhibition of
target gene expression by pools of 4 and 5 siRNA duplexes. The
Y-axis represents the percent expression relative to a control. The
X-axis represents the position of the first siRNA in each pool.
[0089] FIG. 18 is a histogram demonstrating the inhibition of
target gene expression by siRNAs that are ten and twenty basepairs
apart. The Y-axis represents the percent expression relative to a
control. The X-axis represents the position of the first siRNA in
each pool.
[0090] FIG. 19 shows that pools of siRNAs (dark gray bar) work as
well (or better) than the best siRNA in the pool (light gray bar).
The Y-axis represents the percent expression relative to a control.
The-X axis represents the position of the first siRNA in each
pool.
[0091] FIG. 20 shows that the combination of several semifunctional
siRNAs (dark gray) result in a significant improvement of gene
expression inhibition over individual (semi-functional; light gray)
siRNA. The Y-axis represents the percent expression relative to a
control.
[0092] FIG. 21 shows both pools (Library, Lib) and individual
siRNAs in inhibition of gene expression of Beta-Galactosidase,
Renilla Luciferase and SEAP (alkaline phosphatase). Numbers on the
X-axis indicate the position of the 5'-most nucleotide of the sense
strand of the duplex. The Y-axis represents the percent expression
of each gene relative to a control. Libraries contain 19 nucleotide
long siRNAs (not including overhangs) that begin at the following
nucleotides: SEAP: Lib 1: 206, 766, 812,923, Lib 2: 1117, 1280,
1300, 1487, Lib 3: 206, 766, 812, 923, 1117, 1280, 1300,1487, Lib
4: 206, 812, 1117, 1300, Lib 5: 766, 923, 1280, 1487, Lib 6: 206,
1487; Bgal: Lib 1: 979, 1339, 2029, 2590, Lib 2:
1087,1783,2399,3257, Lib 3: 979, 1783, 2590, 3257, Lib 4: 979,
1087, 1339, 1783, 2029, 2399,2590,3257, Lib 5: 979, 1087, 1339,
1783, Lib 6: 2029,2399,2590,3257; Renilla: Lib 1: 174,300,432,568,
Lib 2: 592, 633, 729,867, Lib 3: 174, 300, 432, 568, 592,
633,729,867, Lib 4: 174, 432, 592, 729, Lib 5: 300,568,633,867, Lib
6: 592,568.
[0093] FIG. 22 shows the results of an EGFR and TfnR
internalization assay when single gene knockdowns are performed.
The Y-axis represents percent internalization relative to
control.
[0094] FIG. 23 shows the results of an EGFR and TfnR
internalization assay when multiple genes are knocked down (e.g.,
Rab5a, b, c). The Y-axis represents the percent internalization
relative to control.
[0095] FIG. 24 shows the simultaneous knockdown of four different
genes. siRNAs directed against G6PD, GAPDH, PLK, and UQCwere
simultaneously introduced into cells. Twenty-four hours later,
cultures were harvested and assayed for mRNA target levels for each
of the four genes. A comparison is made between cells transfected
with individual siRNAs vs. a pool of siRNAs directed against all
four genes.
[0096] FIG. 25 shows the functionality of ten siRNAs at 0.3 nM
concentrations.
DETAILED DESCRIPTION
DEFINITIONS
[0097] Unless stated otherwise, the following terms and phrases
have the meanings provided below:
[0098] siRNA
[0099] The term "siRNA" refers to small inhibitory RNA duplexes
that induce the RNA interference (RNAi) pathway. These molecules
can vary in length (generally 18-30 basepairs) and contain varying
degrees of complementarity to their target mRNA in the antisense
strand. Some, but not all, siRNA have unpaired overhanging bases on
the 5' or 3' end of the sense strand and/or the antisense strand.
The term "siRNA" includes duplexes of two separate strands, as well
as single strands that can form hairpin structures comprising a
duplex region.
[0100] siRNA may be divided into five (5) groups (non-functional,
semi-functional, functional, highly functional, and
hyper-functional) based on the level or degree of silencing that
they induce in cultured cell lines. As used herein, these
definitions are based on a set of conditions where the siRNA is
transfected into said cell line at a concentration of 100 nM and
the level of silencing is tested at a time of roughly 24 hours
after transfection, and not exceeding 72 hours after transfection.
In this context, "non-functional siRNA" are defined as those siRNA
that induce less than 50% (<50%) target silencing.
"Semi-functional siRNA" induce 50-79% target silencing. "Functional
siRNA" are molecules that induce 80-95% gene silencing.
"Highly-functional siRNA" are molecules that induce greater than
95% gene silencing. "Hyperfunctional siRNA" are a special class of
molecules. For purposes of this document, hyperfunctional siRNA are
defined as those molecules that: (1) induce greater than 95%
silencing of a specific target when they are transfected at
subnanomolar concentrations (i.e., less than one nanomolar); and/or
(2) induce functional (or better) levels of silencing for greater
than 96 hours. These relative functionalities (though not intended
to be absolutes) may be used to compare siRNAs to a particular
target for applications such as functional genomics, target
identification and therapeutics.
[0101] miRNA
[0102] The term "miRNA" refers to microRNA.
[0103] Gene Silencing
[0104] The phrase "gene silencing" refers to a process by which the
expression of a specific gene product is lessened or attenuated.
Gene silencing can take place by a variety of pathways. Unless
specified otherwise, as used herin, gene silencing refers to
decreases in gene product expression that results from RNA
interference (RNAi), a defined, though partially characterized
pathway whereby small inhibitory RNA (siRNA) act in concert with
host proteins (e.g., the RNA induced silencing complex, RISC) to
degrade messenger RNA (mRNA) in a sequence-dependent fashion. The
level of gene silencing can be measured by a variety of means,
including, but not limited to, measurement of transcript levels by
Northern Blot Analysis, B-DNA techniques, transcription-sensitive
reporter constructs, expression profiling (e.g., DNA chips), and
related technologies. Alternatively, the level of silencing can be
measured by assessing the level of the protein encoded by a
specific gene. This can be accomplished by performing a number of
studies including Western Analysis, measuring the levels of
expression of a reporter protein that has e.g., fluorescent
properties (e.g., GFP) or enzymatic activity (e.g., alkaline
phosphatases), or several other procedures.
[0105] Filters
[0106] The term "filter" refers to one or more procedures that are
performed on sequences that are identified by the algorithm. In
some instances, filtering includes in silico procedures where
sequences identified by the algorithm can be screened to identify
duplexes carrying desirable or undesirable motifs. Sequences
carrying such motifs can be selected for, or selected against, to
obtain a final set with the preferred properties. In other
instances, filtering includes wet lab experiments. For instance,
sequences identified by one or more versions of the algorithm can
be screened using any one of a number of procedures to identify
duplexes that have hyperfunctional traits (e.g., they exhibit a
high degree of silencing at subnanomolar concentrations and/or
exhibit high degrees of silencing longevity).
[0107] Transfection
[0108] The term "transfection" refers to a process by which agents
are introduced into a cell. The list of agents that can be
transfected is large and includes, but is not limited to, siRNA,
sense and/or anti-sense sequences, DNA encoding one or more genes
and organized into an expression plasmid, proteins, protein
fragments, and more. There are multiple methods for transfecting
agents into a cell including, but not limited to, electroporation,
calcium phosphate-based transfections, DEAE-dextran-based
transfections, lipid-based transfections, molecular conjugate-based
transfections (e.g., polylysine-DNA conjugates), microinjection and
others.
[0109] Target
[0110] The term "target" is used in a variety of different forms
throughout this document and is defined by the context in which it
is used. "Target mRNA" refers to a messenger RNA to which a given
siRNA can be directed against. "Target sequence" and "target site"
refer to a sequence within the mRNA to which the sense strand of an
siRNA shows varying degrees of homology and the antisense strand
exhibits varying degrees of complementarity. The phrase "siRNA
target" can refer to the gene, mRNA, or protein against which an
siRNA is directed. Similarly, "target silencing" can refer to the
state of a gene, or the corresponding mRNA or protein.
[0111] Off-Target Silencing and Off-Target Interference
[0112] The phrases "off-target silencing" and "off-target
interference" are defined as degradation of mRNA other than the
intended target mRNA due to overlapping and/or partial homology
with secondary mRNA messages.
[0113] SMARTscore.TM.
[0114] The term "SMARTscore.TM." refers to a number determined by
applying any of the Formulas I-Formula X to a given siRNA sequence.
The phrases "SMART-selected" or "rationally selected" or "rational
selection" refer to siRNA that have been selected on the basis of
their SMARTscores.TM..
[0115] Complementary
[0116] The term "complementary" refers to the ability of
polynucleotides to form base pairs with one another. Base pairs are
typically formed by hydrogen bonds between nucleotide units in
antiparallel polynucleotide strands. Complementary polynucleotide
strands can base pair in the Watson-Crick manner (e.g., A to T, A
to U, C to G), or in any other manner that allows for the formation
of duplexes. As persons skilled in the art are aware, when using
RNA as opposed to DNA, uracil rather than thymine is the base that
is considered to be complementary to adenosine. However, when a U
is denoted in the context of the present invention, the ability to
substitute a T is implied, unless otherwise stated.
[0117] Perfect complementarity or 100% complementarity refers to
the situation in which each nucleotide unit of one polynucleotide
strand can hydrogen bond with a nucleotide unit of a second
polynucleotide strand. Less than perfect complementarity refers to
the situation in which some, but not all, nucleotide units of two
strands can hydrogen bond with each other. For example, for two
20-mers, if only two base pairs on each strand can hydrogen bond
with each other, the polynucleotide strands exhibit 10%
complementarity. In the same example, if 18 base pairs on each
strand can hydrogen bond with each other, the polynucleotide
strands exhibit 90% complementarity. "Substantial complementarity"
refers to polynucleotide strands exhibiting 79% or greater
complementarity, excluding regions of the polynucleotide strands,
such as overhangs, that are selected so as to be noncomplementary.
("Substantial similarity" refers to polynucleotide strands
exhibiting 79% or greater similarity, excluding regions of the
polynucleotide strands, such as overhangs, that are selected so as
not to be similar.) Thus, for example, two polynucleotides of 29
nucleotide units each, wherein each comprises a di-dT at the 3'
terminus such that the duplex region spans 27 bases, and wherein 26
of the 27 bases of the duplex region on each strand are
complementary, are substantially complementary since they are 96.3%
complementary when excluding the di-dT overhangs.
[0118] Deoxynucleotide
[0119] The term "deoxynucleotide" refers to a nucleotide or
polynucleotide lacking a hydroxyl group (OH group) at the 2' and/or
3' position of a sugar moiety. Instead, it has a hydrogen bonded to
the 2' and/or 3' carbon. Within an RNA molecule that comprises one
or more deoxynucleotides, "deoxynucleotide" refers to the lack of
an OH group at the 2' position of the sugar moiety, having instead
a hydrogen bonded directly to the 2' carbon.
[0120] Deoxyribonucleotide
[0121] The terms "deoxyribonucleotide" and "DNA" refer to a
nucleotide or polynucleotide comprising at least one sugar moiety
that has an H, rather than an OH, at its 2' and/or 3' position.
[0122] Substantially Similar
[0123] The phrase "substantially similar" refers to a similarity of
at least 90% with respect to the identity of the bases of the
sequence.
[0124] Duplex Region
[0125] The phrase "duplex region" refers to the region in two
complementary or substantially complementary polynucleotides that
form base pairs with one another, either by Watson-Crick base
pairing or any other manner that allows for a stabilized duplex
between polynucleotide strands that are complementary or
substantially complementary. For example, a polynucleotide strand
having 21 nucleotide units can base pair with another
polynucleotide of 21 nucleotide units, yet only 19 bases on each
strand are complementary or substantially complementary, such that
the "duplex region" has 19 base pairs. The remaining bases may, for
example, exist as 5' and 3' overhangs. Further, within the duplex
region, 100% complementarity is not required; substantial
complementarity is allowable within a duplex region. Substantial
complementarity refers to 79% or greater complementarity. For
example, a mismatch in a duplex region consisting of 19 base pairs
results in 94.7% complementarity, rendering the duplex region
substantially complementary.
[0126] Nucleotide
[0127] The term "nucleotide" refers to a ribonucleotide or a
deoxyribonucleotide or modified form thereof, as well as an analog
thereof. Nucleotides include species that comprise purines, e.g.,
adenine, hypoxanthine, guanine, and their derivatives and analogs,
as well as pyrimidines, e.g., cytosine, uracil, thymine, and their
derivatives and analogs.
[0128] Nucleotide analogs include nucleotides having modifications
in the chemical structure of the base, sugar and/or phosphate,
including, but not limited to, 5-position pyrimidine modifications,
8-position purine modifications, modifications at cytosine
exocyclic amines, and substitution of 5-bromo-uracil; and
2'-position sugar modifications, including but not limited to,
sugar-modified ribonucleotides in which the 2'--OH is replaced by a
group such as an H, OR, R, halo, SH, SR, NH.sub.2, NHR, NR.sub.2,
or CN, wherein R is an alkyl moiety. Nucleotide analogs are also
meant to include nucleotides with bases such as inosine, queuosine,
xanthine, sugars such as 2'-methyl ribose, non-natural
phosphodiester linkages such as methylphosphonates,
phosphorothioates and peptides.
[0129] Modified bases refer to nucleotide bases such as, for
example, adenine, guanine, cytosine, thymine, uracil, xanthine,
inosine, and queuosine that have been modified by the replacement
or addition of one or more atoms or groups. Some examples of types
of modifications that can comprise nucleotides that are modified
with respect to the base moieties include but are not limited to,
alkylated, halogenated, thiolated, aminated, amidated, or
acetylated bases, individually or in combination. More specific
examples include, for example, 5-propynyluridine,
5-propynylcytidine, 6-methyladenine, 6-methylguanine,
N,N,-dimethyladenine, 2-propyladenine, 2-propylguanine,
2-aminoadenine, 1-methylinosine, 3-methyluridine, 5-methylcytidine,
5-methyluridine and other nucleotides having a modification at the
5 position, 5-(2-amino)propyl uridine, 5-halocytidine,
5-halouridine, 4-acetylcytidine, 1-methyladenosine,
2-methyladenosine, 3-methylcytidine, 6-methyluridine,
2-methylguanosine, 7-methylguanosine, 2,2-dimethylguanosine,
5-methylaminoethyluridine, 5-methyloxyuridine, deazanucleotides
such as 7-deaza-adenosine, 6-azouridine, 6-azocytidine,
6-azothymidine, 5-methyl-2-thiouridine, other thio bases such as
2-thiouridine and 4-thiouridine and 2-thiocytidine, dihydrouridine,
pseudouridine, queuosine, archaeosine, naphthyl and substituted
naphthyl groups, any O- and N-alkylated purines and pyrimidines
such as N6-methyladenosine, 5-methylcarbonylmethyluridine, uridine
5-oxyacetic acid, pyridine-4-one, pyridine-2-one, phenyl and
modified phenyl groups such as aminophenol or 2,4,6-trimethoxy
benzene, modified cytosines that act as G-clamp nucleotides,
8-substituted adenines and guanines, 5-substituted uracils and
thymines, azapyrimidines, carboxyhydroxyalkyl nucleotides,
carboxyalkylaminoalkyl nucleotides, and alkylcarbonylalkylated
nucleotides. Modified nucleotides also include those nucleotides
that are modified with respect to the sugar moiety, as well as
nucleotides having sugars or analogs thereof that are not ribosyl.
For example, the sugar moieties may be, or be based on, mannoses,
arabinoses, glucopyranoses, galactopyranoses, 4'-thioribose, and
other sugars, heterocycles, or carbocycles.
[0130] The term nucleotide is also meant to include what are known
in the art as universal bases. By way of example, universal bases
include but are not limited to 3-nitropyrrole, 5-nitroindole, or
nebularine. The term "nucleotide" is also meant to include the N3'
to P5' phosphoramidate, resulting from the substitution of a
ribosyl 3' oxygen with an amine group.
[0131] Further, the term nucleotide also includes those species
that have a detectable label, such as for example a radioactive or
fluorescent moiety, or mass label attached to the nucleotide.
[0132] Polynucleotide
[0133] The term "polynucleotide" refers to polymers of nucleotides,
and includes but is not limited to DNA, RNA, DNA/RNA hybrids
including polynucleotide chains of regularly and/or irregularly
alternating deoxyribosyl moieties and ribosyl moieties (i.e.,
wherein alternate nucleotide units have an --OH, then and --H, then
an --OH, then an --H, and so on at the 2' position of a sugar
moiety), and modifications of these kinds of polynucleotides,
wherein the attachment of various entities or moieties to the
nucleotide units at any position are included.
[0134] Polyribonucleotide
[0135] The term "polyribonucleotide" refers to a polynucleotide
comprising two or more modified or unmodified ribonucleotides
and/or their analogs. The term "polyribonucleotide" is used
interchangeably with the term "oligoribonucleotide."
[0136] Ribonucleotide and Ribonucleic Acid
[0137] The term "ribonucleotide" and the phrase "ribonucleic acid"
(RNA), refer to a modified or unmodified nucleotide or
polynucleotide comprising at least one ribonucleotide unit. A
ribonucleotide unit comprises an hydroxyl group attached to the 2'
position of a ribosyl moiety that has a nitrogenous base attached
in N-glycosidic linkage at the 1' position of a ribosyl moiety, and
a moiety that either allows for linkage to another nucleotide or
precludes linkage.
DETAILED DESCRIPTION OF THE INVENTION
[0138] The present invention is directed to improving the
efficiency of gene silencing by siRNA. Through the inclusion of
multiple siRNA sequences that are targeted to a particular gene
and/or selecting an siRNA sequence based on certain defined
criteria, improved efficiency may be achieved.
[0139] The present invention will now be described in connection
with preferred embodiments. These embodiments are presented in
order to aid in an understanding of the present invention and are
not intended, and should not be construed, to limit the invention
in any way. All alternatives, modifications and equivalents that
may become apparent to those of ordinary skill upon reading this
disclosure are included within the spirit and scope of the present
invention.
[0140] Furthermore, this disclosure is not a primer on RNA
interference. Basic concepts known to persons skilled in the art
have not been set forth in detail.
[0141] The present invention is directed to increasing the
efficiency of RNAi, particularly in mammalian systems. Accordingly,
the present invention provides kits, siRNAs and methods for
increasing siRNA efficacy.
[0142] According to a first embodiment, the present invention
provides a kit for gene silencing, wherein said kit is comprised of
a pool of at least two siRNA duplexes, each of which is comprised
of a sequence that is complementary to a portion of the sequence of
one or more target messenger RNA, and each of which is selected
using non-target specific criteria. Each of the at least two siRNA
duplexes of the kit complementary to a portion of the sequence of
one or more target mRNAs is preferably selected using Formula
X.
[0143] According to a second embodiment, the present invention
provides a method for selecting an siRNA, said method comprising
applying selection criteria to a set of potential siRNA that
comprise 18-30 base pairs, wherein said selection criteria are
non-target specific criteria, and said set comprises at least two
siRNAs and each of said at least two siRNAs contains a sequence
that is at least substantially complementary to a target gene; and
determining the relative functionality of the at least two
siRNAs.
[0144] In one embodiment, the present invention also provides a
method wherein said selection criteria are embodied in a formula
comprising:
(-14)*G.sub.13-13*A.sub.1-12*U.sub.7-11*U.sub.2-10*A.sub.11-10*U.sub.4-10*-
C.sub.3-10*C.sub.5-10*C.sub.6-9*A.sub.10-9*U.sub.9-9*C.sub.18-8*G.sub.10-7-
*U.sub.1-7*U.sub.16-7*C.sub.17-7*C.sub.19+7*U.sub.17+8*A.sub.2+8*A.sub.4+8-
*A.sub.5+8*C.sub.4+9*G.sub.8+10*A.sub.7+10*U.sub.18+11*A.sub.19+11*C.sub.9-
+15*G.sub.1+18*A.sub.3+19*U.sub.10-Tm-3*
(GC.sub.total)-6*(GC.sub.15-19)-3- 0*X; or Formula VIII
(-8)*A1+(-1)*A2+(12)*A3+(7)*A4+(18)*A5+(12)*A6+(19)*A7+(6)*A8+(-4)*A9+(-5)-
*A10+(-2)*A11+(-5)*A12+(17)*A13+(-3)*A14+(4)*A15+(2)*A16+(8)*A17+(11)*A18+-
(30)*A19+(-13)*U1+(-10)*U2+(2)*U3+(-2)*U4+(-5)*U5+(5)*U6+(-2)*U7+(-10)*U8+-
(-5)*U9+(15)*U10+(-1)*U11+(0)*U12+(10)*U13+(-9)*U14+(-13)*U15+(-10)*U16+(3-
)*U17+(9)*U18+(9)*U19+(7)*C1+(3)*C2+(-21)*C3+(5)*C4+(-9)*C5+(-20)*C6+(-18)-
*C7+(-5)*C8+(5)*C9+(1)*C10+(2)*C11+(-5)*C12+(-3)*C13+(-6)*C14+(-2)*C15+(-5-
)*C16+(-3)*C17+(-12)*C18+(-18)*C19+(14)*G1+(8)*G2+(7)*G3+(-10)*G4+(-4)*G5+-
(2)*G6+(1)*G7+(9)*G8+(5)*G9+(-11)*G10+(1)*G11+(9)*G12+(-24)*G13+(18)*G14+(-
11)*G15+(13)*G16+(-7)*G17+(-9)*G18+(-22)*G19+6*(number of A+U in
position 15-19)-3*(number of G+C in whole siRNA), Formula X
[0145] wherein position numbering begins at the 5'-most position of
a sense strand, and
[0146] A.sub.1=1 if A is the base at position 1 of the sense
strand, otherwise its value is 0;
[0147] A.sub.2=1 if A is the base at position 2 of the sense
strand, otherwise its value is 0;
[0148] A.sub.3=1 if A is the base at position 3 of the sense
strand, otherwise its value is 0;
[0149] A.sub.4=1 if A is the base at position 4 of the sense
strand, otherwise its value is 0;
[0150] A.sub.5=1 if A is the base at position 5 of the sense
strand, otherwise its value is 0;
[0151] A.sub.6=1 if A is the base at position 6 of the sense
strand, otherwise its value is 0;
[0152] A.sub.7=1 if A is the base at position 7 of the sense
strand, otherwise its value is 0;
[0153] A.sub.10=1 if A is the base at position 10 of the sense
strand, otherwise its value is 0;
[0154] A.sub.11=1 if A is the base at position 11 of the sense
strand, otherwise its value is 0;
[0155] A.sub.13=1 if A is the base at position 13 of the sense
strand, otherwise its value is 0;
[0156] A.sub.19=1 if A is the base at position 19 of the sense
strand, otherwise if another base is present or the sense strand is
only 18 base pairs in length, its value is 0;
[0157] C.sub.3=1 if C is the base at position 3 of the sense
strand, otherwise its value is 0;
[0158] C.sub.4=1 if C is the base at position 4 of the sense
strand, otherwise its value is 0;
[0159] C.sub.5=1 if C is the base at position 5 of the sense
strand, otherwise its value is 0;
[0160] C.sub.6=1 if C is the base at position 6 of the sense
strand, otherwise its value is 0;
[0161] C.sub.7=1 if C is the base at position 7 of the sense
strand, otherwise its value is 0;
[0162] C.sub.9=1 if C is the base at position 9 of the sense
strand, otherwise its value is 0;
[0163] C.sub.17=1 if C is the base at position 17 of the sense
strand, otherwise its value is 0;
[0164] C.sub.18=1 if C is the base at position 18 of the sense
strand, otherwise its value is 0;
[0165] C.sub.19=1 if C is the base at position 19 of the sense
strand, otherwise if another base is present or the sense strand is
only 18 base pairs in length, its value is 0;
[0166] G.sub.1=1 if G is the base at position 1 on the sense
strand, otherwise its value is 0;
[0167] G.sub.2=1 if G is the base at position 2 of the sense
strand, otherwise its value is 0;
[0168] G.sub.8=1 if G is the base at position 8 on the sense
strand, otherwise its value is 0;
[0169] G.sub.10=1 if G is the base at position 10 on the sense
strand, otherwise its value is 0;
[0170] G.sub.13=1 if G is the base at position 13 on the sense
strand, otherwise its value is 0;
[0171] G.sub.19=1 if G is the base at position 19 of the sense
strand, otherwise if another base is present or the sense strand is
only 18 base pairs in length, its value is 0;
[0172] U.sub.1=1 if U is the base at position 1 on the sense
strand, otherwise its value is 0;
[0173] U.sub.2=1 if U is the base at position 2 on the sense
strand, otherwise its value is 0;
[0174] U.sub.3=1 if U is the base at position 3 on the sense
strand, otherwise its value is 0;
[0175] U.sub.4=1 if U is the base at position 4 on the sense
strand, otherwise its value is 0;
[0176] U.sub.7=1 if U is the base at position 7 on the sense
strand, otherwise its value is 0;
[0177] U.sub.9=1 if U is the base at position 9 on the sense
strand, otherwise its value is 0;
[0178] U .sub.10=1 if U is the base at position 10 on the sense
strand, otherwise its value is 0;
[0179] U.sub.15=1 if U is the base at position 15 on the sense
strand, otherwise its value is 0;
[0180] U.sub.16=1 if U is the base at position 16 on the sense
strand, otherwise its value is 0;
[0181] U.sub.17=1 if U is the base at position 17 on the sense
strand, otherwise its value is 0;
[0182] U.sub.18=1 if U is the base at position 18 on the sense
strand, otherwise its value is 0.
[0183] GC.sub.15-19=the number of G and C bases within positions
15-19 of the sense strand, or within positions 15-18 if the sense
strand is only 18 base pairs in length;
[0184] GC.sub.total=the number of G and C bases in the sense
strand;
[0185] Tm=100 if the siRNA oligo has the internal repeat longer
then 4 base pairs, otherwise its value is 0; and
[0186] X=the number of times that the same nucleotide repeats four
or more times in a row.
[0187] Any of the methods of selecting siRNA in accordance with the
invention can further comprise comparing the internal stability
profiles of the siRNAs to be selected, and selecting those siRNAs
with the most favorable internal stability profiles. Any of the
methods of selecting siRNA can further comprise selecting either
for or against sequences that contain motifs that induce cellular
stress. Such motifs include, for example, toxicity motifs. Any of
the methods of selecting siRNA can further comprise either
selecting for or selecting against sequences that comprise
stability motifs.
[0188] In another embodiment, the present invention provides a
method of gene silencing, comprising introducing into a cell at
least one siRNA selected according to any of the methods of the
present invention. The siRNA can be introduced by allowing passive
uptake of siRNA, or through the use of a vector.
[0189] According to a third embodiment, the invention provides a
method for developing an algorithm for selecting siRNA, said method
comprising: (a) selecting a set of siRNA; (b) measuring gene
silencing ability of each siRNA from said set; (c) determining
relative functionality of each siRNA; (d) determining improved
functionality by the presence or absence of at least one variable
selected from the group consisting of the presence or absence of a
particular nucleotide at a particular position, the total number of
As and Us in positions 15-19, the number of times that the same
nucleotide repeats within a given sequence, and the total number of
Gs and Cs; and (e) developing an algorithm using the information of
step (d).
[0190] In another embodiment, the invention provides a method for
selecting an siRNA with improved functionality, comprising using
the above-mentioned algorithm to identify an siRNA of improved
functionality.
[0191] According to a fourth embodiment, the present invention
provides a kit, wherein said kit is comprised of at least two
siRNAs, wherein said at least two siRNAs comprise a first optimized
siRNA and a second optimized siRNA, wherein said first optimized
siRNA and said second optimized siRNA are optimized according a
formula comprising Formula X.
[0192] According to a fifth embodiment, the present invention
provides a method for identifying a hyperfunctional siRNA,
comprising applying selection criteria to a set of potential siRNA
that comprise 18-30 base pairs, wherein said selection criteria are
non-target specific criteria, and said set comprises at least two
siRNAs and each of said at least two siRNAs contains a sequence
that is at least substantially complementary to a target gene;
determining the relative functionality of the at least two siRNAs
and assigning each of the at least two siRNAs a functionality
score; and selecting siRNAs from the at least two siRNAs that have
a functionality score that reflects greater than 80 percent
silencing at a concentration in the picomolar range, wherein said
greater than 80 percent silencing endures for greater than 120
hours.
[0193] In other embodiments, the invention provides kits and/or
methods wherein the siRNA are comprised of two separate
polynucleotide strands; wherein the siRNA are comprised of a single
contiguous molecule such as, for example, a unimolecular siRNA
(comprising, for example, either a nucleotide or non-nucleotide
loop); wherein the siRNA are expressed from one or more vectors;
and wherein two or more genes are silenced by a single
administration of siRNA.
[0194] According to a sixth embodiment, the present invention
provides a hyperfunctional siRNA that is capable of silencing
Bc12.
[0195] According to a seventh embodiment, the present invention
provides a method for developing an siRNA algorithm for selecting
functional and hyperfunctional siRNAs for a given sequence. The
method comprises:
[0196] (a) selecting a set of siRNAs;
[0197] (b) measuring the gene silencing ability of each siRNA from
said set;
[0198] (c) determining the relative functionality of each
siRNA;
[0199] (d) determining the amount of improved functionality by the
presence or absence of at least one variable selected from the
group consisting of the total GC content, melting temperature of
the siRNA, GC content at positions 15-19, the presence or absence
of a particular nucleotide at a particular position, relative
thermodynamic stability at particular positions in a duplex, and
the number of times that the same nucleotide repeats within a given
sequence; and
[0200] (e) developing an algorithm using the information of step
(d).
[0201] According to this embodiment, preferably the set of siRNAs
comprises at least 90 siRNAs from at least one gene, more
preferably at least 180 siRNAs from at least two different genes,
and most preferably at least 270 and 360 siRNAs from at least three
and four different genes, respectively. Additionally, in step (d)
the determination is made with preferably at least two, more
preferably at least three, even more preferably at least four, and
most preferably all of the variables. The resulting algorithm is
not target sequence specific.
[0202] In another embodiment, the present invention provides
rationally designed siRNAs identified using the formulas above.
[0203] In yet another embodiment, the present invention is directed
to hyperfunctional siRNA.
[0204] The ability to use the above algorithms, which are not
sequence or species specific, allows for the cost-effective
selection of optimized siRNAs for specific target sequences.
Accordingly, there will be both greater efficiency and reliability
in the use of siRNA technologies.
[0205] The methods disclosed herein can be used in conjunction with
comparing internal stability profiles of selected siRNAs, and
designing an siRNA with a desireable internal stability profile;
and/or in conjunction with a selection either for or against
sequences that contain motifs that induce cellular stress, for
example, cellular toxicity.
[0206] Any of the methods disclosed herein can be used to silence
one or more genes by introducing an siRNA selected, or designed, in
accordance with any of the methods disclosed herein. The siRNA(s)
can be introduced into the cell by any method known in the art,
including passive uptake or through the use of one or more
vectors.
[0207] Any of the methods and kits disclosed herein can employ
either unimolecular siRNAs, siRNAs comprised of two separate
polynucleotide strands, or combinations thereof. Any of the methods
disclosed herein can be used in gene silencing, where two or more
genes are silenced by a single administration of siRNA(s). The
siRNA(s) can be directed against two or more target genes, and
administered in a single dose or single transfection, as the case
may be.
[0208] Optimizing siRNA
[0209] According to one embodiment, the present invention provides
a method for improving the effectiveness of gene silencing for use
to silence a particular gene through the selection of an optimal
siRNA. An siRNA selected according to this method may be used
individually, or in conjunction with the first embodiment, i.e.,
with one or more other siRNAs, each of which may or may not be
selected by this criteria in order to maximize their
efficiency.
[0210] The degree to which it is possible to select an siRNA for a
given mRNA that maximizes these criteria will depend on the
sequence of the mRNA itself. However, the selection criteria will
be independent of the target sequence. According to this method, an
siRNA is selected for a given gene by using a rational design. That
said, rational design can be described in a variety of ways.
Rational design is, in simplest terms, the application of a proven
set of criteria that enhance the probability of identifying a
functional or hyperfunctional siRNA. In one method, rationally
designed siRNA can be identified by maximizing one or more of the
following criteria:
[0211] 1. A low GC content, preferably between about 30-52%.
[0212] 2. At least 2, preferably at least 3 A or U bases at
positions 15-19 of the siRNA on the sense strand.
[0213] 3. An A base at position 19 of the sense strand.
[0214] 4. An A base at position 3 of the sense strand.
[0215] 5. A U base at position 10 of the sense strand.
[0216] 6. An A base at position 14 of the sense strand.
[0217] 7. A base other than C at position 19 of the sense
strand.
[0218] 8. A base other than G at position 13 of the sense
strand.
[0219] 9. A Tm, which refers to the character of the internal
repeat that results in inter- or intramolecular structures for one
strand of the duplex, that is preferably not stable at greater than
50.degree. C., more preferably not stable at greater than
37.degree. C., even more preferably not stable at greater than
30.degree. C. and most preferably not stable at greater than
20.degree. C.
[0220] 10. A base other than U at position 5 of the sense
strand.
[0221] 11. A base other than A at position 11 of the sense
strand.
[0222] 12. A base other than an A at position 1 of the sense
strand.
[0223] 13. A base other than an A at position 2 of the sense
strand.
[0224] 14. An A base at position 4 of the sense strand.
[0225] 15. An A base at position 5 of the sense strand.
[0226] 16. An A base at position 6 of the sense strand.
[0227] 17. An A base at position 7 of the sense strand.
[0228] 18. An A base at position 8 of the sense strand.
[0229] 19. A base other than an A at position 9 of the sense
strand.
[0230] 20. A base other than an A at position 10 of the sense
strand.
[0231] 21. A base other than an A at position 11 of the sense
strand.
[0232] 22. A base other than an A at position 12 of the sense
strand.
[0233] 23. An A base at position 13 of the sense strand.
[0234] 24. A base other than an A at position 14 of the sense
strand.
[0235] 25. An A base at position 15 of the sense strand
[0236] 26. An A base at position 16 of the sense strand.
[0237] 27. An A base at position 17 of the sense strand.
[0238] 28. An A base at position 18 of the sense strand.
[0239] 29. A base other than a U at position 1 of the sense
strand.
[0240] 30. A base other than a U at position 2 of the sense
strand.
[0241] 31. A U base at position 3 of the sense strand.
[0242] 32. A base other than a U at position 4 of the sense
strand.
[0243] 33. A base other than a U at position 5 of the sense
strand.
[0244] 34. A U base at position 6 of the sense strand.
[0245] 35. A base other than a U at position 7 of the sense
strand.
[0246] 36. A base other than a U at position 8 of the sense
strand.
[0247] 37. A base other than a U at position 9 of the sense
strand.
[0248] 38. A base other than a U at position 11 of the sense
strand.
[0249] 39. A U base at position 13 of the sense strand.
[0250] 40. A base other than a U at position 14 of the sense
strand.
[0251] 41. A base other than a U at position 15 of the sense
strand.
[0252] 42. A base other than a U at position 16 of the sense
strand.
[0253] 43. A U base at position 17 of the sense strand.
[0254] 44. A U base at position 18 of the sense strand.
[0255] 45. A U base at position 19 of the sense strand.
[0256] 46. A C base at position 1 of the sense strand.
[0257] 47. A C base at position 2 of the sense strand.
[0258] 48. A base other than a C at position 3 of the sense
strand.
[0259] 49. A C base at position 4 of the sense strand.
[0260] 50. A base other than a C at position 5 of the sense
strand.
[0261] 51. A base other than a C at position 6 of the sense
strand.
[0262] 52. A base other than a C at position 7 of the sense
strand.
[0263] 53. A base other than a C at position 8 of the sense
strand.
[0264] 54. A C base at position 9 of the sense strand.
[0265] 55. A C base at position 10 of the sense strand.
[0266] 56. A C base at position 11 of the sense strand.
[0267] 57. A base other than a C at position 12 of the sense
strand.
[0268] 58. A base other than a C at position 13 of the sense
strand.
[0269] 59. A base other than a C at position 14 of the sense
strand.
[0270] 60. A base other than a C at position 15 of the sense
strand.
[0271] 61. A base other than a C at position 16 of the sense
strand.
[0272] 62. A base other than a C at position 17 of the sense
strand.
[0273] 63. A base other than a C at position 18 of the sense
strand.
[0274] 64. A G base at position 1 of the sense strand.
[0275] 65. A G base at position 2 of the sense strand.
[0276] 66. A G base at position 3 of the sense strand.
[0277] 67. A base other than a G at position 4 of the sense
strand.
[0278] 68. A base other than a G at position 5 of the sense
strand.
[0279] 69. A G base at position 6 of the sense strand.
[0280] 70. A G base at position 7 of the sense strand.
[0281] 71. A G base at position 8 of the sense strand.
[0282] 72. A G base at position 9 of the sense strand.
[0283] 73. A base other than a G at position 10 of the sense
strand.
[0284] 74. A G base at position 11 of the sense strand.
[0285] 75. A G base at position 12 of the sense strand.
[0286] 76. A G base at position 14 of the sense strand.
[0287] 77. A G base at position 15 of the sense strand.
[0288] 78. A G base at position 16 of the sense strand.
[0289] 79. A base other than a G at position 17 of the sense
strand.
[0290] 80. A base other than a G at position 18 of the sense
strand.
[0291] 81. A base other than a G at position 19 of the sense
strand.
[0292] The importance of various criteria can vary greatly. For
instance, a C base at position 10 of the sense strand makes a minor
contribution to duplex functionality. In contrast, the absence of a
C at position 3 of the sense strand is very important. Accordingly,
preferably an siRNA will satisfy as many of the aforementioned
criteria as possible.
[0293] With respect to the criteria, GC content, as well as a high
number of AU in positions 15-19 of the sense strand, may be
important for easement of the unwinding of double stranded siRNA
duplex. Duplex unwinding has been shown to be crucial for siRNA
functionality in vivo.
[0294] With respect to criterion 9, the internal structure is
measured in terms of the melting temperature of the single strand
of siRNA, which is the temperature at which 50% of the molecules
will become denatured. With respect to criteria 2-8 and 10-11, the
positions refer to sequence positions on the sense strand, which is
the strand that is identical to the mRNA.
[0295] In one preferred embodiment, at least criteria 1 and 8 are
satisfied. In another preferred embodiment, at least criteria 7 and
8 are satisfied. In still another preferred embodiment, at least
criteria 1, 8 and 9 are satisfied.
[0296] It should be noted that all of the aforementioned criteria
regarding sequence position specifics are with respect to the 5'
end of the sense strand. Reference is made to the sense strand,
because most databases contain information that describes the
information of the mRNA. Because according to the present invention
a chain can be from 18 to 30 bases in length, and the
aforementioned criteria assumes a chain 19 base pairs in length, it
is important to keep the aforementioned criteria applicable to the
correct bases.
[0297] When there are only 18 bases, the base pair that is not
present is the base pair that is located at the 3' of the sense
strand. When there are twenty to thirty bases present, then
additional bases are added at the 5' end of the sense chain and
occupy positions .sup.-1 to .sup.-11. Accordingly, with respect to
SEQ. ID NO. 0001 NNANANNNNUCNAANNNNA and SEQ. ID NO. 0028
GUCNNANANNNNUCNAANNNNA, both would have A at position 3, A at
position 5, U at position 10, C at position 11, A and position 13,
A and position 14 and A at position 19. However, SEQ. ID NO. 0028
would also have C at position -1, U at position -2 and G at
position -3.
[0298] For a 19 base pair siRNA, an optimal sequence of one of the
strands may be represented below, where N is any base, A, C, G, or
U:
1 NNANANNNNUCNAANNNNA. SEQ. ID NO. 0001 NNANANNNNUGNAANNNNA. SEQ.
ID NO. 0001 NNANANNNNUUNAANNNNA. SEQ. ID NO. 0002
NNANANNNNUCNCANNNNA. SEQ. ID NO. 0003 NNANANNNNUGNCANNNNA. SEQ. ID
NO. 0004 NNANANNNNUUNCANNNNA. SEQ. ID NO. 0005 NNANANNNNUCNUANNNNA.
SEQ. ID NO. 0006 NNANANNNNUGNUANNNNA. SEQ. ID NO. 0007
NNANANNNNUUNUANNNNA. SEQ. ID NO. 0008 NNANCNNNNUCNAANNNNA. SEQ. ID
NO. 0010 NNANCNNNNUGNAANNNNA. SEQ. ID NO. 0011 NNANCNNNNUUNAANNNNA.
SEQ. ID NO. 0012 NNANCNNNNUCNCANNNNA. SEQ. ID NO. 0013
NNANCNNNNUGNCANNNNA. SEQ. ID NO. 0014 NNANCNNNNUUNCANNNNA. SEQ. ID
NO. 0015 NANCNNNNUCNUANNNNA. SEQ. ID NO. 0016 NNANCNNNNUGNUANNNNA.
SEQ. ID NO. 0017 NNANCNNNNUUNUANNNNA. SEQ. ID NO. 0018
NNANGNNNNUCNAANNNNA. SEQ. ID NO. 0019 NNANGNNNNUGNAANNNNA. SEQ. ID
NO. 0020 NNANGNNNNUUNAANNNNA. SEQ. ID NO. 0021 NNANGNNNNUCNCANNNNA.
SEQ. ID NO. 0022 NNANGNNNNUGNCANNNNA. SEQ. ID NO. 0023
NNANGNNNNUUNCANNNNA. SEQ. ID NO. 0024 NNANGNNNNUCNUANNNNA. SEQ. ID
NO. 0025 NNANGNNNNUGNUANNNNA. SEQ. ID NO. 0026 NNANGNNNNNUNUANNNNA.
SEQ. ID NO. 0027
[0299] In one embodiment, the sequence used as an siRNA is selected
by choosing the siRNA that score highest according to one of the
following seven algorithms that are represented by Formulas
I-VII:
Relative functionality of
siRNA=-(GC/3)+(AU.sub.15-19)-Tm.sub.20.degree.C.-
)*3-(G.sub.13)*3)-(C.sub.19) Formula I
Relative functionality of
siRNA=-(GC/3)-(AU.sub.15-19)*3-(G.sub.13)*3-(C.s-
ub.19)+(A.sub.19)*2+(A.sub.3) Formula II
Relative functionality of
siRNA=-(GC/3)+(AU.sub.15-19)-(Tm.sub.20.degree.C- .)*3 Formula
III
Relative functionality of
siRNA=GC/2+(AU.sub.15-19)/2-(Tm.sub.20.degree.C.-
)*2-(G.sub.13)*3-(C.sub.19)+(A.sub.19)*2+(A.sub.3)+(U.sub.10)+(A.sub.14)-(-
U.sub.5)-(A.sub.11) Formula IV
Relative functionality of
siRNA=-(G.sub.13)*3-(C.sub.19)+(A.sub.19)*2+(A.s-
ub.3)+(U.sub.10)+(A.sub.14)-(U.sub.5)-(A.sub.11) Formula V
Relative functionality of
siRNA=-(G.sub.13)*3-(C.sub.19)+(A.sub.19)*2+(A.s- ub.3) Formula
VI
Relative functionality of
siRNA=-(GC/2)+(AU.sub.15-19)/2-(Tm.sub.20.degree-
.C.)*1-(G.sub.13)*3-(C.sub.19)+(A.sub.19)*3+(A.sub.3)*3+(U.sub.10)/2+(A.su-
b.14)/2-(U.sub.5)/2-(A.sub.11)/2 Formula VII
[0300] In Formulas I-VII:
[0301] wherein A.sub.19=1 if A is the base at position 19 on the
sense strand, otherwise its value is 0,
[0302] AU.sub.15-19=0-5 depending on the number of A or U bases on
the sense strand at positions 15-19;
[0303] G.sub.13=1 if G is the base at position 13 on the sense
strand, otherwise its value is 0;
[0304] C.sub.19=1 if C is the base at position 19 of the sense
strand, otherwise its value is 0;
[0305] GC=the number of G and C bases in the entire sense
strand;
[0306] Tm.sub.20.degree. C.=1 if the Tm is greater than 20.degree.
C.;
[0307] A.sub.3=1 if A is the base at position 3 on the sense
strand, otherwise its value is 0;
[0308] U.sub.10=1 if U is the base at position 10 on the sense
strand, otherwise its value is 0;
[0309] A.sub.14=1 if A is the base at position 14 on the sense
strand, otherwise its value is 0;
[0310] U.sub.5=1 if U is the base at position 5 on the sense
strand, otherwise its value is 0; and
[0311] A.sub.11=1 if A is the base at position 11 of the sense
strand, otherwise its value is 0.
[0312] Formulas I-VII provide relative information regarding
functionality. When the values for two sequences are compared for a
given formula, the relative functionality is ascertained; a higher
positive number indicates a greater functionality. For example, in
many applications a value of 5 or greater is beneficial.
[0313] Additionally, in many applications, more than one of these
formulas would provide useful information as to the relative
functionality of potential siRNA sequences. However, it is
beneficial to have more than one type of formula, because not every
formula will be able to help to differentiate among potential siRNA
sequences. For example, in particularly high GC mRNAs, formulas
that take that parameter into account would not be useful and
application of formulas that lack GC elements (e.g., formulas V and
VI) might provide greater insights into duplex functionality.
Similarly, formula II might by used in situations where hairpin
structures are not observed in duplexes, and formula IV might be
applicable for sequences that have higher AU content. Thus, one may
consider a particular sequence in light of more than one or even
all of these algorithms to obtain the best differentiation among
sequences. In some instances, application of a given algorithim may
identify an unususally large number of potential siRNA sequences,
and in those cases, it may be appropriate to re-analyze that
sequence with a second algorithm that is, for instance, more
stringent. Alternatively, it is conceivable that analysis of a
sequence with a given formula yields no acceptable siRNA sequences
(i.e., low SMARTscores.TM.). In this instance, it may be
appropriate to re-analyze that sequences with a second algorithm
that is, for instance, less stringent. In still other instances,
analysis of a single sequence with two separate formulas may give
rise to conflicting results (i.e., one formula generates a set of
siRNA with high SMARTscores.TM. while the other formula identifies
a set of siRNA with low SMARTscores.TM.). In these instances, it
may be necessary to determine which weighted factor(s) (e.g., GC
content) are contributing to the discrepancy and assessing the
sequence to decide whether these factors should or should not be
included. Alternatively, the sequence could be analyzed by a third,
fourth, or fifth algorithm to identify a set of rationally designed
siRNA.
[0314] The above-referenced criteria are particularly advantageous
when used in combination with pooling techniques as depicted in
Table I:
2 TABLE I Functional Probability Oligos Pools Criteria >95%
>80% <70% >95% >80% <70% Current 33.0 50.0 23.0 79.5
97.3 0.3 New 50.0 88.5 8.0 93.8 99.98 0.005 (GC) 28.0 58.9 36.0
72.8 97.1 1.6
[0315] The term "current" used in Table I refers to Tuschl's
conventional siRNA parameters (Elbashir, S. M. et al. (2002)
"Analysis of gene function in somatic mammalian cells using small
interfering RNAs" Methods 26: 199-213). "New" refers to the design
parameters described in Formulas I-VII. "GC" refers to criteria
that select siRNA solely on the basis of GC content.
[0316] As Table I indicates, when more functional siRNA duplexes
are chosen, siRNAs that produce <70% silencing drops from 23% to
8% and the number of siRNA duplexes that produce >80% silencing
rises from 50% to 88.5%. Further, of the siRNA duplexes with
>80% silencing, a larger portion of these siRNAs actually
silence >95% of the target expression (the new criteria
increases the portion from 33% to 50%). Using this new criteria in
pooled siRNAs, shows that, with pooling, the amount of silencing
>95% increases from 79.5% to 93.8% and essentially eliminates
any siRNA pool from silencing less than 70%.
[0317] Table II similarly shows the particularly beneficial results
of pooling in combination with the aforementioned criteria.
However, Table II, which takes into account each of the
aforementioned variables, demonstrates even a greater degree of
improvement in functionality.
3TABLE II Functional Probability Oligos Pools Func- Non- Func- Non-
tional Average functional tional Average functional Random 20 40 50
67 97 3 Criteria 1 52 99 0.1 97 93 0.0040 Criteria 4 89 99 0.1 99
99 0.0000
[0318] The terms "functional," "Average," and "Non-functional" used
in Table II, refer to siRNA that exhibit >80%, >50%, and
<50% functionality, respectively. Criteria 1 and 4 refer to
specific criteria described above.
[0319] The above-described algorithms may be used with or without a
computer program that allows for the inputting of the sequence of
the mRNA and automatically outputs the optimal siRNA. The computer
program may, for example, be accessible from a local terminal or
personal computer, over an internal network or over the
Internet.
[0320] In addition to the formulas above, more detailed algorithms
may be used for selecting siRNA. Preferably, at least one RNA
duplex of 18-30 base pairs is selected such that it is optimized
according a formula selected from:
(-14)*G.sub.13-13*A.sub.1-12*U.sub.7-11*U.sub.2-10*A.sub.11-10*U.sub.4-10*-
C.sub.3-10*C.sub.5-10*C.sub.6-9*A.sub.10-9*U.sub.9-9*C.sub.18-8*G.sub.10-7-
*U.sub.1-7*U.sub.16-7*C.sub.17-7*C.sub.19+7*U.sub.17+8*A.sub.2+8*A.sub.4+8-
*A.sub.5+8*C.sub.4+9*G.sub.8+10*A.sub.7+10*U.sub.18+11*A.sub.19+11*C.sub.9-
+15*G.sub.1+18*A.sub.3+19*U.sub.10-Tm-3*
(GC.sub.total)-6*(GC.sub.15-19)-3- 0*X; and Formula VIII
(14.1)*A.sub.3+(14.9)*A.sub.6+(17.6)*A.sub.13+(24.7)*A.sub.19+(14.2)*U.sub-
.10+((10.5)*
C.sub.9+(23.9)*G.sub.1+(16.3)*G.sub.2+(-12.3)*A.sub.11+(-19.3-
)*U.sub.1+(-12.1)*U.sub.2+(-11)*U.sub.3+(-15.2)*U.sub.15+(-11.3)*U.sub.16+-
(-11.8)*C.sub.3+(-17.4)*C.sub.6+(-10.5)*C.sub.7+(-13.7)*G.sub.13+(-25.9)*G-
.sub.19-Tm-3*(GC.sub.total)-6*(GC.sub.15-19)-30*X; and Formula
IX
(-8)*A1+(-1)*A2+(12)*A3+(7)*A4+(18)*A5+(12)*A6+(19)*A7+(6)*A8+(-4)*A9+(-5)-
*A10+(-2)*A11+(-5)*A12+(17)*A13+(-3)*A14+(4)*A15+(2)*A16+(8)*A17+(11)*A18+-
(30)*A19+(-13)*U1+(-10)*U2+(2)*U3+(-2)*U4+(-5)*U5+(5)*U6+(-2)*U7+(-10)*U8+-
(-5)*U9+(15)*U10+(-1)*U11+(0)*U12+(10)*U13+(-9)*U14+(-13)*U15+(-10)*U16+(3-
)*U17+(9)*U18+(9)*U19+(7)*C1+(3)*C2+(-21)*C3+(5)*C4+(-9)*C5+(-20)*C6+(-18)-
*C7+(-5)*C8+(5)*C9+(1)*C10+(2)*C11+(-5)*C12+(-3)*C13+(-6)*C14+(-2)*C15+(-5-
)*C16+(-3)*C17+(-12)*C18+(-18)*C19+(14)*G1+(8)*G2+(7)*G3+(-10)*G4+(-4)*G5+-
(2)*G6+(1)*G7+(9)*G8+(5)*G9+(-11)*G10+(1)*G11+(9)*G12+(-24)*G13+(18)*G14+(-
11)*G15+(13)*G16+(-7)*G17+(-9)*G18+(-22)*G19+6*(number of A+U in
position 15-19)-3*(number of G+C in whole siRNA), Formula X
[0321] wherein
[0322] A.sub.1=1 if A is the base at position 1 of the sense
strand, otherwise its value is 0;
[0323] A.sub.2=1 if A is the base at position 2 of the sense
strand, otherwise its value is 0;
[0324] A.sub.3=1 if A is the base at position 3 of the sense
strand, otherwise its value is 0;
[0325] A.sub.4=1 if A is the base at position 4 of the sense
strand, otherwise its value is 0;
[0326] A.sub.5=1 if A is the base at position 5 of the sense
strand, otherwise its value is 0;
[0327] A.sub.6=1 if A is the base at position 6 of the sense
strand, otherwise its value is 0;
[0328] A.sub.7=1 if A is the base at position 7 of the sense
strand, otherwise its value is 0;
[0329] A.sub.10=1 if A is the base at position 10 of the sense
strand, otherwise its value is 0;
[0330] A.sub.11=1 if A is the base at position 11 of the sense
strand, otherwise its value is 0;
[0331] A.sub.13=1 if A is the base at position 13 of the sense
strand, otherwise its value is 0;
[0332] A.sub.19=1 if A is the base at position 19 of the sense
strand, otherwise if another base is present or the sense strand is
only 18 base pairs in length, its value is 0;
[0333] C.sub.3=1 if C is the base at position 3 of the sense
strand, otherwise its value is 0;
[0334] C.sub.4=1 if C is the base at position 4 of the sense
strand, otherwise its value is 0;
[0335] C.sub.5=1 if C is the base at position 5 of the sense
strand, otherwise its value is 0;
[0336] C.sub.6=1 if C is the base at position 6 of the sense
strand, otherwise its value is 0;
[0337] C.sub.7=1 if C is the base at position 7 of the sense
strand, otherwise its value is 0;
[0338] C.sub.9=1 if C is the base at position 9 of the sense
strand, otherwise its value is 0;
[0339] C.sub.17=1 if C is the base at position 17 of the sense
strand, otherwise its value is 0;
[0340] C.sub.18=1 if C is the base at position 18 of the sense
strand, otherwise its value is 0;
[0341] C.sub.19=1 if C is the base at position 19 of the sense
strand, otherwise if another base is present or the sense strand is
only 18 base pairs in length, its value is 0;
[0342] G.sub.1=1 if G is the base at position 1 on the sense
strand, otherwise its value is 0;
[0343] G.sub.2=1 if G is the base at position 2 of the sense
strand, otherwise its value is 0;
[0344] G.sub.8=1 if G is the base at position 8 on the sense
strand, otherwise its value is 0;
[0345] G.sub.10=1 if G is the base at position 10 on the sense
strand, otherwise its value is 0;
[0346] G.sub.13=1 if G is the base at position 13 on the sense
strand, otherwise its value is 0;
[0347] G.sub.19=1 if G is the base at position 19 of the sense
strand, otherwise if another base is present or the sense strand is
only 18 base pairs in length, its value is 0;
[0348] U.sub.1=1 if U is the base at position 1 on the sense
strand, otherwise its value is 0;
[0349] U.sub.2=1 if U is the base at position 2 on the sense
strand, otherwise its value is 0;
[0350] U.sub.3=1 if U is the base at position 3 on the sense
strand, otherwise its value is 0;
[0351] U.sub.4=1 if U is the base at position 4 on the sense
strand, otherwise its value is 0;
[0352] U.sub.7=1 if U is the base at position 7 on the sense
strand, otherwise its value is 0;
[0353] U.sub.9=1 if U is the base at position 9 on the sense
strand, otherwise its value is 0;
[0354] U.sub.10=1 if U is the base at position 10 on the sense
strand, otherwise its value is 0;
[0355] U.sub.15=1 if U is the base at position 15 on the sense
strand, otherwise its value is 0;
[0356] U.sub.16=1 if U is the base at position 16 on the sense
strand, otherwise its value is 0;
[0357] U.sub.17=1 if U is the base at position 17 on the sense
strand, otherwise its value is 0;
[0358] U.sub.18=1 if U is the base at position 18 on the sense
strand, otherwise its value is 0;
[0359] GC.sub.15-19=the number of G and C bases within positions
15-19 of the sense strand, or within positions 15-18 if the sense
strand is only 18 base pairs in length;
[0360] GC.sub.total=the number of G and C bases in the sense
strand;
[0361] Tm=100 if the siRNA oligo has the internal repeat longer
then 4 base pairs, otherwise its value is 0; and
[0362] X=the number of times that the same nucleotide repeats four
or more times in a row.
[0363] The above formulas VIII, IX, and X, as well as formulas
I-VII, provide methods for selecting siRNA in order to increase the
efficiency of gene silencing. A subset of variables of any of the
formulas may be used, though when fewer variables are used, the
optimization hierarchy becomes less reliable.
[0364] With respect to the variables of the above-referenced
formulas, a single letter of A or C or G or U followed by a
subscript refers to a binary condition. The binary condition is
that either the particular base is present at that particular
position (wherein the value is "1") or the base is not present
(wherein the value is "0"). Because position 19 is optional, i.e.,
there might be only 18 base pairs, when there are only 18 base
pairs, any base with a subscript of 19 in the formulas above would
have a zero value for that parameter. Before or after each variable
is a number followed by *, which indicates that the value of the
variable is to be multiplied or weighed by that number.
[0365] The numbers preceding the variables A, or G, or C, or U in
Formulas VIII, IX, and X (or after the variables in Formula I-VII)
were determined by comparing the difference in the frequency of
individual bases at different positions in functional siRNA and
total siRNA. Specifically, the frequency in which a given base was
observed at a particular position in functional groups was compared
with the frequency that that same base was observed in the total,
randomly selected siRNA set. If the absolute value of the
difference between the functional and total values was found to be
greater than 6%, that parameter was included in the equation. Thus,
for instance, if the frequency of finding a "G" at position 13
(G13) is found to be 6% in a given functional group, and the
frequency of G.sub.13 in the total population of siRNAs is 20%, the
difference between the two values is 6%-20%=-14%. As the absolute
value is greater than six (6), this factor (-14) is included in the
equation. Thus, in Formula VIII, in cases where the siRNA under
study has a G in position 13, the accrued value is (-14)*(1)=-14.
In contrast, when a base other than G is found at position 13, the
accrued value is (-14)*(0)=0.
[0366] When developing a means to optimize siRNAs, the inventors
observed that a bias toward low internal thermodynamic stability of
the duplex at the 5'-antisense (AS) end is characteristic of
naturally occurring miRNA precursors. The inventors extended this
observation to siRNAs for which functionality had been assessed in
tissue culture.
[0367] With respect to the parameter GC.sub.15-19, a value of 0-5
will be ascribed depending on the number of G or C bases at
positions 15 to 19. If there are only 18 base pairs, the value is
between 0 and 4.
[0368] With respect to the criterion GC.sub.total content, a number
from 0-30 will be ascribed, which correlates to the total number of
G and C nucleotides on the sense strand, excluding overhangs.
Without wishing to be bound by any one theory, it is postulated
that the significance of the GC content (as well as AU content at
positions 15-19, which is a parameter for formulas III-VII) relates
to the easement of the unwinding of a double-stranded siRNA duplex.
Duplex unwinding is believed to be crucial for siRNA functionality
in vivo and overall low internal stability, especially low internal
stability of the first unwound base pair is believed to be
important to maintain sufficient processivity of RISC
complex-induced duplex unwinding. If the duplex has 19 base pairs,
those at positions 15-19 on the sense strand will unwind first if
the molecule exhibits a sufficiently low internal stability at that
position. As persons skilled in the art are aware, RISC is a
complex of approximately twelve proteins; Dicer is one, but not the
only, helicase within this complex. Accordingly, although the GC
parameters are believed to relate to activity with Dicer, they are
also important for activity with other RISC proteins.
[0369] The value of the parameter Tm is 0 when there are no
internal repeats longer than (or equal to) four base pairs present
in the siRNA duplex; otherwise the value is 1. Thus for example, if
the sequence ACGUACGU, or any other four nucleotide (or more)
palindrome exists within the structure, the value will be one (1).
Alternatively if the structure ACGGACG, or any other 3 nucleotide
(or less) palindrome exists, the value will be zero (0).
[0370] The variable "X" refers to the number of times that the same
nucleotide occurs contiguously in a stretch of four or more units.
If there are, for example, four contiguous As in one part of the
sequence and elsewhere in the sequence four contiguous Cs, X=2.
Further, if there are two separate contiguous stretches of four of
the same nucleotides or eight or more of the same nucleotides in a
row, then X=2. However, X does not increase for five, six or seven
contiguous nucleotides.
[0371] Again, when applying Formula VIII, Formula IX, or Formula X,
to a given mRNA, (the "target RNA" or "target molecule"), one may
use a computer program to evaluate the criteria for every sequence
of 18-30 base pairs or only sequences of a fixed length, e.g., 19
base pairs. Preferably the computer program is designed such that
it provides a report ranking of all of the potential siRNAs 18-30
base pairs, ranked according to which sequences generate the
highest value. A higher value refers to a more efficient siRNA for
a particular target gene. The computer program that may be used may
be developed in any computer language that is known to be useful
for scoring nucleotide sequences, or it may be developed with the
assistance of commercially available product such as Microsoft's
product.net. Additionally, rather than run every sequence through
one and/or another formula, one may compare a subset of the
sequences, which may be desirable if for example only a subset are
available. For instance, it may be desirable to first perform a
BLAST (Basic Local Alignment Search Tool) search and to identify
sequences that have no homology to other targets. Alternatively, it
may be desirable to scan the sequence and to identify regions of
moderate GC context, then perform relevant calculations using one
of the above-described formulas on these regions. These
calculations can be done manually or with the aid of a
computer.
[0372] As with Formulas I-VII, either Formula VIII, Formula IX, or
Formula X may be used for a given mRNA target sequence. However, it
is possible that according to one or the other formula more than
one siRNA will have the same value. Accordingly, it is beneficial
to have a second formula by which to differentiate sequences.
Formulas IX and X were derived in a similar fashion as Formula
VIII, yet used a larger data set and thus yields sequences with
higher statistical correlations to highly functional duplexes. The
sequence that has the highest value ascribed to it may be referred
to as a "first optimized duplex." The sequence that has the second
highest value ascribed to it may be referred to as a "second
optimized duplex." Similarly, the sequences that have the third and
fourth highest values ascribed to them may be referred to as a
third optimized duplex and a fourth optimized duplex, respectively.
When more than one sequence has the same value, each of them may,
for example, be referred to as first optimized duplex sequences or
co-first optimized duplexes. Formula X is similar to Formula IX,
yet uses a greater numbers of variables and for that reason,
identifies sequences on the basis of slightly different
criteria.
[0373] It should also be noted that the output of a particular
algorithm will depend on several of variables including: (1) the
size of the data base(s) being analyzed by the algorithm, and (2)
the number and stringency of the parameters being applied to screen
each sequence. Thus, for example, in U.S. patent application Ser.
No. 10/714,333, entitled "Functional and Hyperfunctional siRNA,"
filed Nov. 14, 2003, Formula VIII was applied to the known human
genome (ncbi refseq database) through Entrez (efetch). As a result
of these procedures, roughly 1.6 million siRNA sequences were
identified. Application of Formula VIII to the same database in
March of 2004 yielded roughly 2.2 million sequences, a difference
of approximately 600,000 sequences resulting from the growth of the
database over the course of the months that span this period of
time. Application of other formulas (e.g., Formula X) that change
the emphasis of, include, or eliminate different variables can
yield unequal numbers of siRNAs. Alternatively, in cases where
application of one formula to one or more genes fails to yield
sufficient numbers of siRNAs with scores that would be indicative
of strong silencing, said genes can be reassessed with a second
algorithm that is, for instance, less stringent.
[0374] siRNA sequences identified using Formula VIII and Formula X
(minus sequences generated by Formula VIII) are contained within
the enclosed compact disks. The data included on the enclosed
compact disks is described more fully below. The sequences
identified by Formula VIII and Formula X that are disclosed in the
compacts disks may be used in gene silencing applications.
[0375] It should be noted that for Formulas VIII, IX, and X all of
the aforementioned criteria are identified as positions on the
sense strand when oriented in the 5' to 3' direction as they are
identified in connection with Formulas I-VII unless otherwise
specified.
[0376] Formulas I-X, may be used to select or to evaluate one, or
more than one, siRNA in order to optimize silencing. Preferably, at
least two optimized siRNAs that have been selected according to at
least one of these formulas are used to silence a gene, more
preferably at least three and most preferably at least four. The
siRNAs may be used individually or together in a pool or kit.
Further, they may be applied to a cell simultaneously or
separately. Preferably, the at least two siRNAs are applied
simultaneously. Pools are particularly beneficial for many research
applications. However, for therapeutics, it may be more desirable
to employ a single hyperfunctional siRNA as described elsewhere in
this application.
[0377] When planning to conduct gene silencing, and it is necessary
to choose between two or more siRNAs, one should do so by comparing
the relative values when the siRNA are subjected to one of the
formulas above. In general a higher scored siRNA should be
used.
[0378] Useful applications include, but are not limited to, target
validation, gene functional analysis, research and drug discovery,
gene therapy and therapeutics. Methods for using siRNA in these
applications are well known to persons of skill in the art.
[0379] Because the ability of siRNA to function is dependent on the
sequence of the RNA and not the species into which it is
introduced, the present invention is applicable across a broad
range of species, including but not limited to all mammalian
species, such as humans, dogs, horses, cats, cows, mice, hamsters,
chimpanzees and gorillas, as well as other species and organisms
such as bacteria, viruses, insects, plants and C. elegans.
[0380] The present invention is also applicable for use for
silencing a broad range of genes, including but not limited to the
roughly 45,000 genes of a human genome, and has particular
relevance in cases where those genes are associated with diseases
such as diabetes, Alzheimer's, cancer, as well as all genes in the
genomes of the aforementioned organisms.
[0381] The siRNA selected according to the aforementioned criteria
or one of the aforementioned algorithms are also, for example,
useful in the simultaneous screening and functional analysis of
multiple genes and gene families using high throughput strategies,
as well as in direct gene suppression or silencing.
[0382] Development of the Algorithms
[0383] To identify siRNA sequence features that promote
functionality and to quantify the importance of certain currently
accepted conventional factors--such as G/C content and target site
accessibility--the inventors synthesized an siRNA panel consisting
of 270 siRNAs targeting three genes, Human Cyclophilin, Firefly
Luciferase, and Human DBI. In all three cases, siRNAs were directed
against specific regions of each gene. For Human Cyclophilin and
Firefly Luciferase, ninety siRNAs were directed against a 199 bp
segment of each respective mRNA. For DBI, 90 siRNAs were directed
against a smaller, 109 base pair region of the mRNA. The sequences
to which the siRNAs were directed are provided below.
[0384] It should be noted that in certain sequences, "t" is
present. This is because many databases contain information in this
manner. However, the t denotes a uracil residue in mRNA and siRNA.
Any algorithm will, unless otherwise specified, process a t in a
sequence as a u.
[0385] Human cyclophilin: 193-390 M60857
4 gttccaaaaacagtggataattttgtggccttagct SEQ. ID NO. 29
acaggagagaaaggatttggctacaaaaacagcaaa
ttccatcgtgtaatcaaggacttcatgatccagggc
ggagacttcaccaggggagatggcacaggaggaaag
agcatctacggtgagcgcttccccgatgagaacttc
aaactgaagcactacgggcctggctggg:
[0386] Firefly luciferase: 1434-1631, U47298 (pGL3, Promega)
5 tgaacttcccgccgccgttgttgttttggagcacgg SEQ. ID NO. 30
aaagacgatgacggaaaaagagatcgtggattacgt
cgccagtcaagtaacaaccgcgaaaaagttgcgcgg
aggagttgtgtttgtggacgaagtaccgaaaggtct
taccggaaaactcgacgcaagaaaaatcagagagat cctcataaaggccaagaagg:
[0387] DBI, NM.sub.--020548 (202-310) (Every Position)
6 acgggcaaggccaagtgggatgcctggaatgagc SEQ. ID NO. 0031
tgaaagggacttccaaggaagatgccatgaaagc
ttacatcaacaaagtagaagagctaaagaaaaaa tacggg:
[0388] A list of the siRNAs appears in Table III (see Examples
Section, Example II)
[0389] The set of duplexes was analyzed to identify correlations
between siRNA functionality and other biophysical or thermodynamic
properties. When the siRNA panel was analyzed in functional and
non-functional subgroups, certain nucleotides were much more
abundant at certain positions in functional or non-functional
groups. More specifically, the frequency of each nucleotide at each
position in highly functional siRNA duplexes was compared with that
of nonfunctional duplexes in order to assess the preference for or
against any given nucleotide at every position. These analyses were
used to determine important criteria to be included in the siRNA
algorithms (Formulas VIII, IX, and X).
[0390] The data set was also analyzed for distinguishing
biophysical properties of siRNAs in the functional group, such as
optimal percent of GC content, propensity for internal structures
and regional thermodynamic stability. Of the presented criteria,
several are involved in duplex recognition, RISC activation/duplex
unwinding, and target cleavage catalysis.
[0391] The original data set that was the source of the
statistically derived criteria is shown in FIG. 2. Additionally,
this figure shows that random selection yields siRNA duplexes with
unpredictable and widely varying silencing potencies as measured in
tissue culture using HEK293 cells. In the figure, duplexes are
plotted such that each x-axis tick-mark represents an individual
siRNA, with each subsequent siRNA differing in target position by
two nucleotides for Human Cyclophilin B and Firefly Luciferase, and
by one nucleotide for Human DBI. Furthermore, the y-axis denotes
the level of target expression remaining after transfection of the
duplex into cells and subsequent silencing of the target.
[0392] siRNA identified and optimized in this document work equally
well in a wide range of cell types. FIG. 3a shows the evaluation of
thirty siRNAs targeting the DBI gene in three cell lines derived
from different tissues. Each DBI siRNA displays very similar
functionality in HEK293 (ATCC, CRL-1573, human embryonic kidney),
HeLa (ATCC, CCL-2, cervical epithelial adenocarcinoma) and DU145
(HTB-81, prostate) cells as deterimined by the B-DNA assay. Thus,
siRNA functionality is determined by the primary sequence of the
siRNA and not by the intracellular environment. Additionally, it
should be noted that although the present invention provides for a
determination of the functionality of siRNA for a given target, the
same siRNA may silence more than one gene. For example, the
complementary sequence of the silencing siRNA may be present in
more than one gene. Accordingly, in these circumstances, it may be
desirable not to use the siRNA with highest SMARTscore.TM.. In such
circumstances, it may be desirable to use the siRNA with the next
highest SMARTscore.TM..
[0393] To determine the relevance of G/C content in siRNA function,
the G/C content of each duplex in the panel was calculated and the
functional classes of siRNAs (<F50,.gtoreq.F50,
.gtoreq.F80,.gtoreq.F95 where F refers to the percent gene
silencing) were sorted accordingly. The majority of the
highly-functional siRNAs (.gtoreq.F95) fell within the G/C content
range of 36-52% (FIG. 3B). Twice as many non-functional (<F50)
duplexes fell within the high G/C content groups (>57% GC
content) compared to the 36%-52% group. The group with extremely
low GC content (26% or less) contained a higher proportion of
non-functional siRNAs and no highly-functional siRNAs. The G/C
content range of 30%-52% was therefore selected as Criterion I for
siRNA functionality, consistent with the observation that a G/C
range 30%-70% promotes efficient RNAi targeting. Application of
this criterion alone provided only a marginal increase in the
probability of selecting functional siRNAs from the panel:
selection of F50 and F95 siRNAs was improved by 3.6% and 2.2%,
respectively. The siRNA panel presented here permitted a more
systematic analysis and quantification of the importance of this
criterion than that used previously.
[0394] A relative measure of local internal stability is the A/U
base pair (bp) content; therefore, the frequency of A/U bp was
determined for each of the five terminal positions of the duplex
(5' sense (S)/5' antisense (AS)) of all siRNAs in the panel.
Duplexes were then categorized by the number of A/U bp in positions
1-5 and 15-19 of the sense strand. The thermodynamic flexibility of
the duplex 5'-end (positions 1-5; S) did not appear to correlate
appreciably with silencing potency, while that of the 3'-end
(positions 15-19; S) correlated with efficient silencing. No
duplexes lacking A/U bp in positions 15-19 were functional. The
presence of one A/U bp in this region conferred some degree of
functionality, but the presence of three or more A/Us was
preferable and therefore defined as Criterion II. When applied to
the test panel, only a marginal increase in the probability of
functional siRNA selection was achieved: a 1.8% and 2.3% increase
for F50 and F95 duplexes, respectively (Table IV).
[0395] The complementary strands of siRNAs that contain internal
repeats or palindromes may form internal fold-back structures.
These hairpin-like structures exist in equilibrium with the
duplexed form effectively reducing the concentration of functional
duplexes. The propensity to form internal hairpins and their
relative stability can be estimated by predicted melting
temperatures. High Tm reflects a tendency to form hairpin
structures. Lower Tm values indicate a lesser tendency to form
hairpins. When the functional classes of siRNAs were sorted by
T.sub.m (FIG. 3c), the following trends were identified: duplexes
lacking stable internal repeats were the most potent silencers (no
F95 duplex with predicted hairpin structure T.sub.m>60.degree.
C.). In contrast, about 60% of the duplexes in the groups having
internal hairpins with calculated T.sub.m values less than
20.degree. C. were F80. Thus, the stability of internal repeats is
inversely proportional to the silencing effect and defines
Criterion III (predicted hairpin structure
T.sub.m.ltoreq.20.degree. C.).
[0396] Sequence-Based Determinants of siRNA Functionality
[0397] When the siRNA panel was sorted into functional and
non-functional groups, the frequency of a specific nucleotide at
each position in a functional siRNA duplex was compared with that
of a nonfunctional duplex in order to assess the preference for or
against a certain nucleotide. FIG. 4 shows the results of these
queries and the subsequent resorting of the data set (from FIG. 2).
The data is separated into two sets: those duplexes that meet the
criteria, a specific nucleotide in a certain position--grouped on
the left (Selected) and those that do not--grouped on the right
(Eliminated). The duplexes are further sorted from most functional
to least functional with the y-axis of FIG. 4a-e representing the %
expression i.e., the amount of silencing that is elicited by the
duplex (Note: each position on the X-axis represents a different
duplex). Statistical analysis revealed correlations between
silencing and several sequence-related properties of siRNAs. FIG. 4
and Table IV show quantitative analysis for the following five
sequence-related properties of siRNA: (A) an A at position 19 of
the sense strand; (B) an A at position 3 of the sense strand; (C) a
U at position 10 of the sense strand; (D) a base other than G at
position 13 of the sense strand; and (E) a base other than C at
position 19 of the sense strand.
[0398] When the siRNAs in the panel were evaluated for the presence
of an A at position 19 of the sense strand, the percentage of
non-functional duplexes decreased from 20% to 11.8%, and the
percentage of F95 duplexes increased from 21.7% to 29.4% (Table
IV). Thus, the presence of an A in this position defined Criterion
IV.
[0399] Another sequence-related property correlated with silencing
was the presence of an A in position 3 of the sense strand (FIG.
4b). Of the siRNAs with A3, 34.4% were F95, compared with 21.7%
randomly selected siRNAs. The presence of a U base in position 10
of the sense strand exhibited an even greater impact (FIG. 4c). Of
the duplexes in this group, 41.7% were F95. These properties became
criteria V and VI, respectively.
[0400] Two negative sequence-related criteria that were identified
also appear on FIG. 4. The absence of a G at position 13 of the
sense strand, conferred a marginal increase in selecting functional
duplexes (FIG. 4d). Similarly, lack of a C at position 19 of the
sense strand also correlated with functionality (FIG. 4e). Thus,
among functional duplexes, position 19 was most likely occupied by
A, and rarely occupied by C. These rules were defined as criteria
VII and VIII, respectively.
[0401] Application of each criterion individually provided marginal
but statistically significant increases in the probability of
selecting a potent siRNA. Although the results were informative,
the inventors sought to maximize potency and therefore consider
multiple criteria or parameters. Optimization is particularly
important when developing therapeutics. Interestingly, the
probability of selecting a functional siRNA based on each
thermodynamic criteria was 2%-4% higher than random, but 4%-8%
higher for the sequence-related determinates. Presumably, these
sequence-related increases reflect the complexity of the RNAi
mechanism and the multitude of protein-RNA interactions that are
involved in RNAi-mediated silencing.
7TABLE IV Improvement Criterion % Functional over Random I. 30%-52%
G/C content <F50 16.4% -3.6% .gtoreq.F50 83.6% 3.6% .gtoreq.F80
60.4% 4.3% .gtoreq.F95 23.9% 2.2% II. At least 3 A/U bases at
positions <F50 18.2% -1.8% 15-19 of the sense strand .gtoreq.F50
81.8% 1.8% .gtoreq.F80 59.7% 3.6% .gtoreq.F95 24.0% 2.3% III.
Absence of internal repeats, <F50 16.7% -3.3% as measured by
T.sub.m of .gtoreq.F50 83.3% 3.3% secondary structure
.ltoreq.20.degree. C. .gtoreq.F80 61.1% 5.0% .gtoreq.F95 24.6% 2.9%
IV. An A base at position 19 <F50 11.8% -8.2% of the sense
strand .gtoreq.F50 88.2% 8.2% .gtoreq.F80 75.0% 18.9% .gtoreq.F95
29.4% 7.7% V. An A base at position 3 <F50 17.2% -2.8% of the
sense strand .gtoreq.F50 82.8% 2.8% .gtoreq.F80 62.5% 6.4%
.gtoreq.F95 34.4% 12.7% VI. A U base at position 10 <F50 13.9%
-6.1% of the sense strand .gtoreq.F50 86.1% 6.1% .gtoreq.F80 69.4%
13.3% .gtoreq.F95 41.7% 20% VII. A base other than C at <F50
18.8% -1.2% position 19 of the sense strand .gtoreq.F50 81.2% 1.2%
.gtoreq.F80 59.7% 3.6% .gtoreq.F95 24.2% 2.5% VIII. A base other
than G at <F50 15.2% -4.8% position 13 of the sense strand
.gtoreq.F50 84.8% 4.8% .gtoreq.F80 61.4% 5.3% .gtoreq.F95 26.5%
4.8%
[0402] The siRNA Selection Algorithm
[0403] In an effort to improve selection further, all identified
criteria, including but not limited to those listed in Table IV
were combined into the algorithms embodied in Formula VIII, Formula
IX, and Formula X. Each siRNA was then assigned a score (referred
to as a SMARTscore.TM.) according to the values derived from the
formulas. Duplexes that scored higher than 0 or -20 (unadjusted),
for Formulas VIII and IX, respectively, effectively selected a set
of functional siRNAs and excluded all non-functional siRNAs.
Conversely, all duplexes scoring lower than 0 and -20 (minus 20)
according to formulas VIII and IX, respectively, contained some
functional siRNAs but included all non-functional siRNAs. A
graphical representation of this selection is shown in FIG. 5. It
should be noted that the scores derived from the algorithm can also
be provided as "adjusted" scores. To convert Formula VIII
unadjusted scores into adjusted scores it is necessary to use the
following equation:
(160+unadjusted score)/2.25
[0404] When this takes place, an unadjusted score of "0" (zero) is
converted to 75. Similarly, unadjusted scores for Formula X can be
converted to adjusted scores. In this instance, the following
equation is applied:
(228+unadjusted score)/3.56
[0405] When these manipulations take place, an unadjusted score of
38 is converted to an adjusted score of 75.
[0406] The methods for obtaining the seven criteria embodied in
Table IV are illustrative of the results of the process used to
develop the information for Formulas VIII, IX, and X. Thus similar
techniques were used to establish the other variables and their
multipliers. As described above, basic statistical methods were use
to determine the relative values for these multipliers.
[0407] To determine the value for "Improvement over Random" the
difference in the frequency of a given attribute (e.g., GC content,
base preference) at a particular position is determined between
individual functional groups (e.g., <F50) and the total siRNA
population studied (e.g., 270 siRNA molecules selected randomly).
Thus, for instance, in Criterion I (30%-52% GC content) members of
the <F50 group were observed to have GC contents between 30-52%
in 16.4% of the cases. In contrast, the total group of 270 siRNAs
had GC contents in this range, 20% of the time. Thus for this
particular attribute, there is a small negative correlation between
30%-52% GC content and this functional group (i.e.,
16.4%-20%=-3.6%). Similarly, for Criterion VI, (a "U" at position
10 of the sense strand), the >F95 group contained a "U" at this
position 41.7% of the time. In contrast, the total group of 270
siRNAs had a "U" at this position 21.7% of the time, thus the
improvement over random is calculated to be 20% (or
41.7%-21.7%).
[0408] Identifying the Average Internal Stability Profile of Strong
siRNA
[0409] In order to identify an internal stability profile that is
characteristic of strong siRNA, 270 different siRNAs derived from
the cyclophilin B, the diazepam binding inhibitor (DBI), and the
luciferase gene were individually transfected into HEK293 cells and
tested for their ability to induce RNAi of the respective gene.
Based on their performance in the in vivo assay, the sequences were
then subdivided into three groups, (i) >95% silencing; (ii)
80-95% silencing; and (iii) less than 50% silencing. Sequences
exhibiting 51-84% silencing were eliminated from further
consideration to reduce the difficulties in identifying relevant
thermodynamic patterns.
[0410] Following the division of siRNA into three groups, a
statistical analysis was performed on each member of each group to
determine the average internal stability profile (AISP) of the
siRNA. To accomplish this the Oligo 5.0 Primer Analysis Software
and other related statistical packages (e.g., Excel) were exploited
to determine the internal stability of pentamers using the nearest
neighbor method described by Freier et al., (1986) Improved
free-energy parameters for predictions of RNA duplex stability,
Proc Natl. Acad. Sci. U.S.A. 83(24): 9373-7. Values for each group
at each position were then averaged, and the resulting data were
graphed on a linear coordinate system with the Y-axis expressing
the .DELTA.G (free energy) values in kcal/mole and the X-axis
identifying the position of the base relative to the 5' end.
[0411] The results of the analysis identified multiple key regions
in siRNA molecules that were critical for successful gene
silencing. At the 3'-most end of the sense strand (5'antisense),
highly functional siRNA (>95% gene silencing, see FIG. 6a,
>F95) have a low internal stability (AISP of position
19=.about.7.6 kcal/mol). In contrast low-efficiency siRNA (i.e.,
those exhibiting less than 50% silencing, <F50) display a
distinctly different profile, having high .DELTA.G values
(.about.-8.4 kcal/mol) for the same position. Moving in a 5' (sense
strand) direction, the internal stability of highly efficient siRNA
rises (position 12=.about.-8.3 kcal/mole) and then drops again
(position 7=.about.-7.7 kcal/mol) before leveling off at a value of
approximately -8.1 kcal/mol for the 5' terminus. siRNA with poor
silencing capabilities show a distinctly different profile. While
the AISP value at position 12 is nearly identical with that of
strong siRNAs, the values at positions 7 and 8 rise considerably,
peaking at a high of .about.-9.0 kcal/mol. In addition, at the 5'
end of the molecule the AISP profile of strong and weak siRNA
differ dramatically. Unlike the relatively strong values exhibited
by siRNA in the >95% silencing group, siRNAs that exhibit poor
silencing activity have weak AISP values (-7.6, -7.5, and -7.5
kcal/mol for positions 1, 2 and 3 respectively).
[0412] Overall the profiles of both strong and weak siRNAs form
distinct sinusoidal shapes that are roughly 180.degree.
out-of-phase with each other. While these thermodynamic
descriptions define the archetypal profile of a strong siRNA, it
will likely be the case that neither the .DELTA.G values given for
key positions in the profile or the absolute position of the
profile along the Y-axis (i.e., the .DELTA.G-axis) are absolutes.
Profiles that are shifted upward or downward (i.e., having on an
average, higher or lower values at every position) but retain the
relative shape and position of the profile along the X-axis can be
foreseen as being equally effective as the model profile described
here. Moreover, it is likely that siRNA that have strong or even
stronger gene-specific silencing effects might have exaggerated
.DELTA.G values (either higher or lower) at key positions. Thus,
for instance, it is possible that the 5'-most position of the sense
strand (position 19) could have .DELTA.G values of 7.4 kcal/mol or
lower and still be a strong siRNA if, for instance, a
G-C.fwdarw.G-T/U mismatch were substituted at position 19 and
altered duplex stability. Similarly, position 12 and position 7
could have values above 8.3 kcal/mol and below 7.7 kcal/mole,
respectively, without abating the silencing effectiveness of the
molecule. Thus, for instance, at position 12, a stabilizing
chemical modification (e.g., a chemical modification of the 2'
position of the sugar backbone) could be added that increases the
average internal stability at that position. Similarly, at position
7, mismatches similar to those described previously could be
introduced that would lower the .DELTA.G values at that
position.
[0413] Lastly, it is important to note that while functional and
non-functional siRNA were originally defined as those molecules
having specific silencing properties, both broader or more limiting
parameters can be used to define these molecules. As used herein,
unless otherwise specified, "non-functional siRNA" are defined as
those siRNA that induce less than 50% (<50%) target silencing,
"semi-functional siRNA" induce 50-79% target silencing, "functional
siRNA" are molecules that induce 80-95% gene silencing, and
"highly-functional siRNA" are molecules that induce great than 95%
gene silencing. These definitions are not intended to be rigid and
can vary depending upon the design and needs of the application.
For instance, it is possible that a researcher attempting to map a
gene to a chromosome using a functional assay, may identify an
siRNA that reduces gene activity by only 30%. While this level of
gene silencing may be "non-functional" for, e.g., therapeutic
needs, it is sufficient for gene mapping purposes and is, under
these uses and conditions, "functional." For these reasons,
functional siRNA can be defined as those molecules having greater
than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% silencing
capabilities at 100 nM transfection conditions. Similarly,
depending upon the needs of the study and/or application,
non-functional and semi-functional siRNA can be defined as having
different parameters. For instance, semi-functional siRNA can be
defined as being those molecules that induce 20%, 30%, 40%, 50%,
60%, or 70% silencing at 100 nM transfection conditions. Similarly,
non-functional siRNA can be defined as being those molecules that
silence gene expression by less than 70%, 60%, 50%, 40%, 30%, or
less. Nonetheless, unless otherwise stated, the descriptions stated
in the "Definitions" section of this text should be applied.
[0414] Functional attributes can be assigned to each of the key
positions in the AISP of strong siRNA. The low 5' (sense strand)
AISP values of strong siRNAs may be necessary for determining which
end of the molecule enters the RISC complex. In contrast, the high
and low AISP values observed in the central regions of the molecule
may be critical for siRNA-target mRNA interactions and product
release, respectively.
[0415] If the AISP values described above accurately define the
thermodynamic parameters of strong siRNA, it would be expected that
similar patterns would be observed in strong siRNA isolated from
nature. Natural siRNAs exist in a harsh, RNase-rich environment and
it can be hypothesized that only those siRNA that exhibit
heightened affinity for RISC (i.e., siRNA that exhibit an average
internal stability profile similar to those observed in strong
siRNA) would survive in an intracellular environment. This
hypothesis was tested using GFP-specific siRNA isolated from N.
benthamiana. Llave et al. (2002) Endogenous and
Silencing-Associated Small RNAs in Plants, The Plant Cell 14,
1605-1619, introduced long double-stranded GFP-encoding RNA into
plants and subsequently re-isolated GFP-specific siRNA from the
tissues. The AISP of fifty-nine of these GFP-siRNA were determined,
averaged, and subsequently plotted alongside the AISP profile
obtained from the cyclophilin B/DBI/luciferase siRNA having >90%
silencing properties (FIG. 6b). Comparison of the two groups show
that profiles are nearly identical. This finding validates the
information provided by the internal stability profiles and
demonstrates that: (1) the profile identified by analysis of the
cyclophilin B/DBI/luciferase siRNAs are not gene specific; and (2)
AISP values can be used to search for strong siRNAs in a variety of
species.
[0416] Both chemical modifications and base-pair mismatches can be
incorporated into siRNA to alter the duplex's AISP and
functionality. For instance, introduction of mismatches at
positions 1 or 2 of the sense strand destabilized the 5' end of the
sense strand and increases the functionality of the molecule (see
Luc, FIG. 7). Similarly, addition of 2'-O-methyl groups to
positions 1 and 2 of the sense strand can also alter the AISP and
(as a result) increase both the functionality of the molecule and
eliminate off-target effects that results from sense strand
homology with the unrelated targets (FIGS. 8a, 8b).
[0417] Rationale for Criteria in a Biological Context
[0418] The fate of siRNA in the RNAi pathway may be described in 5
major steps: (1) duplex recognition and pre-RISC complex formation;
(2) ATP-dependent duplex unwinding/strand selection and RISC
activation; (3) mRNA target identification; (4) mRNA cleavage, and
(5) product release (FIG. 1). Given the level of nucleic
acid-protein interactions at each step, siRNA functionality is
likely influenced by specific biophysical and molecular properties
that promote efficient interactions within the context of the
multi-component complexes. Indeed, the systematic analysis of the
siRNA test set identified multiple factors that correlate well with
functionality. When combined into a single algorithm, they proved
to be very effective in selecting active siRNAs.
[0419] The factors described here may also be predictive of key
functional associations important for each step in RNAi. For
example, the potential formation of internal hairpin structures
correlated negatively with siRNA functionality. Complementary
strands with stable internal repeats are more likely to exist as
stable hairpins thus decreasing the effective concentration of the
functional duplex form. This suggests that the duplex is the
preferred conformation for initial pre-RISC association. Indeed,
although single complementary strands can induce gene silencing,
the effective concentration required is at least two orders of
magnitude higher than that of the duplex form.
[0420] siRNA-pre-RISC complex formation is followed by an
ATP-dependent duplex unwinding step and "activation" of the RISC.
The siRNA functionality was shown to correlate with overall low
internal stability of the duplex and low internal stability of the
3' sense end (or differential internal stability of the 3' sense
compare to the 5' sense strand), which may reflect strand selection
and entry into the RISC. Overall duplex stability and low internal
stability at the 3' end of the sense strand were also correlated
with siRNA functionality. Interestingly, siRNAs with very high and
very low overall stability profiles correlate strongly with
non-functional duplexes. One interpretation is that high internal
stability prevents efficient unwinding while very low stability
reduces siRNA target affinity and subsequent mRNA cleavage by the
RISC.
[0421] Several criteria describe base preferences at specific
positions of the sense strand and are even more intriguing when
considering their potential mechanistic roles in target recognition
and mRNA cleavage. Base preferences for A at position 19 of the
sense strand but not C, are particularly interesting because they
reflect the same base preferences observed for naturally occurring
miRNA precursors. That is, among the reported miRNA precursor
sequences 75% contain a U at position 1 which corresponds to an A
in position 19 of the sense strand of siRNAs, while G was
under-represented in this same position for miRNA precursors. These
observations support the hypothesis that both miRNA precursors and
siRNA duplexes are processed by very similar if not identical
protein machinery. The functional interpretation of the
predominance of a U/A base pair is that it promotes flexibility at
the 5'antisense ends of both siRNA duplexes and miRNA precursors
and facilitates efficient unwinding and selective strand entrance
into an activated RISC.
[0422] Among the criteria associated with base preferences that are
likely to influence mRNA cleavage or possibly product release, the
preference for U at position 10 of the sense strand exhibited the
greatest impact, enhancing the probability of selecting an F80
sequence by 13.3%. Activated RISC preferentially cleaves target
mRNA between nucleotides 10 and 11 relative to the 5' end of the
complementary targeting strand. Therefore, it may be that U, the
preferred base for most endoribonucleases, at this position
supports more efficient cleavage. Alternatively, a U/A bp between
the targeting siRNA strand and its cognate target mRNA may create
an optimal conformation for the RISC-associated "slicing"
activity.
[0423] Post Algorithm Filters
[0424] According to another embodiment, the output of any one of
the formulas previously listed can be filtered to remove or select
for siRNAs containing undesirable or desirable motifs or
properties, respectively. In one example, sequences identified by
any of the formulas can be filtered to remove any and all sequences
that induce toxicity or cellular stress. Introduction of an siRNA
containing a toxic motif into a cell can induce cellular stress
and/or cell death (apoptosis) which in turn can mislead researchers
into associating a particular (e.g., nonessential) gene with, e.g.,
an essential function. Alternatively, sequences generated by any of
the before mentioned formulas can be filtered to identify and
retain duplexes that contain toxic motifs. Such duplexes may be
valuable from a variety of perspectives including, for instance,
uses as therapeutic molecules. A variety of toxic motifs exist and
can exert their influence on the cell through RNAi and non-RNAi
pathways. Examples of toxic motifs are explained more fully in
commonly assigned U.S. Provisional Patent Application Ser. No.
60/538,874, entitled "Identification of Toxic Sequences," filed
Jan. 23, 2004. Briefly, toxic motifs include A/G UUU A/G/U, G/C AAA
G/C, and GCCA, or a complement of any of the foregoing.
[0425] In another instance, sequences identified by any of the
before mentioned formulas can be filtered to identify duplexes that
contain motifs (or general properties) that provide serum stability
or induce serum instability. In one envisioned application of siRNA
as therapeutic molecules, duplexes targeting disease-associated
genes will be introduced into patients intravenously. As the
half-life of single and double stranded RNA in serum is short,
post-algorithm filters designed to select molecules that contain
motifs that enhance duplex stability in the presence of serum
and/or (conversely) eliminate duplexes that contain motifs that
destabilize siRNA in the presence of serum, would be
beneficial.
[0426] In another instance, sequences identified by any of the
before mentioned formulas can be filtered to identify duplexes that
are hyperfunctional. Hyperfunctional sequences are defined as those
sequences that (1) induce greater than 95% silencing of a specific
target when they are transfected at subnanomolar concentrations
(i.e., less than one nanomolar); and/or (2) induce functional (or
better) levels of silencing for greater than 96 hours. Filters that
identify hyperfunctional molecules can vary widely. In one example,
the top ten, twenty, thirty, or forty siRNA can be assessed for the
ability to silence a given target at, e.g., concentrations of 1 nM
and 0.5 nM to identify hyperfunctional molecules.
[0427] Pooling
[0428] According to another embodiment, the present invention
provides a pool of at least two siRNAs, preferably in the form of a
kit or therapeutic reagent, wherein one strand of each of the
siRNAs, the sense strand comprises a sequence that is substantially
similar to a sequence within a target mRNA. The opposite strand,
the antisense strand, will preferably comprise a sequence that is
substantially complementary to that of the target mRNA. More
preferably, one strand of each siRNA will comprise a sequence that
is identical to a sequence that is contained in the target mRNA.
Most preferably, each siRNA will be 19 base pairs in length, and
one strand of each of the siRNAs will be 100% complementary to a
portion of the target mRNA.
[0429] By increasing the number of siRNAs directed to a particular
target using a pool or kit, one is able both to increase the
likelihood that at least one siRNA with satisfactory functionality
will be included, as well as to benefit from additive or
synergistic effects. Further, when two or more siRNAs directed
against a single gene do not have satisfactory levels of
functionality alone, if combined, they may satisfactorily promote
degradation of the target messenger RNA and successfully inhibit
translation. By including multiple siRNAs in the system, not only
is the probability of silencing increased, but the economics of
operation are also improved when compared to adding different
siRNAs sequentially. This effect is contrary to the conventional
wisdom that the concurrent use of multiple siRNA will negatively
impact gene silencing (e.g., Holen, T. et al. (2003) "Similar
behavior of single strand and double strand siRNAs suggests they
act through a common RNAi pathway." NAR 31: 2401-21407).
[0430] In fact, when two siRNAs were pooled together, 54% of the
pools of two siRNAs induced more than 95% gene silencing. Thus, a
2.5-fold increase in the percentage of functionality was achieved
by randomly combining two siRNAs. Further, over 84% of pools
containing two siRNAs induced more than 80% gene silencing.
[0431] More preferably, the kit is comprised of at least three
siRNAs, wherein one strand of each siRNA comprises a sequence that
is substantially similar to a sequence of the target mRNA and the
other strand comprises a sequence that is substantially
complementary to the region of the target mRNA. As with the kit
that comprises at least two siRNAs, more preferably one strand will
comprise a sequence that is identical to a sequence that is
contained in the mRNA and another strand that is 100% complementary
to a sequence that is contained in the mRNA. During experiments,
when three siRNAs were combined together, 60% of the pools induced
more than 95% gene silencing and 92% of the pools induced more than
80% gene silencing.
[0432] Further, even more preferably, the kit is comprised of at
least four siRNAs, wherein one strand of each siRNA comprises a
sequence that is substantially similar to a region of the sequence
of the target mRNA, and the other strand comprises a sequence that
is substantially complementary to the region of the target mRNA. As
with the kit or pool that comprises at least two siRNAs, more
preferably one strand of each of the siRNA duplexes will comprise a
sequence that is identical to a sequence that is contained in the
mRNA, and another strand that is 100% complementary to a sequence
that is contained in the mRNA.
[0433] Additionally, kits and pools with at least five, at least
six, and at least seven siRNAs may also be useful with the present
invention. For example, pools of five siRNA induced 95% gene
silencing with 77% probability and 80% silencing with 98.8%
probability. Thus, pooling of siRNAs together can result in the
creation of a target-specific silencing reagent with almost a 99%
probability of being functional. The fact that such high levels of
success are achievable using such pools of siRNA, enables one to
dispense with costly and time-consuming target-specific validation
procedures.
[0434] For this embodiment, as well as the other aforementioned
embodiments, each of the siRNAs within a pool will preferably
comprise 18-30 base pairs, more preferably 18-25 base pairs, and
most preferably 19 base pairs. Within each siRNA, preferably at
least 18 contiguous bases of the antisense strand will be 100%
complementary to the target mRNA. More preferably, at least 19
contiguous bases of the antisense strand will be 100% complementary
to the target mRNA. Additionally, there may be overhangs on either
the sense strand or the antisense strand, and these overhangs may
be at either the 5' end or the 3' end of either of the strands, for
example there may be one or more overhangs of 1-6 bases. When
overhangs are present, they are not included in the calculation of
the number of base pairs. The two nucleotide 3' overhangs mimic
natural siRNAs and are commonly used but are not essential.
Preferably, the overhangs should consist of two nucleotides, most
often dTdT or UU at the 3' end of the sense and antisense strand
that are not complementary to the target sequence. The siRNAs may
be produced by any method that is now known or that comes to be
known for synthesizing double stranded RNA that one skilled in the
art would appreciate would be useful in the present invention.
Preferably, the siRNAs will be produced by Dharmacon's proprietary
ACE.RTM. technology. However, other methods for synthesizing siRNAs
are well known to persons skilled in the art and include, but are
not limited to, any chemical synthesis of RNA oligonucleotides,
ligation of shorter oligonucleotides, in vitro transcription of RNA
oligonucleotides, the use of vectors for expression within cells,
recombinant Dicer products and PCR products.
[0435] The siRNA duplexes within the aforementioned pools of siRNAs
may correspond to overlapping sequences within a particular mRNA,
or non-overlapping sequences of the mRNA. However, preferably they
correspond to non-overlapping sequences. Further, each siRNA may be
selected randomly, or one or more of the siRNA may be selected
according to the criteria discussed above for maximizing the
effectiveness of siRNA.
[0436] Included in the definition of siRNAs are siRNAs that contain
substituted and/or labeled nucleotides that may, for example, be
labeled by radioactivity, fluorescence or mass. The most common
substitutions are at the 2' position of the ribose sugar, where
moieties such as H (hydrogen) F, NH.sub.3, OCH.sub.3 and other O-
alkyl, alkenyl, alkynyl, and orthoesters, may be substituted, or in
the phosphorous backbone, where sulfur, amines or hydrocarbons may
be substituted for the bridging of non-bridging atoms in the
phosphodiester bond. Examples of modified siRNAs are explained more
fully in commonly assigned U.S. patent application Ser. No.
10/613,077, filed Jul. 1, 2003.
[0437] Additionally, as noted above, the cell type into which the
siRNA is introduced may affect the ability of the siRNA to enter
the cell; however, it does not appear to affect the ability of the
siRNA to function once it enters the cell. Methods for introducing
double-stranded RNA into various cell types are well known to
persons skilled in the art.
[0438] As persons skilled in the art are aware, in certain species,
the presence of proteins such as RdRP, the RNA-dependent RNA
polymerase, may catalytically enhance the activity of the siRNA.
For example, RdRP propagates the RNAi effect in C. elegans and
other non-mammalian organisms. In fact, in organisms that contain
these proteins, the siRNA may be inherited. Two other proteins that
are well studied and known to be a part of the machinery are
members of the Argonaute family and Dicer, as well as their
homologues. There is also initial evidence that the RISC complex
might be associated with the ribosome so the more efficiently
translated mRNAs will be more susceptible to silencing than
others.
[0439] Another very important factor in the efficacy of siRNA is
mRNA localization. In general, only cytoplasmic mRNAs are
considered to be accessible to RNAi to any appreciable degree.
However, appropriately designed siRNAs, for example, siRNAs
modified with internucleotide linkages or 2'-O-methyl groups, may
be able to cause silencing by acting in the nucleus. Examples of
these types of modifications are described in commonly assigned
U.S. patent application Ser. Nos. 10/431,027 and 10/613,077.
[0440] As described above, even when one selects at least two
siRNAs at random, the effectiveness of the two may be greater than
one would predict based on the effectiveness of two individual
siRNAs. This additive or synergistic effect is particularly
noticeable as one increases to at least three siRNAs, and even more
noticeable as one moves to at least four siRNAs. Surprisingly, the
pooling of the non-functional and semi-functional siRNAs,
particularly more than five siRNAs, can lead to a silencing mixture
that is as effective if not more effective than any one particular
functional siRNA.
[0441] Within the kits of the present invention, preferably each
siRNA will be present in a concentration of between 0.001 and 200
.mu.M, more preferably between 0.01 and 200 nM, and most preferably
between 0.1 and 10 nM.
[0442] In addition to preferably comprising at least four or five
siRNAs, the kits of the present invention will also preferably
comprise a buffer to keep the siRNA duplex stable. Persons skilled
in the art are aware of buffers suitable for keeping siRNA stable.
For example, the buffer may be comprised of 100 mM KCl, 30 mM
HEPES-pH 7.5, and 1 mM MgCl.sub.2. Alternatively, kits might
contain complementary strands that contain any one of a number of
chemical modifications (e.g., a 2'-O-ACE) that protect the agents
from degradation by nucleases. In this instance, the user may (or
may not) remove the modifying protective group (e.g., deprotect)
before annealing the two complementary strands together.
[0443] By way of example, the kits may be organized such that pools
of siRNA duplexes are provided on an array or microarray of wells
or drops for a particular gene set or for unrelated genes. The
array may, for example, be in 96 wells, 384 wells or 1284 wells
arrayed in a plastic plate or on a glass slide using techniques now
known or that come to be known to persons skilled in the art.
Within an array, preferably there will be controls such as
functional anti-lamin A/C, cyclophilin and two siRNA duplexes that
are not specific to the gene of interest.
[0444] In order to ensure stability of the siRNA pools prior to
usage, they may be retained in lyophilized form at minus twenty
degrees (-20.degree. C.) until they are ready for use. Prior to
usage, they should be resuspended; however, even once resuspended,
for example, in the aforementioned buffer, they should be kept at
minus twenty degrees, (-20.degree. C.) until used. The
aforementioned buffer, prior to use, may be stored at approximately
4.degree. C. or room temperature. Effective temperatures at which
to conduct transfections are well known to persons skilled in the
art and include for example, room temperature.
[0445] The kits may be applied either in vivo or in vitro.
Preferably, the siRNA of the pools or kits is applied to a cell
through transfection, employing standard transfection protocols.
These methods are well known to persons skilled in the art and
include the use of lipid-based carriers, electroporation, cationic
carriers, and microinjection. Further, one could apply the present
invention by synthesizing equivalent DNA sequences (either as two
separate, complementary strands, or as hairpin molecules) instead
of siRNA sequences and introducing them into cells through vectors.
Once in the cells, the cloned DNA could be transcribed, thereby
forcing the cells to generate the siRNA. Examples of vectors
suitable for use with the present application include but are not
limited to the standard transient expression vectors, adenoviruses,
retroviruses, lentivirus-based vectors, as well as other
traditional expression vectors. Any vector that has an adequate
siRNA expression and procession module may be used. Furthermore,
certain chemical modifications to siRNAs, including but not limited
to conjugations to other molecules, may be used to facilitate
delivery. For certain applications it may be preferable to deliver
molecules without transfection by simply formulating in a
physiological acceptable solution.
[0446] This embodiment may be used in connection with any of the
aforementioned embodiments. Accordingly, the sequences within any
pool may be selected by rational design.
[0447] Multigene Silencing
[0448] In addition to developing kits that contain multiple siRNA
directed against a single gene, another embodiment includes the use
of multiple siRNA targeting multiple genes. Multiple genes may be
targeted through the use of high- or hyper-functional siRNA. High-
or hyper-functional siRNA that exhibit increased potency, require
lower concentrations to induce desired phenotypic (and thus
therapeutic) effects. This circumvents RISC saturation. It
therefore reasons that if lower concentrations of a single siRNA
are needed for knockout or knockdown expression of one gene, then
the remaining (uncomplexed) RISC will be free and available to
interact with siRNA directed against two, three, four, or more,
genes. Thus in this embodiment, the authors describe the use of
highly functional or hyper-functional siRNA to knock out three
separate genes. More preferably, such reagents could be combined to
knockout four distinct genes. Even more preferably, highly
functional or hyperfunctional siRNA could be used to knock out five
distinct genes. Most preferably, siRNA of this type could be used
to knockout or knockdown the expression of six or more genes.
[0449] Hyperfunctional siRNA
[0450] The term hyperfunctional siRNA (hf-siRNA) describes a subset
of the siRNA population that induces RNAi in cells at low- or
sub-nanomolar concentrations for extended periods of time. These
traits, heightened potency and extended longevity of the RNAi
phenotype, are highly attractive from a therapeutic standpoint.
Agents having higher potency require lesser amounts of the molecule
to achieve the desired physiological response, thus reducing the
probability of side effects due to "off-target" interference. In
addition to the potential therapeutic benefits associated with
hyperfunctional siRNA, hf-siRNA are also desirable from an economic
perspective. Hyperfunctional siRNA may cost less on a per-treatment
basis, thus reducing overall expenditures to both the manufacturer
and the consumer.
[0451] Identification of hyperfunctional siRNA involves multiple
steps that are designed to examine an individual siRNA agent's
concentration- and/or longevity-profiles. In one non-limiting
example, a population of siRNA directed against a single gene are
first analyzed using the previously described algorithm (Formula
VIII). Individual siRNA are then introduced into a test cell line
and assessed for the ability to degrade the target mRNA. It is
important to note that when performing this step it is not
necessary to test all of the siRNA. Instead, it is sufficient to
test only those siRNA having the highest SMARTscores.TM. (i.e.,
SMARTscore.TM.>-10). Subsequently, the gene silencing data is
plotted against the SMARTscores.TM. (see FIG. 9). siRNA that (1)
induce a high degree of gene silencing (i.e., they induce greater
than 80% gene knockdown) and (2) have superior SMARTscores.TM.
(i.e., a SMARTscore.TM. of >-10, suggesting a desirable average
internal stability profile) are selected for further investigations
designed to better understand the molecule's potency and longevity.
In one, non-limiting study dedicated to understanding a molecule's
potency, an siRNA is introduced into one (or more) cell types in
increasingly diminishing concentrations (e.g., 3.0.fwdarw.0.3 nM).
Subsequently, the level of gene silencing induced by each
concentration is examined and siRNA that exhibit hyperfunctional
potency (i.e., those that induce 80% silencing or greater at, e.g.,
picomolar concentrations) are identified. In a second study, the
longevity profiles of siRNA having high (>-10) SMARTscores.TM.
and greater than 80% silencing are examined. In one non-limiting
example of how this is achieved, siRNA are introduced into a test
cell line and the levels of RNAi are measured over an extended
period of time (e.g., 24-168 hrs). siRNAs that exhibit strong RNA
interference patterns (i.e., >80% interference) for periods of
time greater than, e.g., 120 hours, are thus identified. Studies
similar to those described above can be performed on any and all of
the >10.sup.6 siRNA included in this document to further define
the most functional molecule for any given gene. Molecules
possessing one or both properties (extended longevity and
heightened potency) are labeled "hyperfunctional siRNA," and
earmarked as candidates for future therapeutic studies.
[0452] While the example(s) given above describe one means by which
hyperfunctional siRNA can be isolated, neither the assays
themselves nor the selection parameters used are rigid and can vary
with each family of siRNA. Families of siRNA include siRNAs
directed against a single gene, or directed against a related
family of genes.
[0453] The highest quality siRNA achievable for any given gene may
vary considerably. Thus, for example, in the case of one gene (gene
X), rigorous studies such as those described above may enable the
identification of an siRNA that, at picomolar concentrations,
induces 99.sup.+% silencing for a period of 10 days. Yet identical
studies of a second gene (gene Y) may yield an siRNA that at high
nanomolar concentrations (e.g., 100 nM) induces only 75% silencing
for a period of 2 days. Both molecules represent the very optimum
siRNA for their respective gene targets and therefore are
designated "hyperfunctional." Yet due to a variety of factors
including but not limited to target concentration, siRNA stability,
cell type, off-target interference, and others, equivalent levels
of potency and longevity are not achievable. Thus, for these
reasons, the parameters described in the before mentioned assays
can vary. While the initial screen selected siRNA that had
SMARTscore.TM. above -10 and a gene silencing capability of greater
than 80%, selections that have stronger (or weaker) parameters can
be implemented. Similarly, in the subsequent studies designed to
identify molecules with high potency and longevity, the desired
cutoff criteria (i.e., the lowest concentration that induces a
desirable level of interference, or the longest period of time that
interference can be observed) can vary. The experimentation
subsequent to application of the rational criteria of this
application is significantly reduced where one is trying to obtain
a suitable hyperfunctional siRNA for, for example, therapeutic use.
When, for example, the additional experimentation of the type
described herein is applied by one skilled in the art with this
disclosure in hand, a hyperfunctional siRNA is readily
identified.
[0454] The siRNA may be introduced into a cell by any method that
is now known or that comes to be known and that from reading this
disclosure, persons skilled in the art would determine would be
useful in connection with the present invention in enabling siRNA
to cross the cellular membrane. These methods include, but are not
limited to, any manner of transfection, such as, for example,
transfection employing DEAE-Dextran, calcium phosphate, cationic
lipids/liposomes, micelles, manipulation of pressure,
microinjection, electroporation, immunoporation, use of vectors
such as viruses, plasmids, cosmids, bacteriophages, cell fusions,
and coupling of the polynucleotides to specific conjugates or
ligands such as antibodies, antigens, or receptors, passive
introduction, adding moieties to the siRNA that facilitate its
uptake, and the like.
[0455] Having described the invention with a degree of
particularity, examples will now be provided. These examples are
not intended to and should not be construed to limit the scope of
the claims in any way.
EXAMPLES
[0456] General Techniques and Nomenclatures
[0457] siRNA nomenclature. All siRNA duplexes are referred to by
sense strand. The first nucleotide of the 5'-end of the sense
strand is position 1, which corresponds to position 19 of the
antisense strand for a 19-mer. In most cases, to compare results
from different experiments, silencing was determined by measuring
specific transcript mRNA levels or enzymatic activity associated
with specific transcript levels, 24 hours post-transfection, with
siRNA concentrations held constant at 100 nM. For all experiments,
unless otherwise specified, transfection efficiency was ensured to
be over 95%, and no detectable cellular toxicity was observed. The
following system of nomenclature was used to compare and report
siRNA-silencing functionality: "F" followed by the degree of
minimal knockdown. For example, F50 signifies at least 50%
knockdown, F80 means at least 80%, and so forth. For this study,
all sub-F50 siRNAs were considered non-functional.
[0458] Cell culture and transfection. 96-well plates are coated
with 50 .mu.l of 50 mg/ml poly-L-lysine (Sigma) for 1 hr, and then
washed 3.times. with distilled water before being dried for 20 min.
HEK293 cells or HEK293Lucs or any other cell type of interest are
released from their solid support by trypsinization, diluted to
3.5.times.10.sup.5 cells/ml, followed by the addition of 100 .mu.L
of cells/well. Plates are then incubated overnight at 37.degree.
C., 5% CO.sub.2. Transfection procedures can vary widely depending
on the cell type and transfection reagents. In one non-limiting
example, a transfection mixture consisting of 2 mL Opti-MEM I
(Gibco-BRL), 80 .mu.l Lipofectamine 2000 (Invitrogen), 15 .mu.L
SUPERNasin at 20 U/.mu.l (Ambion), and 1.5 .mu.l of reporter gene
plasmid at 1 .mu.g/.mu.l is prepared in 5-ml polystyrene round
bottom tubes. One hundred .mu.l of transfection reagent is then
combined with 100 .mu.l of siRNAs in polystyrene deep-well titer
plates (Beckman) and incubated for 20 to 30 min at room
temperature. Five hundred and fifty microliters of Opti-MEM is then
added to each well to bring the final siRNA concentration to 100
nM. Plates are then sealed with parafilm and mixed. Media is
removed from HEK293 cells and replaced with 95 .mu.l of
transfection mixture. Cells are incubated overnight at 37.degree.
C., 5% CO.sub.2.
[0459] Quantification of gene knockdown. A variety of
quantification procedures can be used to measure the level of
silencing induced by siRNA or siRNA pools. In one non-limiting
example: to measure mRNA levels 24 hrs post-transfection,
QuantiGene branched-DNA (bDNA) kits (Bayer) (Wang, et al,
Regulation of insulin preRNA splicing by glucose. Proc. Natl. Acad.
Sci. USA 1997, 94:4360.) are used according to manufacturer
instructions. To measure luciferase activity, media is removed from
HEK293 cells 24 hrs post-transfection, and 50 .mu.l of Steady-GLO
reagent (Promega) is added. After 5 minutes, plates are analyzed on
a plate reader.
Example I
Sequences Used to Develop the Algorithm
[0460] Anti-Firefly and anti-Cyclophilin siRNAs panels (FIG. 5a, b)
sorted according to using Formula VIII predicted values. All siRNAs
scoring more than 0 (formula VIII) and more then 20 (formula IX)
are fully functional. All ninety sequences for each gene (and DBI)
appear below in Table III.
8TABLE III Cyclo 1 SEQ. ID 0032 GUUCCAAAAACAGUGGAUA Cyclo 2 SEQ. ID
0033 UCCAAAAACAGUGGAUAAU Cyclo 3 SEQ. ID 0034 CAAAAACAGUGGAUAAUUU
Cyclo 4 SEQ. ID 0035 AAAACAGUGGAUAAUUUUG Cyclo 5 SEQ. ID 0036
AACAGUGGAUAAUUUUGUG Cyclo 6 SEQ. ID 0037 CAGUGGAUAAUUUUGUGGC Cyclo
7 SEQ. ID 0038 GUGGAUAAUUUUGUGGCCU Cyclo 8 SEQ. ID 0039
GGAUAAUUUUGUGGCCUUA Cyclo 9 SEQ. ID 0040 AUAAUUUUGUGGCCUUAGC Cyclo
10 SEQ. ID 0041 AAUUUUGUGGCCUUAGCUA Cyclo 11 SEQ. ID 0042
UUUUGUGGCCUUAGCUACA Cyclo 12 SEQ. ID 0043 UUGUGGCCUUAGCUACAGG Cyclo
13 SEQ. ID 0044 GUGGCCUUAGCUACAGGAG Cyclo 14 SEQ. ID 0045
GGCCUUAGCUACAGGAGAG Cyclo 15 SEQ. ID 0046 CCUUAGCUACAGGAGAGAA Cyclo
16 SEQ. ID 0047 UUAGCUACAGGAGAGAAAG Cyclo 17 SEQ. ID 0048
AGCUACAGGAGAGAAAGGA Cyclo 18 SEQ. ID 0049 CUACAGGAGAGAAAGGAUU Cyclo
19 SEQ. ID 0050 ACAGGAGAGAAAGGAUUUG Cyclo 20 SEQ. ID 0051
AGGAGAGAAAGGAUUUGGC Cyclo 21 SEQ. ID 0052 GAGAGAAAGGAUUUGGCUA Cyclo
22 SEQ. ID 0053 GAGAAAGGAUUUGGCUACA Cyclo 23 SEQ. ID 0054
GAAAGGAUUUGGCUACAAA Cyclo 24 SEQ. ID 0055 AAGGAUUUGGCUACAAAAA Cyclo
25 SEQ. ID 0056 GGAUUUGGCUACAAAAACA Cyclo 26 SEQ. ID 0057
AUUUGGCUACAAAAACAGC Cyclo 27 SEQ. ID 0058 UUGGCUACAAAAACAGCAA Cyclo
28 SEQ. ID 0059 GGCUACAAAAACAGCAAAU Cyclo 29 SEQ. ID 0060
CUACAAAAACAGCAAAUUC Cyclo 30 SEQ. ID 0061 ACAAAAACAGCAAAUUCCA Cyclo
31 SEQ. ID 0062 AAAAACAGCAAAUUCCAUC Cyclo 32 SEQ. ID 0063
AAACAGCAAAUUCCAUCGU Cyclo 33 SEQ. ID 0064 ACAGCAAAUUCCAUCGUGU Cyclo
34 SEQ. ID 0065 AGCAAAUUCCAUCGUGUAA Cyclo 35 SEQ. ID 0066
CAAAUUCCAUCGUGUAAUC Cyclo 36 SEQ. ID 0067 AAUUCCAUCGUGUAAUCAA Cyclo
37 SEQ. ID 0068 UUCCAUCGUGUAAUCAAGG Cyclo 38 SEQ. ID 0069
CCAUCGUGUAAUCAAGGAC Cyclo 39 SEQ. ID 0070 AUCGUGUAAUCAAGGACUU Cyclo
40 SEQ. ID 0071 CGUGUAAUCAAGGACUUCA Cyclo 41 SEQ. ID 0072
UGUAAUCAAGGACUUCAUG Cyclo 42 SEQ. ID 0073 UAAUCAAGGACUUCAUGAU Cyclo
43 SEQ. ID 0074 AUCAAGGACUUCAUGAUCC Cyclo 44 SEQ. ID 0075
CAAGGACUUCAUGAUCCAG Cyclo 45 SEQ. ID 0076 AGGACUUCAUGAUCCAGGG Cyclo
46 SEQ. ID 0077 GACUUCAUGAUCCAGGGCG Cyclo 47 SEQ. ID 0078
CUUCAUGAUCCAGGGCGGA Cyclo 48 SEQ. ID 0079 UCAUGAUCCAGGGCGGAGA Cyclo
49 SEQ. ID 0080 AUGAUCCAGGGCGGAGACU Cyclo 50 SEQ. ID 0081
GAUCCAGGGCGGAGACUUC Cyclo 51 SEQ. ID 0082 UCCAGGGCGGAGACUUCAC Cyclo
52 SEQ. ID 0083 CAGGGCGGAGACUUCACCA Cyclo 53 SEQ. ID 0084
GGGCGGAGACUUCACCAGG Cyclo 54 SEQ. ID 0085 GCGGAGACUUCACCAGGGG Cyclo
55 SEQ. ID 0086 GGAGACUUCACCAGGGGAG Cyclo 56 SEQ. ID 0087
AGACUUCACCAGGGGAGAU Cyclo 57 SEQ. ID 0088 ACUUCACCAGGGGAGAUGG Cyclo
58 SEQ. ID 0089 UUCACCAGGGGAGAUGGCA Cyclo 59 SEQ. ID 0090
CACCAGGGGAGAUGGCACA Cyclo 60 SEQ. ID 0091 CCAGGGGAGAUGGCACAGG Cyclo
61 SEQ. ID 0092 AGGGGAGAUGGCACAGGAG Cyclo 62 SEQ. ID 0093
GGGAGAUGGCACAGGAGGA Cyclo 63 SEQ. ID 0094 GAGAUGGCACAGGAGGAAA Cyclo
64 SEQ. ID 0095 GAUGGCACAGGAGGAAAGA Cyclo 65 SEQ. ID 0431
UGGCACAGGAGGAAAGAGC Cyclo 66 SEQ. ID 0096 GCACAGGAGGAAAGAGCAU Cyclo
67 SEQ. ID 0097 ACAGGAGGAAAGAGCAUCU Cyclo 68 SEQ. ID 0098
AGGAGGAAAGAGCAUCUAC Cyclo 69 SEQ. ID 0099 GAGGAAAGAGCAUCUACGG Cyclo
70 SEQ. ID 0100 GGAAAGAGCAUCUACGGUG Cyclo 71 SEQ. ID 0101
AAAGAGCAUCUACGGUGAG Cyclo 72 SEQ. ID 0102 AGAGCAUCUACGGUGAGCG Cyclo
73 SEQ. ID 0103 AGCAUCUACGGUGAGCGCU Cyclo 74 SEQ. ID 0104
CAUCUACGGUGAGCGCUUC Cyclo 75 SEQ. ID 0105 UCUACGGUGAGCGCUUCCC Cyclo
76 SEQ. ID 0106 UACGGUGAGCGCUUCCCCG Cyclo 77 SEQ. ID 0107
CGGUGAGCGCUUCCCCGAU Cyclo 78 SEQ. ID 0108 GUGAGCGCUUCCCCGAUGA Cyclo
79 SEQ. ID 0109 GAGCGCUUCCCCGAUGAGA Cyclo 80 SEQ. ID 0110
GCGCUUCCCCGAUGAGAAC Cyclo 81 SEQ. ID 0111 GCUUCCCCGAUGAGAACUU Cyclo
82 SEQ. ID 0112 UUCCCCGAUGAGAACUUCA Cyclo 83 SEQ. ID 0113
CCCCGAUGAGAACUUCAAA Cyclo 84 SEQ. ID 0114 CCGAUGAGAACUUCAAACU Cyclo
85 SEQ. ID 0115 GAUGAGAACUUCAAACUGA Cyclo 86 SEQ. ID 0116
UGAGAACUUCAAACUGAAG Cyclo 87 SEQ. ID 0117 AGAACUUCAAACUGAAGCA Cyclo
88 SEQ. ID 0118 AACUUCAAACUGAAGCACU Cyclo 89 SEQ. ID 0119
CUUCAAACUGAAGCACUAC Cyclo 90 SEQ. ID 0120 UCAAACUGAAGCACUACGG DB 1
SEQ. ID 0121 ACGGGCAAGGCCAAGUGGG DB 2 SEQ. ID 0122
CGGGCAAGGCCAAGUGGGA DB 3 SEQ. ID 0123 GGGCAAGGCCAAGUGGGAU DB 4 SEQ.
ID 0124 GGCAAGGCCAAGUGGGAUG DB 5 SEQ. ID 0125 GCAAGGCCAAGUGGGAUGC
DB 6 SEQ. ID 0126 CAAGGCCAAGUGGGAUGCC DB 7 SEQ. ID 0127
AAGGCCAAGUGGGAUGCGU DB 8 SEQ. ID 0128 AGGCCAAGUGGGAUGCCUG DB 9 SEQ.
ID 0129 GGCCAAGUGGGAUGCCUGG DB 10 SEQ. ID 0130 GCCAAGUGGGAUGCCUGGA
DB 11 SEQ. ID 0131 CCAAGUGGGAUGCCUGGAA DB 12 SEQ. ID 0132
CAAGUGGGAUGCCUGGAAU DB 13 SEQ. ID 0133 AAGUGGGAUGCCUGGAAUG DB 14
SEQ. ID 0134 AGUGGGAUGCCUGGAAUGA DB 15 SEQ. ID 0135
GUGGGAUGCCUGGAAUGAG DB 16 SEQ. ID 0136 UGGGAUGCCUGGAAUGAGC DB 17
SEQ. ID 0137 GGGAUGCCUGGAAUGAGCU DB 18 SEQ. ID 0138
GGAUGCCUGGAAUGAGCUG DB 19 SEQ. ID 0139 GAUGCCUGGAAUGAGCUGA DB 20
SEQ. ID 0140 AUGCCUGGAAUGAGCUGAA DB 21 SEQ. ID 0141
UGCCUGGAAUGAGCUGAAA DB 22 SEQ. ID 0142 GCCUGGAAUGAGCUGAAAG DB 23
SEQ. ID 0143 CCUGGAAUGAGCUGAAAGG DB 24 SEQ. ID 0144
CUGGAAUGAGCUGAAAGGG DB 25 SEQ. ID 0145 UGGAAUGAGCUGAAAGGGA DB 26
SEQ. ID 0146 GGAAUGAGCUGAAAGGGAC DB 27 SEQ. ID 0147
GAAUGAGCUGAAAGGGACU DB 28 SEQ. ID 0148 AAUGAGCUGAAAGGGACUU DB 29
SEQ. ID 0149 AUGAGCUGAAAGGGACUUC DB 30 SEQ. ID 0150
UGAGCUGAAAGGGACUUCC DB 31 SEQ. ID 0151 GAGCUGAAAGGGACUUCCA DB 32
SEQ. ID 0152 AGCUGAAAGGGACUUCCAA DB 33 SEQ. ID 0153
GCUGAAAGGGACUUCCAAG DB 34 SEQ. ID 0154 CUGAAAGGGACUUCCAAGG DB 35
SEQ. ID 0155 UGAAAGGGACUUCCAAGGA DB 36 SEQ. ID 0156
GAAAGGGACUUCCAAGGAA DB 37 SEQ. ID 0157 AAAGGGACUUCCAAGGAAG DB 38
SEQ. ID 0158 AAGGGACUUCCAAGGAAGA DB 39 SEQ. ID 0159
AGGGACUUCCAAGGAAGAU DB 40 SEQ. ID 0160 GGGACUUCCAAGGAAGAUG DB 41
SEQ. ID 0161 GGACUUCCAAGGAAGAUGC DB 42 SEQ. ID 0162
GACUUCCAAGGAAGAUGCC DB 43 SEQ. ID 0163 ACUUCCAAGGAAGAUGCCA DB 44
SEQ. ID 0164 CUUCCAAGGAAGAUGCCAU DB 45 SEQ. ID 0165
UUCCAAGGAAGAUGCCAUG DB 46 SEQ. ID 0166 UCCAAGGAAGAUGCCAUGA DB 47
SEQ. ID 0167 CCAAGGAAGAUGCCAUGAA DB 48 SEQ. ID 0168
CAAGGAAGAUGCCAUGAAA DB 49 SEQ. ID 0169 AAGGAAGAUGCCAUGAAAG DB 50
SEQ. ID 0170 AGGAAGAUGCCAUGAAAGC DB 51 SEQ. ID 0171
GGAAGAUGCCAUGAAAGCU DB 52 SEQ. ID 0172 GAAGAUGCCAUGAAAGCUU DB 53
SEQ. ID 0173 AAGAUGCCAUGAAAGCUUA DB 54 SEQ. ID 0174
AGAUGCCAUGAAAGGUUAC DB 55 SEQ. ID 0175 GAUGCCAUGAAAGCUUACA DB 56
SEQ. ID 0176 AUGCCAUGAAAGCUUACAU DB 57 SEQ. ID 0177
UGCCAUGAAAGCUUACAUC DB 58 SEQ. ID 0178 GCCAUGAAAGCUUACAUCA DB 59
SEQ. ID 0179 CCAUGAAAGCUUACAUCAA DB 60 SEQ. ID 0180
CAUGAAAGCUUACAUCAAC DB 61 SEQ. ID 0181 AUGAAAGCUUACAUCAACA DB 62
SEQ. ID 0182 UGAAAGCUUACAUCAACAA DB 63 SEQ. ID 0183
GAAAGCUUACAUCAACAAA DB 64 SEQ. ID 0184 AAAGCUUACAUCAACAAAG DB 65
SEQ. ID 0185 AAGCUUACAUCAACAAAGU DB 66 SEQ. ID 0186
AGCUUACAUCAACAAAGUA DB 67 SEQ. ID 0187 GCUUACAUCAACAAAGUAG DB 68
SEQ. ID 0188 CUUACAUCAACAAAGUAGA DB 69 SEQ. ID 0189
UUACAUCAACAAAGUAGAA DB 70 SEQ. ID 0190 UACAUCAACAAAGUAGAAG DB 71
SEQ. ID 0191 ACAUCAACAAAGUAGAAGA DB 72 SEQ. ID 0192
CAUCAACAAAGUAGAAGAG DB 73 SEQ. ID 0193 AUCAACAAAGUAGAAGAGC DB 74
SEQ. ID 0194 UCAACAAAGUAGAAGAGCU DB 75 SEQ. ID 0195
CAACAAAGUAGAAGAGCUA DB 76 SEQ. ID 0196 AACAAAGUAGAAGAGCUAA DB 77
SEQ. ID 0197 ACAAAGUAGAAGAGCUAAA DB 78 SEQ. ID 0198
CAAAGUAGAAGAGCUAAAG DB 79 SEQ. ID 0199 AAAGUAGAAGAGCUAAAGA DB 80
SEQ. ID 0200 AAGUAGAAGAGCUAAAGAA DB 81 SEQ. ID 0201
AGUAGAAGAGCUAAAGAAA DB 82 SEQ. ID 0202 GUAGAAGAGCUAAAGAAAA DB 83
SEQ. ID 0203 UAGAAGAGCUAAAGAAAAA DB 84 SEQ. ID 0204
AGAAGAGCUAAAGAAAAAA DB 85 SEQ. ID 0205 GAAGAGCUAAAGAAAAAAU DB 86
SEQ. ID 0206 AAGAGCUAAAGAAAAAAUA DB 87 SEQ. ID 0207
AGAGCUAAAGAAAAAAUAC DB 88 SEQ. ID 0208 GAGCUAAAGAAAAAAUACG DB 89
SEQ. ID 0209 AGCUAAAGAAAAAAUACGG DB 90 SEQ. ID 0210
GCUAAAGAAAAAAUACGGG Luc 1 SEQ. ID 0211 AUCCUCAUAAAGGCCAAGA Luc 2
SEQ. ID 0212 AGAUCCUCAUAAAGGCCAA Luc 3 SEQ. ID 0213
AGAGAUCCUCAUAAAGGCG Luc 4 SEQ. ID 0214 AGAGAGAUCCUCAUAAAGG Luc 5
SEQ. ID 0215 UCAGAGAGAUCCUCAUAAA Luc 6 SEQ. ID 0216
AAUCAGAGAGAUCCUCAUA Luc 7 SEQ. ID 0217 AAAAUCAGAGAGAUCCUCA Luc 8
SEQ. ID 0218 GAAAAAUCAGAGAGAUCCU Luc 9 SEQ. ID 0219
AAGAAAAAUCAGAGAGAUC Luc 10 SEQ. ID 0220 GCAAGAAAAAUCAGAGAGA Luc 11
SEQ. ID 0221 ACGCAAGAAAAAUCAGAGA Luc 12 SEQ. ID 0222
CGACGCAAGAAAAAUCAGA Luc 13 SEQ. ID 0223 CUCGACGCAAGAAAAAUCA Luc 14
SEQ. ID 0224 AACUCGACGCAAGAAAAAU Luc 15 SEQ. ID 0225
AAAACUCGACGCAAGAAAA Luc 16 SEQ. ID 0226 GGAAAACUCGACGCAAGAA Luc 17
SEQ. ID 0227 CCGGAAAACUCGACGCAAG Luc 18 SEQ. ID 0228
UACCGGAAAACUCGACGCA Luc 19 SEQ. ID 0229 CUUACCGGAAAACUCGACG Luc 20
SEQ. ID 0230 GUCUUACCGGAAAACUCGA Luc 21 SEQ. ID 0231
AGGUCUUACCGGAAAACUC Luc 22 SEQ. ID 0232 AAAGGUCUUACCGGAAAAC Luc 23
SEQ. ID 0233 CGAAAGGUCUUACCGGAAA Luc 24 SEQ. ID 0234
ACCGAAAGGUCUUACCGGA Luc 25 SEQ. ID 0235 GUACCGAAAGGUCUUACCG Luc 26
SEQ. ID 0236 AAGUACCGAAAGGUCUUAC Luc 27 SEQ. ID 0237
CGAAGUACCGAAAGGUCUU Luc 28 SEQ. ID 0238 GACGAAGUACCGAAAGGUC Luc 29
SEQ. ID 0239 UGGACGAAGUACCGAAAGG Luc 30 SEQ. ID 0240
UGUGGACGAAGUACCGAAA Luc 31 SEQ. ID 0241 UUUGUGGACGAAGUACCGA Luc 32
SEQ. ID 0242 UGUUUGUGGACGAAGUACC Luc 33 SEQ. ID 0243
UGUGUUUGUGGACGAAGUA Luc 34 SEQ. ID 0244 GUUGUGUUUGUGGACGAAG Luc 35
SEQ. ID 0245 GAGUUGUGUUUGUGGACGA Luc 36 SEQ. ID 0246
AGGAGUUGUGUUUGUGGAC Luc 37 SEQ. ID 0247 GGAGGAGUUGUGUUUGUGG Luc 38
SEQ. ID 0248 GCGGAGGAGUUGUGUUUGU Luc 39 SEQ. ID 0249
GCGCGGAGGAGUUGUGUUU Luc 40 SEQ. ID 0250 UUGCGCGGAGGAGUUGUGU Luc 41
SEQ. ID 0251 AGUUGCGCGGAGGAGUUGU Luc 42 SEQ. ID 0252
AAAGUUGCGCGGAGGAGUU Luc 43 SEQ. ID 0253 AAAAAGUUGCGCGGAGGAG Luc 44
SEQ. ID 0254 CGAAAAAGUUGCGCGGAGG Luc 45 SEQ. ID 0255
GGCGAAAAAGUUGCGCGGA Luc 46 SEQ. ID 0256 ACCGCGAAAAAGUUGCGCG Luc 47
SEQ. ID 0257 CAACCGCGAAAAAGUUGCG Luc 48 SEQ. ID 0258
AACAACCGCGAAAAAGUUG Luc 49 SEQ. ID 0259 GUAACAACCGCGAAAAAGU Luc 50
SEQ. ID 0260 AAGUAACAACCGCGAAAAA Luc 51 SEQ. ID 0261
UCAAGUAACAACCGCGAAA Luc 52 SEQ. ID 0262 AGUCAAGUAACAACCGCGA Luc 53
SEQ. ID 0263 CCAGUCAAGUAACAACCGC Luc 54 SEQ. ID 0264
CGCCAGUCAAGUAACAACC Luc 55 SEQ. ID 0265 GUCGCCAGUCAAGUAACAA Luc 56
SEQ. ID 0266 ACGUCGCCAGUCAAGUAAC Luc 57 SEQ. ID 0267
UUACGUCGGCAGUCAAGUA Luc 58 SEQ. ID 0268 GAUUACGUCGCCAGUCAAG Luc 59
SEQ. ID 0269 UGGAUUACGUCGCCAGUCA Luc 60 SEQ. ID 0270
CGUGGAUUACGUCGCCAGU Luc 61 SEQ. ID 0271 AUCGUGGAUUACGUCGCCA Luc 62
SEQ. ID 0272 AGAUCGUGGAUUACGUCGC Luc 63 SEQ. ID 0273
AGAGAUCGUGGAUUACGUC Luc 64 SEQ. ID 0274 AAAGAGAUCGUGGAUUACG Luc 65
SEQ. ID 0275 AAAAAGAGAUCGUGGAUUA Luc 66 SEQ. ID 0276
GGAAAAAGAGAUCGUGGAU Luc 67 SEQ. ID 0277 ACGGAAAAAGAGAUCGUGG Luc 68
SEQ. ID 0278 UGACGGAAAAAGAGAUCGU Luc 69 SEQ. ID 0279
GAUGACGGAAAAAGAGAUC Luc 70 SEQ. ID 0280 ACGAUGACGGAAAAAGAGA Luc 71
SEQ. ID 0281 AGACGAUGACGGAAAAAGA Luc 72 SEQ. ID 0282
AAAGACGAUGACGGAAAAA Luc 73 SEQ. ID 0283 GGAAAGACGAUGACGGAAA Luc 74
SEQ. ID 0284 ACGGAAAGACGAUGACGGA Luc 75 SEQ. ID 0285
GCAGGGAAAGACGAUGACG Luc 76 SEQ. ID 0286 GAGCACGGAAAGACGAUGA Luc 77
SEQ. ID 0287 UGGAGCACGGAAAGACGAU Luc 78 SEQ. ID 0288
UUUGGAGCACGGAAAGACG Luc 79 SEQ. ID 0289 GUUUUGGAGCACGGAAAGA Luc 80
SEQ. ID 0290 UUGUUUUGGAGCACGGAAA Luc 81 SEQ. ID 0291
UGUUGUUUUGGAGCACGGA Luc 82 SEQ. ID 0292 GUUGUUGUUUUGGAGCAGG Luc 83
SEQ. ID 0293 CCGUUGUUGUUUUGGAGCA Luc 84 SEQ. ID 0294
CGCCGUUGUUGUUUUGGAG Luc 85 SEQ. ID 0295 GCCGCCGUUGUUGUUUUGG Luc 86
SEQ. ID 0296 CCGGCGCCGUUGUUGUUUU Luc 87 SEQ. ID 0297
UCCCGCCGCCGUUGUUGUU Luc 88 SEQ. ID 0298 CUUCCCGCCGCCGUUGUUG Luc 89
SEQ. ID 0299 AACUUCCCGCCGCCGUUGU Luc 90 SEQ. ID 0300
UGAACUUCCCGCCGCCGUU
Example II
Validation of the Algorithm Using DBI, Luciferase, PLK, EGFR, and
SEAP
[0461] The algorithm (Formula VIII) identified siRNAs for five
genes, human DBI, firefly luciferase (fLuc), renilla luciferase
(rLuc), human PLK, and human secreted alkaline phosphatase (SEAP).
Four individual siRNAs were selected on the basis of their
SMARTscores.TM. derived by analysis of their sequence using Formula
VIII (all of the siRNAs would be selected with Formula IX as well)
and analyzed for their ability to silence their targets'
expression. In addition to the scoring, a BLAST search was
conducted for each siRNA. To minimize the potential for off-target
silencing effects, only those target sequences with more than three
mismatches against un-related sequences were selected. Semizarov,
et al, Specificity of short interfering RNA determined through gene
expression signatures. Proc. Natl. Acad. Sci. U.S.A. 2003,
100:6347. These duplexes were analyzed individually and in pools of
4 and compared with several siRNAs that were randomly selected. The
functionality was measured as a percentage of targeted gene
knockdown as compared to controls. All siRNAs were transfected as
described by the methods above at 100 nM concentration into HEK293
using Lipofectamine 2000. The level of the targeted gene expression
was evaluated by B-DNA as described above and normalized to the
non-specific control. FIG. 10 shows that the siRNAs selected by the
algorithm disclosed herein were significantly more potent than
randomly selected siRNAs. The algorithm increased the chances of
identifying an F50 siRNA from 48% to 91%, and an F80 siRNA from 13%
to 57%. In addition, pools of SMART siRNA silence the selected
target better than randomly selected pools (see FIG. 10F).
Example III
Validation of the Algorithm Using Genes Involved in
Clathrin-Dependent Endocytosis
[0462] Components of clathrin-mediated endocytosis pathway are key
to modulating intracellular signaling and play important roles in
disease. Chromosomal rearrangements that result in fusion
transcripts between the Mixed-Lineage Leukemia gene (MLL) and CALM
(clathrin assembly lymphoid myeloid leukemia gene) are believed to
play a role in leukemogenesis. Similarly, disruptions in Rab7 and
Rab9, as well as HIP1 (Huntingtin-interacting protein), genes that
are believed to be involved in endocytosis, are potentially
responsible for ailments resulting in lipid storage, and neuronal
diseases, respectively. For these reasons, siRNA directed against
clathrin and other genes involved in the clathrin-mediated
endocytotic pathway are potentially important research and
therapeutic tools.
[0463] siRNAs directed against genes involved in the
clathrin-mediated endocytosis pathways were selected using Formula
VIII. The targeted genes were clathrin heavy chain (CHC, accession
# NM.sub.--004859), clathrin light chain A (CLCa, NM.sub.--001833),
clathrin light chain B (CLCb, NM.sub.--001834), CALM (U45976),
.beta.2 subunit of AP-2 (.beta.2, NM.sub.--001282), Eps15
(NM.sub.--001981), Eps15R (NM.sub.--021235), dynamin II (DYNII,
NM.sub.--004945), Rab5a (BC001267), Rab5b (NM.sub.--002868), Rab5c
(AF141304), and EEA.1 (XM.sub.--018197).
[0464] For each gene, four siRNAs duplexes with the highest scores
were selected and a BLAST search was conducted for each of them
using the Human EST database. In order to minimize the potential
for off-target silencing effects, only those sequences with more
than three mismatches against un-related sequences were used. All
duplexes were synthesized at Dharmacon, Inc. as 21-mers with 3'-UU
overhangs using a modified method of 2'-ACE chemistry, Scaringe,
Advanced 5'-silyl-2'-orthoester approach to RNA oligonucleotide
synthesis, Methods Enzymol 2000, 317:3, and the antisense strand
was chemically phosphorylated to insure maximized activity.
[0465] HeLa cells were grown in Dulbecco's modified Eagle's medium
(DMEM) containing 10% fetal bovine serum, antibiotics and
glutamine. siRNA duplexes were resuspended in 1.times. siRNA
Universal buffer (Dharmacon, Inc.) to 20 .mu.M prior to
transfection. HeLa cells in 12-well plates were transfected twice
with 4 .mu.l of 20 .mu.M siRNA duplex in 3 .mu.l Lipofectamine 2000
reagent (Invitrogen, Carlsbad, Calif., USA) at 24-hour intervals.
For the transfections in which 2 or 3 siRNA duplexes were included,
the amount of each duplex was decreased, so that the total amount
was the same as in transfections with single siRNAs. Cells were
plated into normal culture medium 12 hours prior to experiments,
and protein levels were measured 2 or 4 days after the first
transfection.
[0466] Equal amounts of lysates were resolved by electrophoresis,
blotted, and stained with the antibody specific to targeted
protein, as well as antibodies specific to unrelated proteins, PP1
phosphatase and Tsg101 (not shown). The cells were lysed in Triton
X-100/glycerol solubilization buffer as described previously.
Tebar, Bohlander, & Sorkin, Clathrin Assembly Lymphoid Myeloid
Leukemia (CALM) Protein: Localization in Endocytic-coated Pits,
Interactions with Clathrin, and the Impact of Overexpression on
Clathrin-mediated Traffic, Mol. Biol. Cell August 1999, 10:2687.
Cell lysates were electrophoresed, transferred to nitrocellulose
membranes, and Western blotting was performed with several
antibodies followed by detection using enhanced chemiluminescence
system (Pierce, Inc). Several x-ray films were analyzed to
determine the linear range of the chemiluminescence signals, and
the quantifications were performed using densitometry and
AlphaImager v5.5 software (Alpha Innotech Corporation). In
experiments with Eps15R-targeted siRNAs, cell lysates were
subjected to immunoprecipitation with Ab860, and Eps15R was
detected in immunoprecipitates by Western blotting as described
above.
[0467] The antibodies to assess the levels of each protein by
Western blot were obtained from the following sources: monoclonal
antibody to clathrin heavy chain (TD.1) was obtained from American
Type Culture Collection (Rockville, Md., USA); polyclonal antibody
to dynamin II was obtained from Affinity Bioreagents, Inc. (Golden,
Colo., USA); monoclonal antibodies to EEA.1 and Rab5a were
purchased from BD Transduction Laboratories (Los Angeles, Calif.,
USA); the monoclonal antibody to Tsg101 was purchased from Santa
Cruz Biotechnology, Inc. (Santa Cruz, Calif., USA); the monoclonal
antibody to GFP was from ZYMED Laboratories Inc. (South San
Francisco, Calif., USA); the rabbit polyclonal antibodies Ab32
specific to .alpha.-adaptins and Ab20 to CALM were described
previously Sorkin, et al, Stoichiometric Interaction of the
Epidermal Growth Factor Receptor with the Clathrin-associated
Protein Complex AP-2, J. Biol. Chem. January 1995, 270:619, the
polyclonal antibodies to clathrin light chains A and B were kindly
provided by Dr. F. Brodsky (UCSF); monoclonal antibodies to PP1 (BD
Transduction Laboratories) and .alpha.-Actinin (Chemicon) were
kindly provided by Dr. M. Dell'Acqua (University of Colorado);
Eps15 Ab577 and Eps15R Ab860 were kindly provided by Dr. P. P. Di
Fiore (European Cancer Institute).
[0468] FIG. 11 demonstrates the in vivo functionality of 48
individual siRNAs, selected using Formula VIII (most of them will
meet the criteria incorporated by Formula IX as well) targeting 12
genes. Various cell lines were transfected with siRNA duplexes
(Dup1-4) or pools of siRNA duplexes (Pool), and the cells were
lysed 3 days after transfection with the exception of CALM (2 days)
and .beta.2 (4 days).
[0469] Note a .beta.1-adaptin band (part of AP-1 Golgi adaptor
complex) that runs slightly slower than .beta.2 adaptin. CALM has
two splice variants, 66 and 72 kD. The full-length Eps15R (a
doublet of .about.130 kD) and several truncated spliced forms of
.about.100 kD and .about.70 kD were detected in Eps15R
immunoprecipitates (shown by arrows). The cells were lysed 3 days
after transfection. Equal amounts of lysates were resolved by
electrophoresis and blotted with the antibody specific to a
targeted protein (GFP antibody for YFP fusion proteins) and the
antibody specific to unrelated proteins PP1 phosphatase or
.alpha.-actinin, and TSG101. The amount of protein in each specific
band was normalized to the amount of non-specific proteins in each
lane of the gel. Nearly all of them appear to be functional, which
establishes that Formula VIII and IX can be used to predict siRNAs'
functionality in general in a genome wide manner.
[0470] To generate the fusion of yellow fluorescent protein (YFP)
with Rab5b or Rab5c (YFP-Rab5b or YFP-Rab5c), a DNA fragment
encoding the full-length human Rab5b or Rab5c was obtained by PCR
using Pfu polymerase (Stratagene) with a SacI restriction site
introduced into the 5' end and a KpnI site into the 3' end and
cloned into pEYFP-C1 vector (CLONTECH, Palo Alto, Calif., USA).
GFP-CALM and YFP-Rab5a were described previously Tebar, Bohlander,
& Sorkin, Clathrin Assembly Lymphoid Myeloid Leukemia (CALM)
Protein: Localization in Endocytic-coated Pits, Interactions with
Clathrin, and the Impact of Overexpression on Clathrin-mediated
Traffic, Mol. Biol. Cell August 1999, 10:2687.
Example IV
Validation of the Algorithm Using Eg5, GADPH, ATE1, MEK2, MEK1, QB,
LaminA/C, c-myc, Human Cyclophilin, and Mouse Cyclophilin
[0471] A number of genes have been identified as playing
potentially important roles in disease etiology. Expression
profiles of normal and diseased kidneys has implicated Edg5 in
immunoglobulin A neuropathy, a common renal glomerular disease.
Myc1, MEK1/2 and other related kinases have been associated with
one or more cancers, while lamins have been implicated in muscular
dystrophy and other diseases. For these reasons, siRNA directed
against the genes encoding these classes of molecules would be
important research and therapeutic tools.
[0472] FIG. 12 illustrates four siRNAs targeting 10 different genes
(Table V for sequence and accession number information) that were
selected according to the Formula VIII and assayed as individuals
and pools in HEK293 cells. The level of siRNA induced silencing was
measured using the B-DNA assay. These studies demonstrated that
thirty-six out of the forty individual SMART-selected siRNA tested
are functional (90%) and all 10 pools are fully functional.
Example V
Validation of the Algorithm Using Bcl2
[0473] Bcl-2 is a .about.25 kD, 205-239 amino acid, anti-apoptotic
protein that contains considerable homology with other members of
the BCL family including BCLX, MCL1, BAX, BAD, and BIK. The protein
exists in at least two forms (Bcl2a, which has a hydrophobic tail
for membrane anchorage, and Bcl2b, which lacks the hydrophobic
tail) and is predominantly localized to the mitochondrial membrane.
While Bcl2 expression is widely distributed, particular interest
has focused on the expression of this molecule in B and T cells.
Bcl2 expression is down-regulated in normal germinal center B cells
yet in a high percentage of follicular lymphomas, Bcl2 expression
has been observed to be elevated. Cytological studies have
identified a common translocation ((14;18)(q32;q32)) amongst a high
percentage (>70%) of these lymphomas. This genetic lesion places
the Bcl2 gene in juxtaposition to immunoglobulin heavy chain gene
(IgH) encoding sequences and is believed to enforce inappropriate
levels of gene expression, and resistance to programmed cell death
in the follicle center B cells. In other cases, hypomethylation of
the Bcl2 promoter leads to enhanced expression and again,
inhibition of apoptosis. In addition to cancer, dysregulated
expression of Bcl-2 has been correlated with multiple sclerosis and
various neurological diseases.
[0474] The correlation between Bcl-2 translocation and cancer makes
this gene an attractive target for RNAi. Identification of siRNA
directed against the bcl2 transcript (or Bcl2-IgH fusions) would
further our understanding Bcl2 gene function and possibly provide a
future therapeutic agent to battle diseases that result from
altered expression or function of this gene.
[0475] In Silico Identification of Functional siRNA.
[0476] To identify functional and hyperfunctional siRNA against the
Bcl2 gene, the sequence for Bcl-2 was downloaded from the NCBI
Unigene database and analyzed using the Formula VIII algorithm. As
a result of these procedures, both the sequence and SMARTscores.TM.
of the Bcl2 siRNA were obtained and ranked according to their
functionality. Subsequently, these sequences were BLAST'ed
(database) to insure that the selected sequences were specific and
contained minimal overlap with unrealated genes. The
SMARTscores.TM. for the top 10 Bcl-2 siRNA are identified in FIG.
13.
[0477] In Vivo Testing of Bcl-2 SiRNA
[0478] Bcl-2 siRNAs having the top ten SMARTscores.TM. were
selected and tested in a functional assay to determine silencing
efficiency. To accomplish this, each of the ten duplexes were
synthesized using 2'-O-ACE chemistry and transfected at 100 nM
concentrations into cells. Twenty-four hours later assays were
performed on cell extracts to assess the degree of target
silencing. Controls used in these experiments included mock
transfected cells, and cells that were transfected with a
non-specific siRNA duplex.
[0479] The results of these experiments are presented below (and in
FIG. 14) and show that all ten of the selected siRNA induce 80% or
better silencing of the Bcl2 message at 100 nM concentrations.
These data verify that the algorithm successfully identified
functional Bcl2 siRNA and provide a set of functional agents that
can be used in experimental and therapeutic environments.
9 siRNA 1 GGGAGAUAGUGAUGAAGUA SEQ. ID NO. 301 siRNA 2
GAAGUACAUCCAUUAUAAG SEQ. ID NO. 302 siRNA 3 GUACGACAACCGGGAGAUA
SEQ. ID NO. 303 siRNA 4 AGAUAGUGAUGAAGUACAU SEQ. ID NO. 304 siRNA 5
UGAAGACUCUGCUCAGUUU SEQ. ID NO. 305 siRNA 6 GCAUGCGGCCUCUGUUUGA
SEQ. ID NO. 306 siRNA 7 UGCGGCCUCUGUUUGAUUU SEQ. ID NO. 307 siRNA 8
GAGAUAGUGAUGAAGUACA SEQ. ID NO. 308 siRNA 9 GGAGAUAGUGAUGAAGUAC
SEQ. ID NO. 309 siRNA 10 GAAGACUCUGCUCAGUUUG SEQ. ID NO. 310
[0480] Bcl2 siRNA: Sense Strand, 5'.fwdarw.3'
Example VI
Sequences Selected by the Algorithm
[0481] Sequences of the siRNAs selected using Formulas (Algorithms)
VIII and IX with their corresponding ranking, which have been
evaluated for the silencing activity in vivo in the present study
(Formula VIII and IX, respectively) are shown in Table V. It should
be noted that the "t" residues in Table V, and elsewhere, when
referring to siRNA, should be replaced by "u" residues.
10TABLE V Gene Accession Formula Formula Name Number SEQ. ID NO.
FTllSeqTence VIII IX CLTC NM_004859 SEQ. ID NO. 2400
GAAAGAATCTGTAGAGAAA 76 94.2 CLTC NM_004859 SEQ. ID NO. 2401
GCAATGAGCTGTTTGAAGA 65 39.9 CLTC NM_004859 SEQ. ID NO. 2402
TGACAAAGGTGGATAAATT 57 38.2 CLTC NM_004859 SEQ. ID NO. 2403
GGAAATGGATCTCTTTGAA 54 49.4 CLTA NM_001833 SEQ. ID NO. 2404
GGAAAGTAATGGTCCAACA 22 55.5 CLTA NM_001833 SEQ. ID NO. 2405
AGACAGTTATGCAGCTATT 4 22.9 CLTA NM_001833 SEQ. ID NO. 2406
CCAATTCTCGGAAGCAAGA 1 17 CLTA NM_001833 SEQ. ID NO. 2407
GAAAGTAATGGTCCAACAG -1 -13 CLTB NM_001834 SEQ. ID NO. 2408
GCGCCAGAGTGAACAAGTA 17 57.5 CLTB NM_001834 SEQ. ID NO. 2409
GAAGGTGGCCCAGCTATGT 15 -8.6 CLTB NM_001834 SEQ. ID NO. 0311
GGAACCAGCGCCAGAGTGA 13 40.5 CLTB NM_001834 SEQ. ID NO. 0312
GAGCGAGATTGCAGGCATA 20 61.7 CALM U45976 SEQ. ID NO. 0313
GTTAGTATCTGATGACTTG 36 -34.6 CALM U45976 SEQ. ID NO. 0314
GAAATGGAACCACTAAGAA 33 46.1 CALM U45976 SEQ. ID NO. 0315
GGAAATGGAACCACTAAGA 30 61.2 CALM U45976 SEQ. ID NO. 0316
CAACTACACTTTCCAATGC 28 6.8 EPS15 NM_001981 SEQ. ID NO. 0317
CCACCAAGATTTCATGATA 48 25.2 EPS15 NM_001981 SEQ. ID NO. 0318
GATCGGAACTCCAACAAGA 43 49.3 EPS15 NM_001981 SEQ. ID NO. 0319
AAACGGAGCTACAGATTAT 39 11.5 EPS15 NM_001981 SEQ. ID NO. 0320
CCACACAGCATTCTTGTAA 33 -23.6 EPS15R NM_021235 SEQ. ID NO. 0321
GAAGTTACCTTGAGCAATC 48 33 EPS15R NM_021235 SEQ. ID NO. 0322
GGACTTGGCCGATCCAGAA 27 33 EPS15R NM_021235 SEQ. ID NO. 0323
GCACTTGGATCGAGATGAG 20 1.3 EPS15R NM_021235 SEQ. ID NO. 0324
CAAAGACCAATTCGCGTTA 17 27.7 DNM2 NM_004945 SEQ. ID NO. 0325
CCGAATCAATCGCATCTTC 6 -29.6 DNM2 NM_004945 SEQ. ID NO. 0326
GACATGATCCTGCAGTTCA 5 -14 DNM2 NM_004945 SEQ. ID NO. 0327
GAGCGAATCGTCACCACTT 5 24 DNM2 NM_004945 SEQ. ID NO. 0328
CCTCCGAGCTGGCGTCTAC -4 -63.6 ARF6 AF93885 SEQ. ID NO. 0329
TCACATGGTTAACCTCTAA 27 -21.1 ARF6 AF93885 SEQ. ID NO. 0330
GATGAGGGACGCCATAATC 7 -38.4 ARF6 AF93885 SEQ. ID NO. 0331
CCTCTAACTACAAATCTTA 4 16.9 ARF6 AF93885 SEQ. ID NO. 0332
GGAAGGTGCTATCCAAAAT 4 11.5 RAB5A BC001267 SEQ. ID NO. 0333
GCAAGCAAGTCCTAACATT 40 25.1 RAB5A BC001267 SEQ. ID NO. 0334
GGAAGAGGAGTAGACCTTA 17 50.1 RAB5A BC001267 SEQ. ID NO. 0335
AGGAATCAGTGTTGTAGTA 16 11.5 RAB5A BC001267 SEQ. ID NO. 0336
GAAGAGGAGTAGACCTTAC 12 7 RAB5B NM_002868 SEQ. ID NO. 0337
GAAAGTCAAGCCTGGTATT 14 18.1 RAB5B NM_002868 SEQ. ID NO. 0338
AAAGTCAAGCCTGGTATTA 6 -17.8 RAB5B NM_002868 SEQ. ID NO. 0339
GGTATGAACGTGAATGATC 3 -21.1 RAB5B NM_002868 SEQ. ID NO. 0340
CAAGCCTGGTATTACGTTT -7 -37.5 RAB5C AF141304 SEQ. ID NO. 0341
GGAACAAGATCTGTCAATT 38 51.9 RAB5C AF141304 SEQ. ID NO. 0342
GCAATGAACGTGAACGAAA 29 43.7 RAB5C AF141304 SEQ. ID NO. 0343
CAATGAACGTGAACGAAAT 18 43.3 RAB5C AF141304 SEQ. ID NO. 0344
GGACAGGAGCGGTATCACA 6 18.2 EEA1 XM_018197 SEQ. ID NO. 0345
AGACAGAGCTTGAGAATAA 67 64.1 EEA1 XM_018197 SEQ. ID NO. 0346
GAGAAGATCTTTATGCAAA 60 48.7 EEA1 XM_018197 SEQ. ID NO. 0347
GAAGAGAAATCAGCAGATA 58 45.7 EEA1 XM_018197 SEQ. ID NO. 0348
GCAAGTAACTCAACTAACA 56 72.3 AP2B1 NM_001282 SEQ. ID NO. 0349
GAGCTAATCTGCCACATTG 49 -12.4 AP2B1 NM_001282 SEQ. ID NO. 0350
GCAGATGAGTTACTAGAAA 44 48.9 AP2B1 NM_001282 SEQ. ID NO. 0351
CAACTTAATTGTCCAGAAA 41 28.2 AP2B1 NM_001282 SEQ. ID NO. 0352
CAACACAGGATTCTGATAA 33 -5.8 PLK NM_005030 SEQ. ID NO. 0353
AGATTGTGCCTAAGTCTCT -35 -3.4 PLK NM_005030 SEQ. ID NO. 0354
ATGAAGATCTGGAGGTGAA 0 -4.3 PLK NM_005030 SEQ. ID NO. 0355
TTTGAGACTTCTTGCCTAA -5 -27.7 PLK NM_005030 SEQ. ID NO. 0356
AGATCACCCTCCTTAAATA 15 72.3 GAPDH NM_002046 SEQ. ID NO. 0357
CAACGGATTTGGTCGTATT 27 -2.8 GAPDH NM_002046 SEQ. ID NO. 0358
GAAATCCCATCACCATCTT 24 3.9 GAPDH NM_002046 SEQ. ID NO. 0359
GACCTCAACTACATGGTTT 22 -22.9 GAPDH NM_002046 SEQ. ID NO. 0360
TGGTTTACATGTTCCAATA 9 9.8 c-Myc SEQ. ID NO. 0361
GAAGAAATCGATGTTGTTT 31 -11.7 c-Myc SEQ. ID NO. 0362
ACACAAACTTGAAGAGCTA 22 51.3 c-Myc SEQ. ID NO. 0363
GGAAGAAATCGATGTTGTT 18 26 c-Myc SEQ. ID NO. 0364
GAAACGACGAGAACAGTTG 18 -8.9 MAP2K1 NM_002755 SEQ. ID NO. 0365
GCACATGGATGGAGGTTCT 26 16 MAP2K1 NM_002755 SEQ. ID NO. 0366
GCAGAGAGAGCAGATTTGA 16 0.4 MAP2K1 NM_002755 SEQ. ID NO. 0367
GAGGTTCTCTGGATCAAGT 14 15.5 MAP2K1 NM_002755 SEQ. ID NO. 0368
GAGCAGATTTGAAGCAACT 14 18.5 MAP2K2 NM_030662 SEQ. ID NO. 0369
CAAAGACGATGACTTCGAA 37 26.4 MAP2K2 NM_030662 SEQ. ID NO. 0370
GATCAGCATTTGCATGGAA 24 -0.7 MAP2K2 NM_030662 SEQ. ID NO. 0371
TCCAGGAGTTTGTCAATAA 17 -4.5 MAP2K2 NM_030662 SEQ. ID NO. 0372
GGAAGCTGATCCACCTTGA 16 59.2 KNSL1(EG5) NM_004523 SEQ. ID NO. 0373
GCAGAAATCTAAGGATATA 53 35.8 KNSL1(EG5) NM_004523 SEQ. ID NO. 0374
CAACAAGGATGAAGTCTAT 50 18.3 KNSL1(EGS) NM_004523 SEQ. ID NO. 0375
CAGCAGAAATCTAAGGATA 41 32.7 KNSL1(EG5) NM_004523 SEQ. ID NO. 0376
CTAGATGGCTTTCTCAGTA 39 3.9 CyclophilinA_ NM_021130 SEQ. ID NO. 0377
AGACAAGGTCCCAAAGACA -16 58.1 CyclophilinA_ NM_021130 SEQ. ID NO.
0378 GGAATGGCAAGACCAGCAA -6 36 CyclophilinA_ NM_021130 SEQ. ID NO.
0379 AGAATTATTCCAGGGTTTA -3 16.1 CyclophilinA_ NM_021130 SEQ. ID
NO. 0380 GCAGACAAGGTCCCAAAGA 8 8.9 LAMIN A/C NM_170707 SEQ. ID NO.
0381 AGAAGCAGCTTCAGGATGA 31 38.8 LAMIN A/C NM_170707 SEQ. ID NO.
0382 GAGCTTGACTTCCAGAAGA 33 22.4 LAMIN A/C NM_170707 SEQ. ID NO.
0383 CCACCGAAGTTCACCCTAA 21 27.5 LAMIN A/C NM_170707 SEQ. ID NO.
0384 GAGAAGAGCTCCTCCATCA 55 30.1 CyclophilinB M60857 SEQ. ID NO.
0385 GAAAGAGCATCTACGGTGA 41 83.9 CyclophilinB M60857 SEQ. ID NO.
0386 GAAAGGATTTGGCTACAAA 53 59.1 CyclophilinB M60857 SEQ. ID NO.
0387 ACAGCAAATTCCATCGTGT -20 28.8 CyclophilinB M60857 SEQ. ID NO.
0388 GGAAAGACTGTTCCAAAAA 2 27 DBI1 NM_020548 SEQ. ID NO. 0389
CAACACGCCTCATCCTCTA 27 -7.6 DBI2 NM_020548 SEQ. ID NO. 0390
CATGAAAGCTTACATCAAC 25 -30.8 DBI3 NM_020548 SEQ. ID NO. 0391
AAGATGCCATGAAAGCTTA 17 22 DBI4 NM_020548 SEQ. ID NO. 0392
GCACATACCGCCTGAGTCT 15 3.9 rLUC1 SEQ. ID NO. 0393
GATCAAATCTGAAGAAGGA 57 49.2 rLUC2 SEQ. ID NO. 0394
GCCAAGAAGTTTCCTAATA 50 13.7 rLUC3 SEQ. ID NO. 0395
CAGCATATCTTGAACCATT 41 -2.2 rLUC4 SEQ. ID NO. 0396
GAACAAAGGAAACGGATGA 39 29.2 SeAP1 NM_031313 SEQ. ID NO. 0397
CGGAAACGGTCCAGGCTAT 6 26.9 SeAP2 NM_031313 SEQ. ID NO. 0398
GCTTCGAGCAGACATGATA 4 -11.2 SeAP3 NM_031313 SEQ. ID NO. 0399
CCTACACGGTCCTCCTATA 4 4.9 SeAP4 NM_031313 SEQ. ID NO. 0400
GCCAAGAACCTCATCATCT 1 -9.9 fLUC1 SEQ. ID NO. 0401
GATATGGGCTGAATACAAA 54 40.4 fLUC2 SEQ. ID NO. 0402
GCACTCTGATTGACAAATA 47 54.7 fLUC3 SEQ. ID NO. 0403
TGAAGTCTCTGATTAAGTA 46 34.5 fLUC4 SEQ. ID NO. 0404
TCAGAGAGATCCTCATAAA 40 11.4 mCyclo_1 NM_008907 SEQ. ID NO. 0405
GCAAGAAGATCACCATTTC 52 46.4 mCyclo_2 NM_008907 SEQ. ID NO. 0406
GAGAGAAATTTGAGGATGA 36 70.7 mCyclo_3 NM_008907 SEQ. ID NO. 0407
GAAAGGATTTGGCTATAAG 35 -1.5 mCyclo_4 NM_008907 SEQ. ID NO. 0408
GAAAGAAGGCATGAACATT 27 10.3 BCL2_1 NM_000633 SEQ. ID NO. 0409
GGGAGATAGTGATGAAGTA 21 72 BCL2_2 NM_000633 SEQ. ID NO. 0410
GAAGTACATCCATTATAAG 1 3.3 BCL2_3 NM_000633 SEQ. ID NO. 0411
GTACGACAACCGGGAGATA 1 35.9 BCL2_4 NM_000633 SEQ. ID NO. 0412
AGATAGTGATGAAGTACAT -12 22.1 BCL2_5 NM_000633 SEQ. ID NO. 0413
TGAAGACTCTGCTCAGTTT 36 19.1 BCL2_6 NM_000633 SEQ. ID NO. 0414
GCATGCGGCCTCTGTTTGA 5 -9.7 QB1 NM_003365.1 SEQ. ID NO. 0415
GCACACAGCUUACUACAUC 52 -4.8 QB2 NM_003365.1 SEQ. ID NO. 0416
GAAAUGCCCUGGUAUCUCA 49 22.1 QB3 NM_003365.1 SEQ. ID NO. 0417
GAAGGAACGUGAUGUGAUC 34 22.9 QB4 NM_003365.1 SEQ. ID NO. 0418
GCACUACUCCUGUGUGUGA 28 20.4 ATE1-1 NM_007041 SEQ. ID NO. 0419
GAACCGAGCUGGAGAACUU 45 15.5 ATE1-2 NM_007041 SEQ. ID NO. 0420
GAUAUACAGUGUGAUCUUA 40 12.2 ATE1-3 NM_007041 SEQ. ID NO. 0421
GUACUACGAUCCUGAUUAU 37 32.9 ATE1-4 NM_007041 SEQ. ID NO. 0422
GUGCCGACCUUUACAAUUU 35 18.2 EGFR-1 NM_005228 SEQ. ID NO. 0423
GAAGGAAACTGAATTCAAA 68 79.4 EGFR-1 NM_005228 SEQ. ID NO. 0424
GGAAATATGTACTACGAAA 49 49.5 EGFR-1 NM_005228 SEQ. ID NO. 0425
CCACAAAGCAGTGAATTTA 41 7.6 EGFR-1 NM 005228 SEQ. ID NO. 0426
GTAACAAGCTCACGCAGTT 40 25.9
Example VII
Genome-Wide Application of Formula VIII or Formula X
[0482] The examples described above demonstrate that the
algorithm(s) can successfully identify functional siRNA and that
these duplexes can be used to induce the desirable phenotype of
transcriptional knockdown or knockout. Each gene or family of genes
in each organism plays an important role in maintaining
physiological homeostasis and the algorithm can be used to develop
functional, highly functional, or hyperfunctional siRNA to each
gene. In one example of how this is accomplished, the entire online
ncbi refseq, locuslink, and/or unigene database for the human
genome is first downloaded to local servers. Concommitantly, the
most current version of the BLAST algorithm/program is also
downloaded to enable analysis of all siRNA identified by the
algorithm. Prior to applying the algorithm, sequences are filtered
to eliminate all non-coding sequences (e.g., 3' and 5' UTRs) and
sequences that contain single nucleotide polymorphisms (SNPs). In
addition, in one version of the siRNA selection process, only those
sequences that are associated with all isoforms (e.g., splice
variants) of a given gene are reserved and considered for
targeting. Subsequently, a list of all potential siRNAs (including
a 19 basepair "core" sequence with two basepair 3' overhangs) is
generated for each gene sequence. This group is then filtered to
eliminate sequences that contain any one of a number of undesirable
traits including, but not limited to: 1) sequences that contain
more than two GC basepairs in the last 5 nucleotides of the 3' end
of the sense strand, and 2) sequences that contained internal
repeats that could potentially form hairpin structures. The output
of these procedures are then submitted for scoring by the
algorithm. In this example, the pre-filtered database was processed
with Formula VIII or Formula X and the top 5-100 siRNAs having
scores of 75 (adjusted) or greater were selected. If desired, the
sequences of these siRNA can be BLAST'ed against the Unigene
database containing all sequences in the genome of choice (e.g.,
the human genome) to eliminate any duplexes that show undesirable
degrees of homology to sequences other than the intended target.
The sequences of the (roughly) top 100 sequences for each gene are
provided on the enclosed CDs in electronic form. In this example,
the Formula X sequences were first generated using the procedures
described above and subsequently compared to Formula VIII generated
sequences. Formula VIII sequences that were also identified by
Formula X were then removed (subtracted) from this database (Table
XIII) to eliminate duplications.
[0483] With respect to the material on disk which is part of this
disclosure, there are two tables provided in text format. Table
XII, which is located in a file entitled table-xii.txt, created 26
Apr. 2004, with a file size of 110,486 kb, provides a list of the
5-100 sequences for each target, identified by Formula VIII as
having the highest relative SMARTscores.TM. for the target
analyzed. Table XIII, which is located in a file entitled
table-xiii.txt, created 26 Apr. 2004, with a file size of 23,146
kb, provides a list of the 5-100 sequences for each target
identified by Formula X. In addition, each table provides
information concerning: the gene name, an NCBI accession number, an
adjusted SMARTscore, and a sequence ID number. Any of the provided
sequences can be used for gene silencing either alone or in
combination with other sequences. The information contained on the
disks is part of this patent application and is incorporated into
the specification by reference. One may use these tables in order
to identify functional siRNAs for the gene provided therein, by
simply looking for the gene of interest and an siRNA that is listed
as functional. Preferably, one would select one or more of the
siRNAs that is most optimized for the target of interest and is
denoted as a pool pick.
Table XII: siRNA Selected by Formula VIII
[0484] See data submitted herewith on a CD-ROM in accordance with
PCT Administrative Instructions Part 8. Table XII is included on
the compact disk labeled COPY 1--TABLES PART DISK 1/1, TABLES XII
and XIII (provided in triplicate, which copies are identical), in a
file entitled table-xii.txt, date of creation 26 Apr. 2004, with a
size of 110,486 kb.
Table XIII: siRNA Selected by Formula X
[0485] See data submitted herewith on a CD-ROM in accordance with
PCT Administrative Instructions Part 8. Table XIII is included on
the compact disk labeled COPY 1--TABLES PART DISK 1/1, TABLES XII
and XIII (provided in triplicate, which copies are identical), in
file entitled table-xiii.txt, date of creation 26 Apr. 2004, with a
size of 23,146 kb.
[0486] Many of the genes to which the described siRNA are directed
play critical roles in disease etiology. For this reason, the
siRNAs listed in the accompanying compact disk may potentially act
as therapeutic agents. A number of prophetic examples follow and
should be understood in view of the siRNA that are identified on
the accompanying CD. To isolate these siRNAs, the appropriate
message sequence for each gene is analyzed using one of the before
mentioned formulas (preferably formula VIII) to identify potential
siRNA targets. Subsequently these targets are BLAST'ed to eliminate
homology with potential off-targets.
[0487] The list of potential disease targets is extensive. For
instance, over-expression of Bcl10 has been implicated in the
development of MALT lymphoma (mucosa associated lymphoid tissue
lymphoma) and thus, functional, highly functional, or
hyperfunctional siRNA directed against that gene (e.g., SEQ. ID NO.
0427: GGAAACCUCUCAUUGCUAA; SEQ. ID NO. 0428: GAAAGAACCUUGCCGAUCA;
SEQ. ID NO. 0429: GGAAAUACAUCAGAGCUUA, or SEQ. ID NO. 0430:
GAAAGUAUGUGUCUUAAGU) may contribute to treatment of this
disorder.
[0488] In another example, studies have shown that molecules that
inhibit glutamine:fructose-6-phosphate aminotransferase (GFA) may
act to limit the symptoms suffered by Type II diabetics. Thus,
functional, highly functional, or hyperfunctional siRNA directed
against GFA (also known as GFPT1: siRNA=SEQ. ID NO. 0433
UGAAACGGCUGCCUGAUUU; SEQ. ID NO. 0434 GAAGUUACCUCUUACAUUU; SEQ. ID
NO. 0435 GUACGAAACUGUAUGAUUA; SEQ. ID NO. 0436 GGACGAGGCUAUCAUUAUG)
may contribute to treatment of this disorder.
[0489] In another example, the von Hippel-Lindau (VHL) tumor
suppressor has been observed to be inactivated at a high frequency
in sporadic clear cell renal cell carcinoma (RCC) and RCCs
associated with VHL disease. The VHL tumor suppressor targets
hypoxia-inducible factor-1 alpha (HIF-1 alpha), a transcription
factor that can induce vascular endothelial growth factor (VEGF)
expression, for ubiquitination and degradation. Inactivation of VHL
can lead to increased levels of HIF-1 alpha, and subsequent VEGF
over expression. Such over expression of VEGF has been used to
explain the increased (and possibly necessary) vascularity observed
in RCC. Thus, functional, highly functional, or hyperfunctional
siRNAs directed against either HIF-1 alpha (SEQ. ID NO. 0437
GAAGGAACCUGAUGCUUUA; SEQ. ID NO. 0438 GCAUAUAUCUAGAAGGUAU; SEQ. ID
NO. 0439 GAACAAAUACAUGGGAUUA; SEQ. ID NO. 0440 GGACACAGAUUUAGACUUG)
or VEGF (SEQ. ID NO. 0441 GAACGUACUUGCAGAUGUG; SEQ. ID NO. 0442
GAGAAAGCAUUUGUUUGUA; SEQ. ID NO. 0443 GGAGAAAGCAUUUGUUUGU; SEQ. ID
NO. 0444 CGAGGCAGCUUGAGUUAAA) may be useful in the treatment of
renal cell carcinoma.
[0490] In another example, gene expression of platelet derived
growth factor A and B (PDGF-A and PDGF-B) has been observed to be
increased 22- and 6-fold, respectively, in renal tissues taken from
patients with diabetic nephropathy as compared with controls. These
findings suggest that over expression of PDGF A and B may play a
role in the development of the progressive fibrosis that
characterizes human diabetic kidney disease. Thus, functional,
highly functional, or hyperfunctional siRNAs directed against
either PDGF A
[0491] (SEQ. ID NO. 0445: GGUAAGAUAUUGUGCUUUA;
[0492] SEQ. ID NO. 0446: CCGCAAAUAUGCAGAAUUA;
[0493] SEQ. ID NO. 0447: GGAUGUACAUGGCGUGUUA;
[0494] SEQ. ID NO. 0448: GGUGAAGUUUGUAUGUUUA) or
[0495] PDGF B
[0496] (SEQ. ID NO. 0449: CCGAGGAGCUUUAUGAGAU;
[0497] SEQ. ID NO. 0450: GCUCCGCGCUUUCCGAUUU;
[0498] SEQ. ID NO. 0451 GAGCAGGAAUGGUGAGAUG;
[0499] SEQ. ID NO. 0452: GAACUUGGGAUAAGAGUGU;
[0500] SEQ. ID NO. 0453 CCGAGGAGCUUUAUGAGAU;
[0501] SEQ. ID NO. 0454 UUUAUGAGAUGCUGAGUGA) may be useful in the
treatment of this form of kidney disorder.
[0502] In another example, a strong correlation exists between the
over-expression of glucose transporters (e.g., GLUT12) and cancer
cells. It is predicted that cells undergoing uncontrolled cell
growth up-regulate GLUT molecules so that they can cope with the
heightened energy needs associated with increased rates of
proliferation and metastasis. Thus, siRNA-based therapies that
target the molecules such as GLUT1 (also known as SLC2A1:
siRNA=
[0503] SEQ. ID NO.: 0455 GCAAUGAUGUCCAGAAGAA;
[0504] SEQ. ID NO.: 0456 GAAGAAUAUUCAGGACUUA;
[0505] SEQ. ID NO.: 0457 GAAGAGAGUCGGCAGAUGA;
[0506] SEQ. ID NO.: 0458 CCAAGAGUGUGCUAAAGAA)
[0507] GLUT12 (also known as SLCA12: siRNA=
[0508] SEQ. ID NO. 0459: GAGACACUCUGAAAUGAUA;
[0509] SEQ. ID NO. 0460: GAAAUGAUGUGGAUAAGAG;
[0510] SEQ. ID NO. 0461: GAUCAAAUCCUCCCUGAAA;
[0511] SEQ. ID NO. 0462: UGAAUGAGCUGAUGAUUGU) and other related
transporters, may be of value in treating a multitude of
malignancies.
[0512] The siRNA sequences listed above are presented in a
5'.fwdarw.3' sense strand direction. In addition, siRNA directed
against the targets listed above as well as those directed against
other targets and listed in the accompanying compact disk may be
useful as therapeutic agents.
Example VIII
Evidence for the Benefits of Pooling
[0513] Evidence for the benefits of pooling have been demonstrated
using the reporter gene, luciferase. Ninety siRNA duplexes were
synthesized using Dharmacon proprietary ACE.RTM. chemistry against
one of the standard reporter genes: firefly luciferase. The
duplexes were designed to start two base pairs apart and to cover
approximately 180 base pairs of the luciferase gene (see sequences
in Table III). Subsequently, the siRNA duplexes were co-transfected
with a luciferase expression reporter plasmid into HEK293 cells
using standard transfection protocols and luciferase activity was
assayed at 24 and 48 hours.
[0514] Transfection of individual siRNAs showed standard
distribution of inhibitory effect. Some duplexes were active, while
others were not. FIG. 15 represents a typical screen of ninety
siRNA duplexes (SEQ. ID NO. 0032-0120) positioned two base pairs
apart. As the figure suggests, the functionality of the siRNA
duplex is determined more by a particular sequence of the
oligonucleotide than by the relative oligonucleotide position
within a gene or excessively sensitive part of the mRNA, which is
important for traditional anti-sense technology.
[0515] When two continuous oligonucleotides were pooled together, a
significant increase in gene silencing activity was observed. (See
FIG. 16) A gradual increase in efficacy and the frequency of pools
functionality was observed when the number of siRNAs increased to 3
and 4. (FIGS. 16, 17). Further, the relative positioning of the
oligonucleotides within a pool did not determine whether a
particular pool was functional (see FIG. 18, in which 100% of pools
of oligonucleotides distanced by 2, 10 and 20 base pairs were
functional).
[0516] However, relative positioning may nonetheless have an
impact. An increased functionality may exist when the siRNA are
positioned continuously head to toe (5' end of one directly
adjacent to the 3' end of the others).
[0517] Additionally, siRNA pools that were tested performed at
least as well as the best oligonucleotide in the pool, under the
experimental conditions whose results are depicted in FIG. 19.
Moreover, when previously identified non-functional and marginally
(semi) functional siRNA duplexes were pooled together in groups of
five at a time, a significant functional cooperative action was
observed. (See FIG. 20) In fact, pools of semi-active
oligonucleotides were 5 to 25 times more functional than the most
potent oligonucleotide in the pool. Therefore, pooling several
siRNA duplexes together does not interfere with the functionality
of the most potent siRNAs within a pool, and pooling provides an
unexpected significant increase in overall functionality
Example IX
Additional Evidence of the Benefits of Pooling
[0518] Experiments were performed on the following genes:
.beta.-galactosidase, Renilla luciferase, and Secreted alkaline
phosphatase, which demonstrates the benefits of pooling. (see FIG.
21). Individual and pools of siRNA (described in Figure legend 21)
were transfected into cells and tested for silencing efficiency.
Approximately 50% of individual siRNAs designed to silence the
above-specified genes were functional, while 100% of the pools that
contain the same siRNA duplexes were functional.
Example X
Highly Functional siRNA
[0519] Pools of five siRNAs in which each two siRNAs overlap to
10-90% resulted in 98% functional entities (>80% silencing).
Pools of siRNAs distributed throughout the mRNA that were evenly
spaced, covering an approximate 20-2000 base pair range, were also
functional. When the pools of siRNA were positioned continuously
head to tail relative to mRNA sequences and mimicked the natural
products of Dicer cleaved long double stranded RNA, 98% of the
pools evidenced highly functional activity (>95% silencing).
Example XI
Human Cyclophilin B
[0520] Table III above lists the siRNA sequences for the human
cyclophilin B protein. A particularly functional siRNA may be
selected by applying these sequences to any of Formula I to VII
above.
[0521] Alternatively, one could pool 2, 3, 4, 5 or more of these
sequences to create a kit for silencing a gene. Preferably, within
the kit there would be at least one sequence that has a relatively
high predicted functionality when any of Formulas I-VII is
applied.
Example XII
Sample Pools of siRNAs and Their Application to Human Disease
[0522] The genetic basis behind human disease is well documented
and siRNA may be used as both research or diagnostic tools and
therapeutic agents, either individually or in pools. Genes involved
in signal transduction, the immune response, apoptosis, DNA repair,
cell cycle control, and a variety of other physiological functions
have clinical relevance and therapeutic agents that can modulate
expression of these genes may alleviate some or all of the
associated symptoms. In some instances, these genes can be
described as a member of a family or class of genes and siRNA
(randomly, conventionally, or rationally designed) can be directed
against one or multiple members of the family to induce a desired
result.
[0523] To identify rationally designed siRNA to each gene, the
sequence was analyzed using Formula VIII or Formula X to identify
rationally designed siRNA. To confirm the activity of these
sequences, the siRNA are introduced into a cell type of choice
(e.g., HeLa cells, HEK293 cells) and the levels of the appropriate
message are analyzed using one of several art proven techniques.
siRNA having heightened levels of potency can be identified by
testing each of the before mentioned duplexes at increasingly
limiting concentrations. Similarly, siRNA having increased levels
of longevity can be identified by introducing each duplex into
cells and testing functionality at 24, 48, 72, 96, 120, 144, 168,
and 192 hours after transfection. Agents that induce >95%
silencing at sub-nanomolar concentrations and/or induce functional
levels of silencing for >96 hours are considered
hyperfunctional.
Example XIII
[0524] The information presented in Tables XII and XIII provides
the siRNA sequence (sense strand), the gene name, the NCBI
accession number, the adjusted algorithm score, and the sequence ID
number. All sequences have an adjusted score of 75 or above. For
Table XIII, Formula X derived sequences were compared with Formula
VIII sequences. Sequences that were in common with both were
eliminated from Table XIII. Pool picks are typically identified as
gene specific siRNA that have the hightest adjusted scores.
[0525] The following are non-limiting examples of families of
proteins to which siRNA described in this document are targeted
against:
[0526] Transporters, Pumps, and Channels
[0527] Transporters, pumps, and channels represent one class of
genes that are attractive targets for siRNAs. One major class of
transporter molecules are the ATP-binding cassette (ABC)
transporters. To date, nearly 50 human ABC-transporter genes have
been characterized and have been shown to be involved in a variety
of physiological functions including transport of bile salts,
nucleosides, chloride ions, cholesterol, toxins, and more.
Predominant among this group are MDR1 (which encodes the
P-glycoprotein, NP.sub.--000918), the MDR-related proteins
(MRP1-7), and the breast cancer resistance protein (BCRP). In
general, these transporters share a common structure, with each
protein containing a pair of ATP-binding domains (also known as
nucleotide binding folds, NBF) and two sets of transmembrane (TM)
domains, each of which typically contains six membrane-spanning
.alpha.-helices. The genes encoding this class of transporter are
organized as either full transporters (i.e., containing two TM and
two NBF domains) or as half transporters that assemble as either
homodimers or heterodimers to create functional transporters. As a
whole, members of the family are widely dispersed throughout the
genome and show a high degree of amino acid sequence identify among
eukaryotes.
[0528] ABC-transporters have been implicated in several human
diseases. For instance, molecular efflux pumps of this type play a
major role in the development of drug resistance exhibited by a
variety of cancers and pathogenic microorganisms. In the case of
human cancers, increased expression of the MDR1 gene and related
pumps have been observed to generate drug resistance to a broad
collection of commonly used chemotherapeutics including
doxorubicin, daunorubicin, vinblastine, vincristine, colchicines.
In addition to the contribution these transporters make to the
development of multi-drug resistance, there are currently 13 human
genetic diseases associated with defects in 14 different
transporters. The most common of these conditions include cystic
fibrosis, Stargardt disease, age-related macular degeneration,
adrenoleukodystrophy, Tangier disease, Dubin-Johnson syndrome and
progressive familial intrahepatic cholestasis. For this reason,
siRNAs directed against members of this, and related, families are
potentially valuable research and therapeutic tools.
[0529] With respect to channels, analysis of Drosophila mutants has
enabled the initial molecular isolation and characterization of
several distinct channels including (but not limited to) potassium
(K+) channels. This list includes shaker (Sh), which encodes a
voltage activated K.sup.+ channel, slowpoke (Slo), a Ca.sup.2+
activated K.sup.+ channel, and ether-a-go-go (Eag). The Eag family
is further divided into three subfamilies: Eag, Elk (eag-like K
channels), and Erg (Eag related genes).
[0530] The Erg subfamily contains three separate family members
(Erg1-3) that are distantly related to the sh family of voltage
activated K.sup.+ channels. Like sh, erg polypetides contain the
classic six membrane spanning architecture of K.sup.+ channels
(S1-S6) but differ in that each includes a segment associated with
the C-terminal cytoplasmic region that is homologous to cyclic
nucleotide binding domains (cNBD). Like many isolated ion channel
mutants, erg mutants are temperature-sensitive paralytics, a
phenotype caused by spontaneous repetitive firing (hyperactivity)
in neurons and enhanced transmitter release at the neuromuscular
junction.
[0531] Initial studies on the tissue distribution of all three
members of the erg subfamily show two general patterns of
expression. Erg1 and erg3 are broadly expressed throughout the
nervous system and are observed in the heart, the superior
mesenteric ganglia, the celiac ganglia, the retina, and the brain.
In contrast, erg2 shows a much more restricted pattern of
expression and is only observed in celiac ganglia and superior
mesenteric ganglia. Similarly, the kinetic properties of the three
erg potassium channels are not homogeneous. Erg1 and erg2 channels
are relatively slow activating delayed rectifiers whereas the erg3
current activates rapidly and then exhibits a predominantly
transient component that decays to a sustained plateau. The current
properties of all three channels are sensitive to
methanesulfonanilides, suggesting a high degree of conservation in
the pore structure of all three proteins.
[0532] Recently, the erg family of K.sup.+ channels has been
implicated in human disease. Consistent with the observation that
erg1 is expressed in the heart, single strand conformation
polymorphism and DNA sequence analyses have identified HERG (human
erg1) mutations in six long-QT-syndrome (LQT) families, an
inherited disorder that results in sudden death from a ventricular
tachyarrythmia. Thus siRNA directed against this group of molecules
(e.g., KCNH1-8) will be of extreme therapeutic value.
[0533] Another group of channels that are potential targets of
siRNAs are the CLCA family that mediate a Ca.sup.2+-activated
Cl.sup.- conductance in a variety of tissues. To date, two bovine
(bCLC1; bCLCA2 (Lu-ECAM-1)), three mouse (mCLCA1; mCLCA2; mCLCA3)
and four human (hCLCA1; hCLCA2; hCLCA3; hCLCA4) CLCA family members
have been isolated and patch-clamp studies with transfected human
embryonic kidney (HEK-293) cells have shown that bCLCA1, mCLCA1,
and hCLCA1 mediate a Ca.sup.2+-activated Cl.sup.- conductance that
can be inhibited by the anion channel blocker DIDS and the reducing
agent dithiothreitol (DTT).
[0534] The protein size, structure, and processing seem to be
similar among different CLCA family members and has been studied in
greatest detail for Lu-ECAM-1. The Lu-ECAM-1 open reading frame
encodes a precursor glycoprotein of 130 kDa that is processed to a
90-kDa amino-terminal cleavage product and a group of 30- to 40-kDa
glycoproteins that are glycosylation variants of a single
polypeptide derived from its carboxy terminus. Both subunits are
associated with the outer cell surface, but only the 90-kDa subunit
is thought to be anchored to the cell membrane via four
transmembrane domains.
[0535] Although the protein processing and function appear to be
conserved among CLCA homologs, significant differences exist in
their tissue expression patterns. For example, bovine Lu-ECAM-1 is
expressed primarily in vascular endothelia, bCLCA1 is exclusively
detected in the trachea, and hCLCA1 is selectively expressed in a
subset of human intestinal epithelial cells. Thus the emerging
picture is that of a multigene family with members that are highly
tissue specific, similar to the ClC family of voltage-gated
Cl.sup.- channels. The human channel, hCLCA2, is particular
interesting from a medical and pharmacological standpoint. CLCA2 is
expressed on the luminal surface of lung vascular endothelia and
serves as an adhesion molecule for lung metastatic cancer cells,
thus mediating vascular arrest and lung colonization. Expression of
this molecule in normal mammary epithelium is consistently lost in
human breast cancer and in nearly all tumorigenic breast cancer
cell lines. Moreover, re-expression of hCLCA2 in human breast
cancer cells abrogates tumorigenicity in nude mice, implying that
hCLCA2 acts as a tumour suppressor in breast cancer. For these
reasons, siRNA directed against CLCA family members and related
channels may prove to be valuable in research and therapeutic
venues.
[0536] Transporters Involved in Synaptic Transmission
[0537] Synaptic transmission involves the release of a
neurotransmitter into the synaptic cleft, interaction of that
transmitter with a postsynaptic receptor, and subsequent removal of
the transmitter from the cleft. In most synapses the signal is
terminated by a rapid reaccumulation of the neurotransmitter into
presynaptic terminals. This process is catalyzed by specific
neurotransmitter transporters that are often energized by the
electrochemical gradient of sodium across the plasma membrane of
the presynaptic cells.
[0538] Aminobutyric acid (GABA) is the major inhibitory
neurotransmitter in the central nervous system. The inhibitory
action of GABA, mediated through GABA.sub.A/GABA.sub.B receptors,
and is regulated by GABA transporters (GATs), integral membrane
proteins located perisynaptically on neurons and glia. So far four
different carriers (GAT1-GAT4) have been cloned and their cellular
distribution has been partly worked out. Comparative sequence
analysis has revealed that GABA transporters are related to several
other proteins involved in neurotransmitter uptake including
gamma-aminobutyric acid transporters, monoamine transporters, amino
acid transporters, certain "orphan" transporters, and the recently
discovered bacterial transporters. Each of these proteins has a
similar 12 transmembrane helices topology and relies upon the
Na+/Cl- gradient for transport function. Transport rates are
dependent on substrate concentrations, with half-maximal effective
concentrations for transport frequently occurring in the
submicromolar to low micromolar range. In addition, transporter
function is bidirectional, and non-vesicular efflux of transmitter
may contribute to ambient extracellular transmitter levels.
[0539] Recent evidence suggests that GABA transporters, and
neurotransmitter transporters in general, are not passive players
in regulating neuronal signaling; rather, transporter function can
be altered by a variety of initiating factors and signal
transduction cascades. In general, this functional regulation
occurs in two ways, either by changing the rate of transmitter flux
through the transporter or by changing the number of functional
transporters on the plasma membrane. A recurring theme in
transporter regulation is the rapid redistribution of the
transporter protein between intracellular locations and the cell
surface. In general, this functional modulation occurs in part
through activation of second messengers such as kinases,
phosphatases, arachidonic acid, and pH. However, the mechanisms
underlying transporter phosphorylation and transporter
redistribution have yet to be fully elucidated.
[0540] GABA transporters play a pathophysiological role in a number
of human diseases including temporal lobe epilepsy and are the
targets of pharmacological interventions. Studies in seizure
sensitive animals show some (but not all) of the GAT transporters
have altered levels of expression at times prior to and post
seizure, suggesting this class of transporter may affect
epileptogenesis, and that alterations following seizure may be
compensatory responses to modulate seizure activity. For these
reasons, siRNAs directed against members of this family of genes
(including but not limited to SLCG6A1-12) may prove to be valuable
research and therapeutic tools.
[0541] Organic Ion Transporters
[0542] The human body is continuously exposed to a great variety of
xenobiotics, via food, drugs, occupation, and environment.
Excretory organs such as kidney, liver, and intestine defend the
body against the potentially harmful effects of these compounds by
transforming them into less active metabolites that are
subsequently secreted from the system.
[0543] Carrier-mediated transport of xenobiotics and their
metabolites exist for the active secretion of organic anions and
cations. Both systems are characterized by a high clearance
capacity and tremendous diversity of substances accepted,
properties that result from the existance of multiple transporters
with overlapping substrate specificities. The class of organic
anion transporters plays a critical role in the elimination of a
large number of drugs (e.g., antibiotics, chemotherapeutics,
diuretics, nonsteroidal anti-inflammatory drugs, radiocontrast
agents, cytostatics); drug metabolites (especially conjugation
products with glutathione, glucuronide, glycine, sulfate, acetate);
and toxicants and their metabolites (e.g., mycotoxins, herbicides,
plasticizers, glutathione S-conjugates of polyhaloalkanes,
polyhaloalkenes, hydroquinones, aminophenols), many of which are
specifically harmful to the kidney.
[0544] Over the past couple of years the number of identified anion
transporting molecules has grown tremendously. Uptake of organic
anions (OA.sup.-) across the basolateral membrane is mediated by
the classic sodium-dependent organic anion transport system, which
includes .alpha.-ketoglutarate (.alpha.-KG.sup.2-)/OA.sup.-
exchange via the organic anion transporter (OAT1) and
sodium-ketoglutarate cotransport via the Na.sup.+/dicarboxylate
cotransporter (SDCT2). The organic anion transporting polypetide,
Oatp1, and the kidney-specific OAT-K1 and OAT-K2 are seen as
potential molecules that mediate facilitated OA.sup.- efflux but
could also be involved in reabsorption via an exchange mechanism.
Lastly the PEPT1 and PEPT2 mediate luminal uptake of peptide drugs,
whereas CNT1 and CNT2 are involved in reabsorption of
nucleosides.
[0545] The organic anion-transporting polypeptide 1 (Oatp1) is a
Na.sup.+- and ATP-independent transporter originally cloned from
rat liver. The tissue distribution and transport properties of the
Oatp1 gene product are complex. Oatp1 is localized to the
basolateral membrane of hepatocytes, and is found on the apical
membrane of S3 proximal tubules. Studies with transiently
transfected cells (e.g., HeLa cells) have indicated that Oatp1
mediates transport of a variety of molecules including
taurocholate, estrone-3-sulfate, aldosterone, cortisol, and others.
The observed uptake of taurocholate by Oatp1 expressed in X. laevis
oocytes is accompanied by efflux of GSH, suggesting that transport
by this molecule may be glutathione dependent.
[0546] Computer modeling suggests that members of the Oatp family
are highly conserved, hydrophobic, and have 12 transmembrane
domains. Decreases in expression of Oatp family members have been
associated with cholestatic liver diseases and human
hepatoblastomas, making this family of proteins of key interest to
researchers and the medical community. For these reasons, siRNAs
directed against OAT family members (including but not limited to
SLC21A2, 3, 6, 8, 9, 11, 12, 14, 15, and related transporters) are
potentially useful as research and therapeutic tools.
[0547] Nucleoside Transporters
[0548] Nucleoside transporters play key roles in physiology and
pharmacology. Uptake of exogenous nucleosides is a critical first
step of nucleotide synthesis in tissues such as bone marrow and
intestinal epithelium and certain parasitic organisms that lack de
novo pathways for purine biosynthesis. Nucleoside transporters also
control the extracellular concentration of adenosine in the
vicinity of its cell surface receptors and regulate processes such
as neurotransmission and cardiovascular activity. Adenosine itself
is used clinically to treat cardiac arrhythmias, and nucleoside
transport inhibitors such as dipyridamole, dilazep, and draflazine
function as coronary vasodilators.
[0549] In mammals, plasma membrane transport of nucleosides is
brought about by members of the concentrative, Na.sup.+-dependent
(CNT) and equilibrative, Na.sup.+-independent (ENT) nucleoside
transporter families. CNTs are expressed in a tissue-specific
fashion; ENTs are present in most, possibly all, cell types and are
responsible for the movement of hydrophilic nucleosides and
nucleoside analogs down their concentration gradients. In addition,
structure/function studies of ENT family members have predicted
these molecules to contain eleven transmembrane helical segments
with an amino terminus that is intracellular and a carboxyl
terminus that is extracellular. The proteins have a large
glycosylated loop between TMs 1 and 2 and a large cytoplasmic loop
between TMs 6 and 7. Recent investigations have implicated the TM
3-6 region as playing a central role in solute recognition. The
medical importance of the ENT family of proteins is broad. In
humans adenosine exerts a range of cardioprotective effects and
inhibitors of ENTs are seen as being valuable in alleviating a
variety of cardio/cardiovascular ailments. In addition, responses
to nucleoside analog drugs has been observed to vary considerably
amongst, e.g., cancer patients. While some forms of drug resistance
have been shown to be tied to the up-regulation of ABC-transporters
(e.g., MDR1), resistance may also be the result of reduced drug
uptake (i.e., reduced ENT expression). Thus, a clearer
understanding of ENT transporters may aid in optimizing drug
treatments for patients suffering a wide range of malignancies. For
these reasons, siRNAs directed against this class of molecules
(including SLC28A1-3, SLC29A1-4, and related molecules) may be
useful as therapeutic and research tools.
[0550] Sulfate Transporters
[0551] All cells require inorganic sulfate for normal function.
Sulfate is the fourth most abundant anion in human plasma and is
the major source of sulfur in many organisms. Sulfation of
extracellular matrix proteins is critical for maintaining normal
cartilage metabolism and sulfate is an important constituent of
myelin membranes found in the brain
[0552] Because sulfate is a hydrophilic anion that cannot passively
cross the lipid bilayer of cell membranes, all cells require a
mechanism for sulfate influx and efflux to ensure an optimal
supply. To date, a variety of sulfate transporters have been
identified in tissues from many origins. These include the renal
sulfate transporters (NaSi-1 and Sat-1), the ubiquitously expressed
diastrophic dysplasia sulfate transporter (DTDST), the intestinal
sulfate transporter (DRA), and the erythrocyte anion exchanger
(AE1). Most, if not all, of these molecules contain the classic 12
transmembrane spanning domain architecture commonly found amongst
members of the anion transporter superfamily.
[0553] Recently three different sulfate transporters have been
associated with specific human genetic diseases. Family members
SLC26A2, SLC26A3, and SLC26A4 have been recognized as the disease
genes mutated in diastrophic dysplasia, congenital chloride
diarrhea (CLD), and Pendred syndrome (PDS), respectively. DTDST is
a particularly complex disorder. The gene encoding this molecule
maps to chromosome 5q, and encodes two distinct transcripts due to
alternative exon usage. In contrast to other sulfate transporters
(e.g., Sat-1) anion movement by the DTDST protein is markedly
inhibited by either extracellular chloride or bicarbonate. Impaired
function of the DTDST gene product leads to undersulfation of
proteoglycans and a complex family of recessively inherited
osteochondrodysplasias (achondrogenesis type 1B, atelosteogenesis
type II, and diastrophic dysplasia) with clinical features
including but not limited to, dwarfism, spinal deformation, and
specific joint abnormalities. Interestingly, while epidemiological
studies have shown that the disease occurs in most populations, it
is particularly prevalent in Finland owing to an apparent founder
effect. For these reasons, siRNAs directed against this class of
genes (including but not limited to SLC26A1-9, and related
molecules) may be potentially helpful in both therapeutic and
research venues.
[0554] Ion Exchangers
[0555] Intracellular pH regulatory mechanisms are critical for the
maintenance of countless cellular processes. For instance, in
muscle cells, contractile processes and metabolic reactions are
influenced by pH. During periods of increased energy demands and
ischemia, muscle cells produce large amounts of lactic acid that,
without quick and efficient disposal, would lead to acidification
of the sarcoplasm.
[0556] Several different transport mechanisms have evolved to
maintain a relatively constant intracellular pH. The relative
contribution of each of these processes varies with cell type, the
metabolic requirements of the cell, and the local environmental
conditions. Intracellular pH regulatory processes that have been
characterized functionally include but are not limited to the
Na.sup.+/H.sup.+ exchange, the Na(HCO.sub.3).sub.n cotransport, and
the Na.sup.+-dependent and -independent Cl.sup.-/base exchangers.
As bicarbonate and CO.sub.2 comprise the major pH buffer of
biological fluids, sodium biocarbonate cotransporters (NBCs) are
critical. Studies have shown that these molecules exist in numerous
tissues including the kidney, brain, liver, cornea, heart, and
lung, suggesting that NBCs play an important role in mediating
HCO.sub.3.sup.- transport in both epithelial as well as
nonepithelial cells.
[0557] Recent molecular cloning experiments have identified the
existence of four NBC isoforms (NBC1, 2, 3 and 4) and two
NBC-related proteins, AE4 and NCBE (Anion Exchanger 4 and
Na-dependent Chloride-Bicarbonate Exchanger). The secondary
structure analyses and hydropathy profile of this family predict
them to be intrinsic membrane proteins with 12 putative
transmembrane domains and several family members exhibit N-linked
glycosylation sites, protein kinases A and C, casein kinase II, and
ATP/GTP-binding consensus phosphorylation sites, as well as
potential sites for myristylation and amidation. AE4 is a
relatively recent addition to this family of proteins and shows
between 30-48% homology with the other family members. When
expressed in COS-7 cells and Xenopus oocytes AE4 exhibits
sodium-independent and DIDS-insensitive anion exchanger activity.
Exchangers have been shown to be responsible for a variety of human
diseases. For instance, mutations in three genes of the anion
transporter family (SLC) are believed to cause known hereditary
diseases, including chondrodysplasia (SLC26A2, DTD), diarrhea (A3,
down-regulated in adenoma/chloride-losing diarrhea protein:
DRA/CLD), and goiter/deafness syndrome (A4, pendrin). Moreover,
mutations in Na+/HCO3 co-transporters have also been associated
with various human maladies. For these reasons, siRNAs directed
against these sorts of genes (e.g., SLC4A4-10, and related genes)
may be useful for therapeutic and research purposes.
[0558] Receptors Involved in Synaptic Transmission
[0559] In all vertebrates, fast inhibitory synaptic transmission is
the result of the interaction between the neurotransmitters glycine
(Gly) and .gamma.-aminobutyric acid (GABA) and their respective
receptors. The strychnine-sensitive glycine receptor is especially
important in that it acts in the mammalian spinal cord and brain
stem and has a well-established role in the regulation of locomotor
behavior.
[0560] Glycine receptors display significant sequence homology to
several other receptors including the nicotinic acetylcholine
receptor, the aminobutyric acid receptor type A (GABA.sub.AR), and
the serotonin receptor type 3 (5-HT.sub.3R) subunits. As members of
the superfamily of ligand-gated ion channels, these polypeptides
share common topological features. The glycine receptor is composed
of two types of glycosylated integral membrane proteins
(.alpha.1-.alpha.4 and .beta.) arranged in a pentameric
suprastructure. The alpha subunit encodes a large extracellular,
N-terminal domain that carries the structural determinants
essential for agonist and antagonist binding, followed by four
transmembrane spanning regions (TM1-TM4), with TM2 playing the
critical role of forming the inner wall of the chloride
channel.
[0561] The density, location, and subunit composition of glycine
neurotransmitter receptors changes over the course of development.
It has been observed that the amount of GlyR gene translation
(assessed by the injection of developing rat cerebral cortex mRNA
into Xenopus oocytes) decreases with age, whereas that of GABARs
increases. In addition, the type and location of mRNAs coding for
GlyR changes over the course of development. For instance in a
study of the expression of alpha 1 and alpha 2 subunits in the rat,
it was observed that (in embryonic periods E11-18) the mantle zone
was scarce in the alpha 1 mRNA, but the germinal zone (matrix
layer) at E11-14 expressed higher levels of the message. At
postnatal day 0 (P0), the alpha 1 signals became manifested
throughout the gray matter of the spinal cord. By contrast, the
spinal tissues at P0 exhibited the highest levels of alpha 2 mRNA,
which decreased with the postnatal development.
[0562] In both, man and mouse mutant lines, mutations of GlyR
subunit genes result in hereditary motor disorders characterized by
exaggerated startle responses and increased muscle tone.
Pathological alleles of the Glra1 gene are associated with the
murine phenotypes oscillator (spd.sup.ot) and spasmodic (spd).
Similarly, a mutant allele of Glrb has been found to underly the
molecular pathology of the spastic mouse (spa). Resembling the
situation in the mouse, a variety of GLRA1 mutant alleles have been
shown to be associated with the human neurological disorder
hyperekplexia or startle disease. For these reasons, siRNA directed
against glycine receptors (GLRA1-3, GLRB, and related molecules),
glutamate receptors, GABA receptors, ATP receptors, and related
neurotransmitter receptor molecules may be valuable therapeutic and
research reagents.
[0563] Proteases
[0564] Kallikreins
[0565] One important class of proteases are the kallikreins, serine
endopeptidases that split peptide substrates preferentially on the
C-terminal side of internal arginyl and lysyl residues. Kallikreins
are generally divided into two distinct groups, plasma kallikreins
and tissue kallikreins. Tissue kallikreins represent a large group
of enzymes that have substantial similarities at both the gene and
protein level. The genes encoding this group are frequently found
on a single chromosome, are organized in clusters, and are
expressed in a broad range of tissues (e.g., pancreas, ovaries,
breast). In contrast, the plasma form of the enzyme is encoded by a
single gene (e.g., KLK3) that has been localized to chromosome
4q34-35 in humans. The gene encoding plasma kallikrein is expressed
solely in the liver, contains 15 exons, and encodes a glycoprotein
that is translated as a preprotein called prekallikrein.
[0566] Kallikreins are believed to play an important role in a host
of physiological events. For instance, the immediate consequence of
plasma prekallikrein activation is the cleavage of high molecular
weight kininogen (HK) and the subsequent liberation of bradykinin,
a nine amino acid vasoactive peptide that is an important mediator
of inflammatory responses. Similarly, plasma kallikrein promotes
single-chain urokinase activation and subsequent plasminogen
activation, events that are critical to blood coaggulation and
wound healing.
[0567] Disruptions in the function of kallikreins have been
implicated in a variety of pathological processes including
imbalances in renal function and inflammatory processes. For these
reasons, siRNAs directed against this class of genes (e.g.,
KLK1-15) may prove valuable in both research and therapeutic
settings.
[0568] ADAM Proteins
[0569] The process of fertilization takes place in a series of
discrete steps whereby the sperm interacts with, i) the cumulus
cells and the hyaluronic acid extracellular matrix (ECM) in which
they are embedded, ii) the egg's own ECM, called the zona pellucida
(ZP), and iii) the egg plasma membrane. During the course of these
interactions, the "acrosome reaction," the exocytosis of the
acrosome vesicle on the head of the sperm, is induced, allowing the
sperm to penetrate the ZP and gain access to the perivitelline
space. This process exposes new portions of the sperm membrane,
including the inner acrosomal membrane and the equatorial segment,
regions of the sperm head that can participate in initial gamete
membrane binding.
[0570] The interactions of the gamete plasma membranes appear to
involve multiple ligands and receptors and are frequently compared
to leukocyte-endothelial interactions. These interactions lead to a
series of signal transduction events in the egg, known as
collectively as egg activation and include the initiation of
oscillations in intracellular calcium concentration, the exit from
meiosis, the entry into the first embryonic mitosis, and the
formation of a block to polyspermy via the release of ZP-modifying
enzymes from the egg's cortical granules. Ultimately, sperm and egg
not only adhere to each other but also go on to undergo membrane
fusion, making one cell (the zygote) from two.
[0571] Studies on the process of sperm-egg interactions have
identified a number of proteins that are crucial for fertilization.
One class of proteins, called the ADAM family (A Disintegrin And
Metalloprotease), has been found to be important in spermatogenesis
and fertilization, as well as various developmental systems
including myogenesis and neurogenesis. Members of the family
contain a disintegrin and metalloprotease domain (and therefore
have (potentially) both cell adhesion and protease activities), as
well as cysteine-rich regions, epidermal growth factor (EGF)-like
domains, a transmembrane region, and a cytoplasmic tail. Currently,
the ADAM gene family has 29 members and constituents are widely
distributed in many tissues including the brain, testis,
epididymis, ovary, breast, placenta, liver, heart, lung, bone, and
muscle.
[0572] One of the best-studied members of the ADAM family is
fertilin, a heterodimeric protein comprised of at least two
subunits, fertilin alpha and fertilin beta. The fertilin beta gene
(ADAM2) has been disrupted with a targeting gene construct
corresponding to the exon encoding the fertilin beta disintegrin
domain. Sperm from males homozygous for disruptions in this region
exhibit defects in multiple facets of sperm function including
reduced levels of sperm transit from the uterus to the oviduct,
reduced sperm-ZP binding, and reduced sperm-egg binding, all of
which contribute to male infertility.
[0573] Recently, four new ADAM family members (ADAM 24-27) have
been isolated. The deduced amino acid sequences show that all four
contain the complete domain organization common to ADAM family
members and Northern Blot analysis has shown all four to be
specific to the testes. siRNAs directed against this class of genes
(e.g., ADAM2 and related proteins) may be useful as research tools
and therapeutics directed toward fertility and birth control.
[0574] Aminopeptidases
[0575] Aminopeptidases are proteases that play critical roles in
processes such as protein maturation, protein digestion in its
terminal stage, regulation of hormone levels, selective or
homeostatic protein turnover, and plasmid stabilization. These
enzymes generally have broad substrate specificity, occur in
several forms and play a major role in physiological homeostasis.
For instance, the effects of bradykinin, angiotensin converting
enzyme (ACE), and other vasoactive molecules are muted by one of
several peptidases that cleave the molecule at an internal position
and eliminate its ability to bind its cognate receptor (e.g., for
bradykinin, the B2-receptor).
[0576] Among the enzymes that can cleave bradykinin is the membrane
bound aminopeptidase P, also referred to as aminoacylproline
aminopeptidase, proline aminopeptidase; X-Pro aminopeptidase
(eukaryote) and XPNPEP2. Aminopeptidase P is an aminoacylproline
aminopeptidase specific for NH.sub.2-terminal Xaa-proline bonds.
The enzyme i) is a mono-zinc-containing molecule that lacks any of
the typical metal binding motifs found in other zinc
metalloproteases, ii) has an active-site configuration similar to
that of other members of the MG peptidase family, and iii) is
present in a variety of tissues including but not limited to the
lung, kidney, brain, and intestine.
[0577] Aminopeptidases play an important role in a diverse set of
human diseases. Low plasma concentrations of aminopeptidase P are a
potential predisposing factor for development of angio-oedema in
patients treated with ACE inhibitors, and inhibitors of
aminopeptidase P may act as cardioprotectors against other forms of
illness including, but not limited to myocardial infarction. For
these reasons, siRNAs directed against this family of proteins
(including but not limited to XPNPEP1 and related proteins) may be
useful as research and therapeutic tools.
[0578] Serine Proteases
[0579] One important class of proteases are the serine proteases.
Serine proteases share a common catalytic triad of three amino
acids in their active site (serine (nucleophile), aspartate
(electrophile), and histidine (base)) and can hydrolyze either
esters or peptide bonds utilizing mechanisms of covalent catalysis
and preferential binding of the transition state. Based on the
position of their introns serine proteases have been classified
into a minimum of four groups including those in which 1) the gene
has no introns interrupting the exon coding for the catalytic triad
(e.g., the haptoglobin gene,); 2) each gene contains an intron just
downstream from the codon for the histidine residue at the active
site, a second intron downstream from the exon containing the
aspartic acid residue of the active site and a third intron just
upstream from the exon containing the serine of the active site
(e.g., trypsinogen, chymotrypsinogen, kallikrein and proelastase);
3) the genes contain seven introns interrupting the exons coding
the catalytic region (e.g., complement factor B gene); and 4) the
genes contain two introns resulting in a large exon that contains
both the active site aspartatic acid and serine residues (e.g.,
factor X, factor IX and protein C genes).
[0580] Cytotoxic lymphocytes (e.g., CD8(+) cytotoxic T cells and
natural killer cells) form the major defense of higher organisms
against virus-infected and transformed cells. A key function of
these cells is to detect and eliminate potentially harmful cells by
inducing them to undergo apoptosis. This is achieved through two
principal pathways, both of which require direct but transient
contact between the killer cell and its target. The first pathway
involves ligation of TNF receptor-like molecules such as Fas/CD95
to their cognate ligands, and results in mobilization of
conventional, programmed cell-death pathways centered on activation
of pro-apoptotic caspases. The second mechanism consists of a
pathway whereby the toxic contents of a specialized class of
secretory vesicles are introduced into the target cell. Studies
over the last two decades have identified the toxic components as
Granzymes, a family of serine proteases that are expressed
exclusively by cytotoxic T lymphocytes and natural killer (NK)
cells. These agents are stored in specialized lytic granules and
enter the target cell via endocytosis. Like caspases, cysteine
proteases that play an important role in apoptosis, granzymes can
cleave proteins after acidic residues, especially aspartic acid,
and induce apoptosis in the recipient cell.
[0581] Granzymes have been grouped into three subfamilies according
to substrate specificity. Members of the granzyme family that have
enzymatic activity similar to the serine protease chymotrypsin are
encoded by a gene cluster termed the `chymase locus`. Similarly,
granzymes with trypsin-like specificities are encoded by the
`tryptase locus`, and a third subfamily cleaves after unbranched
hydrophobic residues, especially methionine, and are encoded by the
`Met-ase locus`. All granzymes are synthesized as zymogens and,
after clipping of the leader peptide, obtain maximal enzymatic
activity subsequent to the removal of an amino-terminal
dipeptide.
[0582] Granzymes have been found to be important in a number of
important biological functions including defense against
intracellular pathogens, graft versus host reactions, the
susceptibility to transplantable and spontaneous malignancies,
lymphoid homeostasis, and the tendency toward auto-immune diseases.
For these reasons, siRNAs directed against granszymes (e.g., GZMA,
GZMB, GZMH, GZHK, GZMM) and related serine proteases may be useful
research and therapeutic reagents.
[0583] Kinases
[0584] Protein Kinases (PKs) have been implicated in a number of
biological processes. Kinase molecules play a central role in
modulating cellular physiology and developmental decisions, and
have been implicated in a large list of human maladies including
cancer, diabetes, and others.
[0585] During the course of the last three decades, over a hundred
distinct protein kinases have been identified, all with presumed
specific cellular functions. A few of these enzymes have been
isolated to sufficient purity to perform in vitro studies, but most
remain intractable due to the low abundance of these molecules in
the cell. To counter this technical difficulty, a number of protein
kinases have been isolated by molecular cloning strategies that
utilize the conserved sequences of the catalytic domain to isolate
closely related homologs. Alternatively, some kinases have been
purified (and subsequently studied) based on their interactions
with other molecules.
[0586] p58 is a member of the p34cdc2-related supergene family and
contains a large domain that is highly homologous to the cell
division control kinase, cdc2. This new cell division
control-related protein kinase was originally identified as a
component of semipurified galactosyltransferase; thus, it has been
denoted galactosyltransferase-as- sociated protein kinase
(GTA-kinase). GTA-kinase has been found to be expressed in both
adult and embryonic tissues and is known to phosphorylate a number
of substrates, including histone H1, and casein. Interestingly
enough, over expression of this molecule in CHO cells has shown
that elevated levels of p58 result in a prolonged late telophase
and an early G1 phase, thus hinting of an important role for
GTA-kinase in cell cycle regulation.
[0587] Cyclin Dependent Kinases
[0588] The cyclin-dependent kinases (Cdks) are a family of highly
conserved serine/threonine kinases that mediate many of the cell
cycle transitions that occur during duplication. Each of these Cdk
catalytic subunits associates with a specific subset of regulatory
subunits, termed cyclins, to produce a distinct Cdk.cyclin kinase
complex that, in general, functions to execute a unique cell cycle
event.
[0589] Activation of the Cdk.cyclin kinases during cellular
transitions is controlled by a variety of regulatory mechanisms.
For the Cdc2.cyclin B complex, inhibition of kinase activity during
S phase and G.sub.2 is accomplished by phosphorylation of two Cdc2
residues, Thr.sup.14 and Tyr.sup.15, which are positioned within
the ATP-binding cleft. Phosphorylation of Thr.sup.14 and/or
Tyr.sup.15 suppresses the catalytic activity of the molecule by
disrupting the orientation of the ATP present within this cleft. In
contrast, the abrupt dephosphorylation of these residues by the
Cdc25 phosphatase results in the rapid activation of Cdc2.cyclin B
kinase activity and subsequent downstream mitotic events. While the
exact details of this pathway have yet to be elucidated, it has
been proposed that Thr.sup.14/Tyr.sup.15 phosphorylation functions
to permit a cell to attain a critical concentration of inactive
Cdk.cyclin complexes, which, upon activation, induces a rapid and
complete cell cycle transition. Furthermore, there is evidence in
mammalian cells that Thr.sup.14/Tyr.sup.15 phosphorylation also
functions to delay Cdk activation after DNA damage.
[0590] The Schizosaccharomyces pombe wee1 gene product was the
first kinase identified that is capable of phosphorylating
Tyr.sup.15 in Cdc2. Homologs of the Wee1 kinase have been
subsequently identified and biochemically characterized from a wide
range of species including human, mouse, frog, Saccharomyces
cerevisiae, and Drosophila. In vertebrate systems, where Thr.sup.14
in Cdc2 is also phosphorylated, the Wee1 kinase was capable of
phosphorylating Cdc2 on Tyr.sup.15, but not Thr.sup.14, indicating
that another kinase was responsible for Thr.sup.14 phosphorylation.
This gene, Myt1 kinase, was recently isolated from the membrane
fractions of Xenopus egg extracts and has been shown to be capable
of phosphorylating Thr.sup.14 and, to a lessor extent, Tyr.sup.15
in Cdc2. A human Myt1 homolog displaying similar properties has
been isolated, as well as a non-membrane-associated molecule with
Thr.sup.14 kinase activity.
[0591] In the past decade it has been shown that cancer can
originate from overexpression of positive regulators, such as
cyclins, or from underexpression of negative regulators (e.g., p16
(INK4a), p15 (INK4b), p21 (Cip1)). Inhibitors such as Myt1 are the
focus of much cancer research because they are capable of
controlling cell cycle proliferation, now considered the Holy Grail
for cancer treatment. For these reasons, siRNA directed against
kinases and kinase inhibitors including but not limited to ABL1,
ABL2, ACK1, ALK, AXL, BLK, BMX, BTK, C20orf64, CSF1R, SCK, DDR1,
DDR2, DKFZp761P1010, EGFR, EPHA1, EPHA2, EPHA3, EPHA4, EPHA7,
EPHA8, EPHB1, EPHB2, EPHB3, EPHB4. EPHB6, ERBB2, ERBB3, ERBB4, FER,
FES, FGFR1, FGFR2, FGFR3, FGFR4, FGR, FLT1, FLT3, FLT4, FRK, FYN,
HCK, IGF1R, INSR, ITK, JAK1, JAK2, JAK3, KDR, KIAA1079, KIT, LCK,
LTK, LYN, MATK, MERTK, MET, MST1R, MUSK, NTRK1, NTRK2, NTRK3,
PDGFRA, PDGFRB, PTK2, PTK2B, PTK6, PTK7, PTK9, PTK9L, RET, ROR1,
ROR2, ROS1, RYK, SRC, SYK, TEC, TEK, TIE, TNK1, TXK, TYK2, TYRO3,
YES 1, and related proteins, may be useful for research and
therapeutic purposes.
[0592] G Protein Coupled Receptors
[0593] One important class of genes to which siRNAs can be directed
are G-protein coupled receptors (GPCRs). GPCRs constitute a
superfamily of seven transmembrane spanning proteins that respond
to a diverse array of sensory and chemical stimuli, such as light,
odor, taste, pheromones, hormones and neurotransmitters. GPCRs play
a central role in cell proliferation, differentiation, and have
been implicated in the etiology of disease.
[0594] The mechanism by which G protein-coupled receptors translate
extracellular signals into cellular changes was initially
envisioned as a simple linear model: activation of the receptor by
agonist binding leads to dissociation of the heterotrimeric
GTP-binding G protein (Gs, Gi, or Gq) into its alpha and beta/gamma
subunits, both of which can activate or inhibit various downstream
effector molecules. More specifically, activation of the GPCR
induces a conformational change in the G.alpha. subunit, causing
GDP to be released and GTP to be bound in its place. The G.alpha.
and G.beta..alpha. subunits then dissociate from the receptor and
interact with a variety of effector molecules. For instance in the
case of the Gs family, the primary function is to stimulate the
intracellular messenger adenylate cyclase (AC), which catalyzes the
conversion of cytoplasmic ATP into the secondary messenger cyclic
AMP (cAMP). In contrast, the Gi family inhibits this pathway and
the Gq family activates phospholipases C (PLC), which cleaves
phosphatidylinositol 4,5, bisphosphate (PIP2) to generate
inositol-1,4,5-phosphate (IP3) and diacylglycerol (DAG).
[0595] More recently, studies have shown that the functions of
GPCRs are not limited to their actions on G-proteins and that
considerable cross-talk exists between this diverse group of
receptor molecules and a second class of membrane bound proteins,
the receptor tyrosine kinases (RTKs). A number of GPCRs such as
endothelin-1, thrombin, bombesin, and dopamine receptors can
activate MAPKs, a downstream effector of the RTK/Ras pathway.
Interestingly, the interaction between these two families is not
unidirectional and RTKs can also modulate the activity of signaling
pathways traditionally thought to be controlled exclusively by
ligands that couple to GPCRs. For instance, EGF, which normally
activates the MAPK cascade via the EGF receptor can stimulate
adenylate cyclase activity by activating G.alpha.s.
[0596] There are dozens of members of the G Protein-Coupled
Receptor family that have emerged as prominent drug targets in the
last decade. One non-limiting list of potential GPCR-siRNA targets
is as follows:
[0597] CMKLR1
[0598] CML1/CMKLR1 (Accession No. Q99788) is a member of the
chemokine receptor family of GPCRs that may play a role in a number
of diseases including those involved in inflammation and
immunological responses (e.g., asthma, arthritis). For this reason,
siRNA directed against this protein may prove to be important
therapeutic reagents.
[0599] Studies of juvenile-onset neuronal ceroid lipofuscinosis
(JNCL, Batten disease), the most common form of childhood
encephalopathy that is characterized by progressive neural
degeneration, show that it is brought on by mutations in a novel
lysosomal membrane protein (CLN3). In addition to being implicated
in JNCL, CLN3 (GPCR-like protein, Accession No. A57219) expression
studies have shown that the CLN3 mRNA and protein are highly
over-expressed in a number of cancers (e.g., glioblastomas,
neuroblastomas, as well as cancers of the prostate, ovaries,
breast, and colon) suggesting a possible contribution of this gene
to tumor growth. For this reason, siRNA directed against this
protein may prove to be important therapeutic reagents.
[0600] CLACR
[0601] The calcitonin receptor (CTR/CALCR, Accession No.
NM.sub.--001742) belongs to "family B" of GPCRs which typically
recognized regulatory peptides such as parathyroid hormone,
secretin, glucagons and vasoactive intestinal polypeptide. Although
the CT receptor typically binds to calcitonin (CT), a 32 amino acid
peptide hormone produced primarily by the thyroid, association of
the receptor with RAMP (Receptor Activity Modulating Protein)
enables it to readily bind other members of the calcitonin peptide
family including amylin (AMY) and other CT gene-related peptides
(e.g., .alpha.CGRP and .beta.CGRP). While the primary function of
the calcitonin receptor pertains to regulating osteoclast mediated
bone resorption and enhanced Ca.sup.+2 excretion by the kidney,
recent studies have shown that CT and CTRs may play an important
role in a variety of processes as wide ranging as embryonic/fetal
development and sperm function/physiology. In addition, studies
have shown that patients with particular CTR genotypes may be at
higher risk to lose bone mass and that this GPCR may contribute to
the formation of calcium oxalate urinary stones. For this reason,
siRNA directed against CTR may be useful as therapeutic
reagents.
[0602] OXTR
[0603] The human oxytocin receptor (OTR, OXTR) is a 389 amino acid
polypeptide that exhibits the seven transmembrane domain structure
and belongs to the Class-I (rhodopsin-type) family of G-protein
coupled receptors. OTR is expressed in a wide variety of tissues
throughout development and mediates physiological changes through
G(q) proteins and phospholipase C-beta. Studies on the functions of
oxytocin and the oxytocin receptor have revealed a broad list of
duties. OT and OTR play a role in a host of sexual, maternal and
social behaviors that include egg-laying, birth, milk-letdown,
feeding, grooming, memory and learning. In addition, it has been
hypothesized that abnormalities in the functionality of
oxytocin-OTR receptor-ligand system can lead to a host of
irregularities including compulsive behavior, eating disorders
(such as anorexia), depression, and various forms of
neurodegenerative diseases. For these reasons, siRNA directed
against this gene (NM.sub.--000916) may play an important role in
combating OTR-associated illnesses.
[0604] EDG GPCRs
[0605] Lysophosphatidic acid and other lipid-based hormones/growth
factors induce their effects by activating signaling pathways
through the G-protein coupled receptors (GPCRs) and have been
observed to play important roles in a number of human diseases
including cancer, asthma, and vascular pathologies. For instance,
during studies of immunoglobulin A nephropathy (IgAN), researchers
have observed an enhanced expression of EDG5 (NP.sub.--004221)
suggesting a contribution of this gene product in the development
of IgAN. For that reason, siRNA directed against Edg5
(NM.sub.--004230), Edg4 (NM.sub.--004720), Edg7 (Nm.sub.--012152)
and related genes may play an important role in combating human
disease.
[0606] Genes Involved in Cholesterol Signaling and Biosynthesis
[0607] Studies on model genetic organisms such as Drosophila and C.
elegans have led to the identification of a plethora of genes that
are essential for early development. Mutational analysis and
ectopic expression studies have allowed many of these genes to be
grouped into discreet signal transduction pathways and have shown
that these elements play critical roles in pattern formation and
cell differentiation. Disruption of one or more of these genes
during early stages of development frequently leads to birth
defects whereas as alteration of gene function at later stages in
life can result in tumorigenesis.
[0608] One critical set of interactions known to exist in both
invertebrates and vertebrates is the Sonic Hedgehog-Patched-Gli
pathway. Originally documented as a Drosophila segmentation mutant,
several labs have recently identified human and mouse orthologs of
many of the pathways members and have successfully related
disruptions in these genes to known diseases. Pathway activation is
initiated with the secretion of Sonic hedgehog. There are three
closely related members of the Shh family (Sonic hedgehog, Desert,
and Indian) with Shh being the most widely expressed form of the
group. The Shh gene product is secreted as a small pro-signal
molecule. To successfully initiate its developmental role, Shh is
first cleaved, whereupon the N-terminal truncated fragment is
covalently modified with cholesterol. The addition of the sterol
moiety promotes the interaction between Shh and its cognate
membrane bound receptor, Patched (Ptch). There are at least two
isoforms of the Patched gene, Ptch1 and Ptch2. Both isoforms
contain a sterol-sensing domain (SSD); a roughly 180 amino acid
cluster that is found in at least seven different classes of
molecules including those involved in cholesterol biosynthesis,
vesicular traffic, signal transduction, cholesterol transport, and
sterol homeostasis. In the absence of Shh, the Patched protein is a
negative regulator of the pathway. In contrast, binding of
Shh-cholesterol to the Patched receptor releases the negative
inhibition which that molecule enforces on a G-protein coupled
receptor known as Smoothened. Subsequent activation of Smoothened
(directly or indirectly) leads to the triggering of a trio of
transcription factors that belong to the Gli family. All three
factors are relatively large, contain a characteristic C2-H2
zinc-finger pentamer, and recognize one of two consensus sequences
(SEQ. ID NO. 0463 GACCACCCA or SEQ. ID NO. 0464 GAACCACCCA). In the
absence of Shh, Gli proteins are cleaved by the proteosome and the
C-terminally truncated fragment translocates to the nucleus and
acts as a dominant transcription repressor. In the presence of
Shh-cholesterol, Gli repressor formation is inhibited and
full-length Gli functions as a transcriptional activator.
[0609] Shh and other members of the Shh-PTCH-Gli pathway are
expressed in a broad range of tissues (e.g., the notochord, the
floorplate of the neural tube, the brain, and the gut) at early
stages in development. Not surprisingly, mutations that lead to
altered protein expression or function have been shown to induce
developmental abnormalities. Defects in the human Shh gene have
been shown to cause holoprosencephaly, a midline defect that
manifests itself as cleft lip or palate, CNS septation, and a wide
range of other phenotypes. Interestingly, defects in cholesterol
biosynthesis generate similar Shh-like disorders (e.g.,
Smith-Lemli-Opitz syndrome) suggesting that cholesterol
modification of the Shh gene product is crucial for pathway
function. Both the Patched and Smoothened genes have also been
shown to be clinically relevant with Smoothened now being
recognized as an oncogene that, like PTCH-1 and PTCH-2, is believed
to be the causative agent of several forms of adult tumors. For
these reasons, siRNA directed against Smoothened (SMO,
NM.sub.--005631), Patched (PTCH, nm.sub.--000264), and additional
genes that participate in cholesterol signaling, biosynthesis, and
degradation, have potentially useful research and therapeutic
applications.
[0610] Targeted Pathways.
[0611] In addition to targeting siRNA against one or more members
of a family of proteins, siRNA can be directed against members of a
pathway. Thus, for instance, siRNA can be directed against members
of a signal transduction pathway (e.g., the insulin pathway,
including AKT1-3, CBL, CBLB, EIF4EBP1, FOXO1A, FOXO3A, FRAP1,
GSK3A, GSK3B, IGF1, IGF1R, INPP5D, INSR, IRS1, MLLT7, PDPK1,
PIK3CA, PIK3CB, PIK3R1, PIK3R2, PPP2R2B, PTEN, RPS6, RPS6KA1,
RPX6KA3, SGK, TSC1, TSC2, AND XPO1), an apoptotic pathway
(CASP3,6,7,8,9, DSH1/2, P110, P85, PDK1/2, CATENIN, HSP90, CDC37,
P23, BAD, BCLXL, BCL2, SMAC, and others), pathways, involved in DNA
damage, cell cycle, and other physiological (p53,MDM2, CHK1/2,
BRCA1/2, ATM, ATR, P15INK4, P27, P21, SKP2, CDC25C/A, 14-3-3, PLK,
RB, CDK4, GLUT4, Inos, Mtor, FKBP, PPAR, RXR, ER). Similarly, genes
involved in immune system function including TNFR1, IL-IR, IRAK1/2,
TRAF2, TRAF6, TRADD, FADD, IKK.epsilon., IKK.gamma., IKK.beta.,
IKK.alpha., IkB.alpha., IkB.beta., p50, p65, Rac, RhoA, Cdc42,
ROCK, Pak1/2/3/4/5/6, cIAP, HDAC1/2, CBP, .beta.-TrCP, Rip2/4, and
others are also important targets for the siRNAs described in this
document and may be useful in treating immune system disorders.
Genes involved in apoptosis, such as Dsh1/2,PTEN, P110 (pan), P85,
PDK1/2, Akt1, Akt2, Akt (pan), p70.sup.S6K, GSK3.beta., PP2A (cat),
.beta.-catenin, HSP90, Cdc37/p50, P23, Bad, BclxL, Bcl2,
Smac/Diablo, and Ask1 are potentially useful in the treatment of
diseases that involve defects in programmed cell death (e.g.,
cancer), while siRNA agents directed against p53, MDM2, Chk1/2,
BRCA1/2, ATM, ATR, p15.sup.INK4, P27, P21, Skp2, Cdc25C/A,
14-3-3.sigma./.epsilon., PLK, Rb, Cdk4, Glut4, iNOS, mTOR, FKBP,
PPAR.gamma., RXR.alpha., ER.alpha. and related genes may play a
critical role in combating diseases associated with disruptions in
DNA repair, and cell cycle abnormalities.
[0612] Tables VI-Table X below provide examples of useful pools for
inhibiting different genes in the human insulin pathway and
tyrosine kinase pathways, proteins involved in the cell cycle, the
production of nuclear receptors, and other genes. These particular
pools are particularly useful in humans, but would be useful in any
species that generates an appropriately homologous mRNA. Further,
within each of the listed pools any one sequence maybe used
independently but preferably at least two of the listed sequences,
more preferably at least three, and most preferably all of the
listed sequences for a given gene is present.
11TABLE VI Gene SEQ. Name Acc # GI L.L. Duplex # Sequence ID NO.
AKT1 NM_005163 4885060 207 D-003000-05 GACAAGGACGGGCACATTA 465 AKT1
NM_005163 4885060 207 D-003000-06 GGACAAGGACGGGCACATT 466 AKT1
NM_005163 4885060 207 D-003000-07 GCTACTTCCTCCTCAAGAA 467 AKT1
NM_005163 4885060 207 D-003000-08 GACCGCCTCTGCTTTGTCA 468 AKT2 AKT2
NM_001626 6715585 208 D-003001-05 GTACTTCGATGATGAATTT 469 AKT2
NM_001626 6715585 208 D-003001-06 GCAAAGAGGGCATCAGTGA 470 AKT2
NM_001626 6715585 208 D-003001-07 GGGCTAAAGTGACCATGAA 471 AKT2
NM_001626 6715585 208 D-003001-08 GCAGAATGCCAGCTGATGA 472 AKT3 AKT3
NM_005465 32307164 10000 D-003002-05 GGAGTAAACTGGCAAGATG 473 AKT3
NM_005465 32307164 10000 D-003002-06 GACATTAAATTTCCTCGAA 474 AKT3
NM_005465 32307164 10000 D-003002-07 GACCAAAGCCAAACACATT 475 AKT3
NM_005465 32307164 10000 D-003002-08 GAGGAGAGAATGAATTGTA 476 CBL
CBL NM_005188 4885116 867 D-003003-05 GGAGACACATTTCGGATTA 477 CBL
NM_005188 4885116 867 D-003003-06 GATCTGACCTGCAATGATT 478 CBL
NM_005188 4885116 867 D-003003-07 GACAATCCCTCACAATAAA 479 CBL
NM_005188 4885116 867 D-003003-08 CCAGAAAGCTTTGGTCATT 480 CBLB CBLB
NM_170662 29366807 868 D-003004-05 GACCATACCTCATAACAAG 481 CBLB
NM_170662 29366807 868 D-003004-06 TGAAAGACCTCCACCAATC 482 CBLB
NM_170662 29366807 868 D-003004-07 GATGAAGGCTCCAGGTGTT 483 CBLB
NM_170662 29366807 868 D-003004-08 TATCAGCATTTACGACTTA 484 EIF4EBP1
EIF4EBP1 NM_004095 20070179 1978 D-003005-05 GCAATAGCCCAGAAGATAA
485 EIF4EBP1 NM_004095 20070179 1978 D-003005-06
CGCAATAGCCCAGAAGATA 486 EIF4EBP1 NM_004095 20070179 1978
D-003005-07 GAGATGGACATTTAAAGCA 487 EIF4EBP1 NM_004095 20070179
1978 D-003005-08 CAATAGCCCAGAAGATAAG 488 FOXO1A FOXO1A NM_002015
9257221 2308 D-003006-05 CCAGGCATCTCATAACAAA 489 FOXO1A NM_002015
9257221 2308 D-003006-06 CCAGATGCCTATACAAACA 490 FOXO1A NM_002015
9257221 2308 D-003006-07 GGAGGTATGAGTCAGTATA 491 FOXO1A NM_002015
9257221 2308 D-003006-08 GAGGTATGAGTCAGTATAA 492 FOXO3A FOXO3A
NM_001455 4503738 2309 D-003007-01 CAATAGCAACAAGTATACC 493 FOXO3A
NM_001455 4503738 2309 D-003007-02 TGAAGTCCAGGACGATGAT 494 FOXO3A
NM_001455 4503738 2309 D-003007-03 TGTCACACTATGGTAACCA 495 FOXO3A
NM_001455 4503738 2309 D-003007-04 TGTTCAATGGGAGCTTGGA 496 FRAP1
FRAP1 NM_004958 19924298 2475 D-003008-05 GAGAAGAAATGGAAGAAAT 497
FRAP1 NM_004958 19924298 2475 D-003008-06 CCAAAGTGCTGCAGTACTA 498
FRAP1 NM_004958 19924298 2475 D-003008-07 GAGCATGCCGTCAATAATA 499
FRAP1 NM_004958 19924298 2475 D-003008-08 GGTCTGAACTGAATGAAGA 500
GSK3A GSK3A NM_019884 11995473 2931 D-003009-05 GGACAAAGGTGTTCAAATC
501 GSK3A NM_019884 11995473 2931 D-003009-06 GAACCCAGCTGCCTAACAA
502 GSK3A NM_019884 11995473 2931 D-003009-07 GCGCACAGCTTCTTTGATG
503 GSK3A NM_019884 11995473 2931 D-003009-08 GCTCTAGCCTGCTGGAGTA
504 GSK3B GSK3B NM_002093 21361339 2932 D-003010-05
GAAGAAAGATGAGGTCTAT 505 GSK3B NM_002093 21361339 2932 D-003010-06
GGACCCAAATGTCAAACTA 506 GSK3B NM_002093 21361339 2932 D-003010-07
GAAATGAACCCAAACTACA 507 GSK3B NM_002093 21361339 2932 D-003010-08
GATGAGGTCTATCTTAATC 508 IGF1 IGF1 NM_000618 D-003011-05
GGAAGTACATTTGAAGAAC 509 IGF1 NM_000618 D-003011-06
AGAAGGAAGTACATTTGAA 510 IGF1 NM_000618 D-003011-07
CCTCAAGCCTGCCAAGTCA 511 IGF1 NM_000618 D-003011-08
GGTGGATGCTCTTCAGTTC 512 IGF1R IGF1R NM_000875 11068002 3480
D-003012-05 CAACGAAGCTTCTGTGATG 513 IGF1R NM_000875 11068002 3480
D-003012-06 GGCCAGAAATGGAGAATAA 514 IGF1R NM_000875 11068002 3480
D-003012-07 GAAGCACCCTTTAAGAATG 515 IGF1R NM_000875 11068002 3480
D-003012-08 GCAGACACCTACAACATCA 516 INPP5D INPP5D NM_005541 5031798
3635 D-003013-05 GGAATTGCGTTTACACTTA 517 INPP5D NM_005541 5031798
3635 D-003013-06 GGAAACTGATCATTAAGAA 518 INPP5D NM_005541 5031798
3635 D-003013-07 CGACAGGGATGAAGTACAA 519 INPP5D NM_005541 5031798
3635 D-003013-08 AAACGCAGCTGCCCATCTA 520 INSR INSR NM_000208
4557883 3643 D-003014-05 GGAAGACGTTTGAGGATTA 521 INSR NM_000208
4557883 3643 D-003014-06 GAACAAGGCTCCCGAGAGT 522 INSR NM_000208
4557883 3643 D-003014-07 GGAGAGACCTTGGAAATTG 523 INSR NM_000208
4557883 3643 D-003014-08 GGACGGAACCCACCTATTT 524 IRS1 IRS1
NM_005544 5031804 3667 D-003015-05 AAAGAGGTCTGGCAAGTGA 525 IRS1
NM_005544 5031804 3667 D-003015-06 GAACCTGATTGGTATCTAC 526 IRS1
NM_005544 5031804 3667 D-003015-07 CCACGGCGATCTAGTGCTT 527 IRS1
NM_005544 5031804 3667 D-003015-08 GTCAGTCTGTCGTCCAGTA 528 MLLT7
MLLT7 NM_005938 5174578 4303 D-003016-05 GGACTGGACTTCAACTTTG 529
MLLT7 NM_005938 5174578 4303 D-003016-06 CCACGAAGCAGTTCAAATG 530
MLLT7 NM_005938 5174578 4303 D-003016-07 GAGAAGCGACTGACACTTG 531
MLLT7 NM_005938 5174578 4303 D-003016-08 GACCAGAGATCGCTAACCA 532
PDPK1 PDPK1 NM_002613 4505694 5170 D-003017-05 CAAGAGACCTCGTGGAGAA
533 PDPK1 NM_002613 4505694 5170 D-003017-06 GACCAGAGGCCAAGAATTT
534 PDPK1 NM_002613 4505694 5170 D-003017-07 GGAAACGAGTATCTTATAT
535 PDPK1 NM_002613 4505694 5170 D-003017-08 GAGAAGCGACATATCATAA
536 PIK3CA PIK3CA NM_006218 5453891 5290 D-003018-05
GCTATCATCTGAACAATTA 537 PIK3CA NM_006218 5453891 5290 D-003018-06
GGATAGAGGCCAAATAATA 538 PIK3CA NM_006218 5453891 5290 D-003018-07
GGACAACTGTTTCATATAG 539 PIK3CA NM_006218 5453891 5290 D-003018-08
GCCAGTACCTCATGGATTA 540 PIK3CB PIK3CB NM_006219 5453893 5291
D-003019-05 CGACAAGACTGCCGAGAGA 541 PIK3CB NM_006219 5453893 5291
D-003019-06 TCAAGTGTCTCCTAATATG 542 PIK3CB NM_006219 5453893 5291
D-003019-07 GGATTCAGTTGGAGTGATT 543 PIK3CB NM_006219 5453893 5291
D-003019-08 TTTCAAGTGTCTCCTAATA 544 PIK3R1 PIK3R1 NM_181504
32455251 5295 D-003020-05 GGAAATATGGCTTCTCTGA 545 PIK3R1 NM_181504
32455251 5295 D-003020-06 GAAAGACGAGAGACCAATA 546 PIK3R1 NM_181504
32455251 5295 D-003020-07 GTAAAGCATTGTGTCATAA 547 PIK3R1 NM_181504
32455251 5295 D-003020-08 GGATCAAGTTGTCAAAGAA 548 PIK3R2 PIK3R2
NM_005027 4826907 5296 D-003021-05 GGAAAGGCGGGAACAATAA 549 PIK3R2
NM_005027 4826907 5296 D-003021-06 GATGAAGCGTACTGCAATT 550 PIK3R2
NM_005027 4826907 5296 D-003021-07 GGACAGCGAATCTCACTAC 551 PIK3R2
NM_005027 4826907 5296 D-003021-08 GCAAGATCCGAGACCAGTA 552 PPP2R2B
PPP2R2B NM_004576 4758953 5521 D-003022-05 GAATGCAGCTTACTTTCTT 553
PPP2R2B NM_004576 4758953 5521 D-003022-06 GACCGAAGCTGACATTATC 554
PPP2R2B NM_004576 4758953 5521 D-003022-07 TCGATTACCTGAAGAGTTT 555
PPP2R2B NM_004576 4758953 5521 D-003022-08 CCTGAAGAGTTTAGAAATA 556
PTEN PTEN NM_000314 4506248 5728 D-003023-05 GTGAAGATCTTGACCAATG
557 PTEN NM_000314 4506248 5728 D-003023-06 GATCAGCATACACAAATTA 558
PTEN NM_000314 4506248 5728 D-003023-07 GGCGCTATGTGTATTATTA 559
PTEN NM_000314 4506248 5728 D-003023-08 GTATAGAGCGTGCAGATAA 560
RPS6 RPS6 NM_001010 17158043 6194 D-003024-05 GCCAGAAACTCATTGAAGT
561 RPS6 NM_001010 17158043 6194 D-003024-06 GGATATTCCTGGACTGACT
562 RPS6 NM_001010 17158043 6194 D-003024-07 CCAAGGAGAACTGGAGAAA
563 RPS6 NM_001010 17158043 6194 D-003024-08 GCGTATGGCCACAGAAGTT
564 RPS6KA1 RPS6KA1 NM_002953 20149546 6195 D-003025-05
GATGACACCTTCTACTTTG 565 RPS6KA1 NM_002953 20149546 6195 D-003025-06
GAGAATGGGCTCCTCATGA 566 RPS6KA1 NM_002953 20149546 6195 D-003025-07
CAAGCGGGATCCTTCAGAA 567 RPS6KA1 NM_002953 20149546 6195 D-003025-08
CCACCGGCCTGATGGAAGA 568 RPS6KA3 RPS6KA3 NM_004586 4759049 6197
D-003026-05 GAAGGGAAGTTGTATCTTA 569 RPS6KA3 NM_004586 4759049 6197
D-003026-06 GAAAGTATGTGTATGTAGT 570 RPS6KA3 NM_004586 4759049 6197
D-003026-07 GGACAGCATCCAAACATTA 571 RPS6KA3 NM_004586 4759049 6197
D-003026-08 GGAGGTGAATTGCTGGATA 572 SGK SGK NM_005627 5032090 6446
D-003027-01 TTAATGGTGGAGAGTTGTT 573 SGK NM_005627 5032090 6446
D-003027-04 ATTAACTGGGATGATCTCA 574 SGK NM_005627 25168262 6446
D-003027-05 GAAGAAAGCAATCCTGAAA 575 SGK NM_005627 25168262 6446
D-003027-06 AAACACAGCTGAAATGTAC 576 TSC1 TSC1 NM_000368 24475626
7248 D-003028-05 GAAGATGGCTATTCTGTGT 577 TSC1 NM_000368 24475626
7248 D-003028-06 TATGAAGGCTCGAGAGTTA 578 TSC1 NM_000368 24475626
7248 D-003028-07 CGACACGGCTGATAACTGA 579 TSC1 NM_000368 24475626
7248 D-003028-08 CGGCTGATGTTGTTAAATA 580 TSC2 TSC2 NM_000548
10938006 7249 D-003029-05 GCATTAATCTCTTACCATA 581 TSC2 NM_000548
10938006 7249 D-003029-06 CCAATGTCCTCTTGTCTTT 582 TSC2 NM_000548
10938006 7249 D-003029-07 GGAGACACATCACCTACTT 583 TSC2 NM_000548
10938006 7249 D-003029-08 TCACCAGGCTCATCAAGAA 584 XPO1 XPO1
NM_003400 8051634 7514 D-003030-05 GAAAGTCTCTGTCAAAATA 585 XPO1
NM_003400 8051634 7514 D-003030-06 GCAATAGGCTCCATTAGTG 586 XPO1
NM_003400 8051634 7514 D-003030-07 GGAACATGATCAACTTATA 587 XPO1
NM_003400 8051634 7514 D-003030-08 GGATACAGATTCCATAAAT 588
[0613]
12TABLE VII Gene SEQ. Name Acc # GI L.L. Duplex # Sequence ID NO.
ABL1 ABL1 NM_007313 6382057 25 D-003100-05 GGAAATCAGTGACATAGTG 589
ABL1 NM_007313 6382057 25 D-003100-06 GGTCCACACTGCAATGTTT 590 ABL1
NM_007313 6382057 25 D-003100-07 GAAGGAAATCAGTGACATA 591 ABL1
NM_007313 6382057 25 D-003100-08 TCACTGAGTTCATGACCTA 592 ABL2 ABL2
NM_007314 6382061 27 D-003101-05 GAAATGGAGCGAACAGATA 593 ABL2
NM_007314 6382061 27 D-003101-06 GAGCCAAATTTCCTATTAA 594 ABL2
NM_007314 6382061 27 D-003101-07 GTAATAAGCCTACAGTCTA 595 ABL2
NM_007314 6382061 27 D-003101-08 GGAGTGAAGTTCGCTCTAA 596 ACK1 ACK1
NM_005781 8922074 10188 D-003102-05 AAACGCAAGTCGTGGATGA 597 ACK1
NM_005781 8922074 10188 D-003102-06 GCAAGTCGTGGATGAGTAA 598 ACK1
NM_005781 8922074 10188 D-003102-07 GAGCACTACCTCAGAATGA 599 ACK1
NM_005781 8922074 10188 D-003102-08 TCAGCAGCACCCACTATTA 600 ALK ALK
NM_004304 29029631 238 D-003103-05 GACAAGATCCTGCAGAATA 601 ALK
NM_004304 29029631 238 D-003103-06 GGAAGAGTCTGGCAGTTGA 602 ALK
NM_004304 29029631 238 D-003103-07 GCACGTGGCTCGGGACATT 603 ALK
NM_004304 29029631 238 D-003103-08 GAACTGCAGTGAAGGAACA 604 AXL AXL
NM_021913 21536465 558 D-003104-05 GGTCAGAGCTGGAGGATTT 605 AXL
NM_021913 21536465 558 D-003104-06 GAAAGAAGGAGACCCGTTA 606 AXL
NM_021913 21536465 558 D-003104-07 CCAAGAAGATCTACAATGG 607 AXL
NM_021913 21536465 558 D-003104-08 GGAACTGCATGCTGAATGA 608 BLK BLK
NM_001715 4502412 640 D-003105-05 GAGGATGCCTGCTGGATTT 609 BLK
NM_001715 4502412 640 D-003105-06 ACATGAAGGTGGCCATTAA 610 BLK
NM_001715 4502412 640 D-003105-07 GGTCAGCGCCCAAGACAAG 611 BLK
NM_001715 4502412 640 D-003105-08 GAAACTCGGGTCTGGACAA 612 BMX BMX
NM_001721 21359831 660 D-003106-05 AAACAAACCTTTCCTACTA 613 BMX
NM_001721 21359831 660 D-003106-06 GAAGGAGCATTTATGGTTA 614 BMX
NM_001721 21359831 660 D-003106-07 GAGAAGAGATTACCTTGTT 615 BMX
NM_001721 21359831 660 D-003106-08 GTAAGGCTGTGAATGATAA 616 BTK BTK
NM_000061 4557376 695 D-003107-05 GAACAGGAATGGAAGCTTA 617 BTK
NM_000061 4557376 695 D-003107-06 GCTATGGGCTGCCAAATTT 618 BTK
NM_000061 4557376 695 D-003107-07 GAAAGCAACTTACCATGGT 619 BTK
NM_000061 4557376 695 D-003107-08 GGTAAACGATCAAGGAGTT 620 C20orf64
C20orf64 NM_033550 19923655 11285 D-003108-05 CAACTTAGCCAAGACAATT
621 C20orf64 NM_033550 19923655 11285 D-003108-06
GAAATTGAAGGCTCAGTGA 622 C20orf64 NM_033550 19923655 11285
D-003108-07 TGGAACAGCTGAACATTGT 623 C20orf64 NM_033550 19923655
11285 D-003108-08 GCTTCCAACTGCTTATATA 624 CSF1R CSF1R NM_005211
27262658 1436 D-003109-05 GGAGAGCTCTGACGTTTGA 625 CSF1R NM_005211
27262658 1436 D-003109-06 CAACAACGCTACCTTCCAA 626 CSF1R NM_005211
27262658 1436 D-003109-07 CCACGCAGCTGCCTTACAA 627 CSF1R NM_005211
27262658 1436 D-003109-08 GGAACAACCTGCAGTTTGG 628 CSK CSK NM_004383
4758077 1445 D-003110-05 CAGAATGTATTGCCAAGTA 629 CSK NM_004383
4758077 1445 D-003110-06 GAACAAAGTCGCCGTCAAG 630 CSK NM_004383
4758077 1445 D-003110-07 GCGAGTGCCTTATCCAAGA 631 CSK NM_004383
4758077 1445 D-003110-08 GGAGAAGGGCTACAAGATG 632 DDR1 DDR1
NM_013994 7669484 780 D-003111-05 GGAGATGGAGTTTGAGTTT 633 DDR1
NM_013994 7669484 780 D-003111-06 CAGAGGCCCTGTCATCTTT 634 DDR1
NM_013994 7669484 780 D-003111-07 GCTGGTAGCTGTCAAGATC 635 DDR1
NM_013994 7669484 780 D-003111-08 TGAAAGAGGTGAAGATCAT 636 DDR2 DDR2
NM_006182 5453813 4921 D-003112-05 GGTAAGAACTACACAATCA 637 DDR2
NM_006182 5453813 4921 D-003112-06 GAACGAGAGTGCCACCAAT 638 DDR2
NM_006182 5453813 4921 D-003112-07 ACACCAATCTGAAGTTTAT 639 DDR2
NM_006182 5453813 4921 D-003112-08 CAACAAGAATGCCAGGAAT 640 DKFZp761
P1010 DKFZp761 NM_018423 8922178 55359 D-003113-05
CCTAGAAGCTGCCATTAAA 641 P1010 DKFZp761 NM_018423 8922178 55359
D-003113-06 GATTAGGCCTGGCTTATGA 642 P1010 DKFZp761 NM_018423
8922178 55359 D-003113-07 CCCAGTAGCTGCACACATA 643 P1010 DKFZp761
NM_018423 8922178 55359 D-003113-08 GGTGGTACCTGAACTGTAT 644 P1010
EGFR EGFR NM_005228 4885198 1956 D-003114-05 GAAGGAAACTGAATTCAAA
645 EGFR NM_005228 4885198 1956 D-003114-06 GGAAATATGTACTACGAAA 646
EGFR NM_005228 4885198 1956 D-003114-07 CCACAAAGCAGTGAATTTA 647
EGFR NM_005228 4885198 1956 D-003114-08 GTAACAAGCTCACGCAGTT 648
EPHA1 EPHA1 NM_005232 4885208 2041 D-003115-05 GACCAGAGCTTCACCATTC
649 EPHA1 NM_005232 4885208 2041 D-003115-06 GCAAGACTGTGGCCATTAA
650 EPHA1 NM_005232 4885208 2041 D-003115-07 GGGCGAACCTGACCTATGA
651 EPHA1 NM_005232 4885208 2041 D-003115-08 GATTGTAGCCGTCATCTTT
652 EPHA2 EPHA2 NM_004431 4758277 1969 D-003116-05
GGAGGGATCTGGCAACTTG 653 EPHA2 NM_004431 4758277 1969 D-003116-06
GCAGCAAGGTGCACGAATT 654 EPHA2 NM_004431 4758277 1969 D-003116-07
GGAGAAGGATGGCGAGTTC 655 EPHA2 NM_004431 4758277 1969 D-003116-08
GAAGTTCACTACCGAGATC 656 EPHA3 EPHA3 NM_005233 21361240 2042
D-003117-05 GATCGGACCTCCAGAAATA 657 EPHA3 NM_005233 21361240 2042
D-003117-06 GAACTCAGCTCAGAAGATT 658 EPHA3 NM_005233 21361240 2042
D-003117-07 GCAAGAGGCACAAATGTTA 659 EPHA3 NM_005233 21361240 2042
D-003117-08 GAGCATCAGTTTACAAAGA 660 EPHA4 EPHA4 NM_004438 4758279
2043 D-003118-05 GGTCTGGGATGAAGTATTT 661 EPHA4 NM_004438 4758279
2043 D-003118-06 GAATGAAGTTACCTTATTG 662 EPHA4 NM_004438 4758279
2043 D-003118-07 GAACTTGGGTGGATAGCAA 663 EPHA4 NM_004438 4758279
2043 D-003118-08 GAGATTAAATTCACCTTGA 664 EPHA7 EPHA7 NM_004440
4758281 2045 D-003119-05 GAAAAGAGATGTTGCAGTA 665 EPHA7 NM_004440
4758281 2045 D-003119-06 CTAGATGCCTCCTGTATTA 666 EPHA7 NM_004440
4758281 2045 D-003119-07 AGAAGAAGGTTATCGTTTA 667 EPHA7 NM_004440
4758281 2045 D-003119-08 TAGCAAAGCTGACCAAGAA 668 EPHA8 EPHA8
NM_020526 18201903 2046 D-003120-05 GAAGATGCACTATCAGAAT 669 EPHA8
NM_020526 18201903 2046 D-003120-06 GAGAAGATGCACTATCAGA 670 EPHA8
NM_020526 18201903 2046 D-003120-07 AACCTGATCTCCAGTGTGA 671 EPHA8
NM_020526 18201903 2046 D-003120-08 TCTCAGACCTGGGCTATGT 672 EPHB1
EPHB1 NM_004441 21396502 2047 D-003121-05 GCGATAAGCTCCAGCATTA 673
EPHB1 NM_004441 21396502 2047 D-003121-06 GAAACGGGCTTATAGCAAA 674
EPHB1 NM_004441 21396502 2047 D-003121-07 GGATGAAGATCTACATTGA 675
EPHB1 NM_004441 21396502 2047 D-003121-08 GCACGTCTCTGTCAACATC 676
EPHB2 EPHB2 NM_017449 17975764 2048 D-003122-05 ACTATGAGCTGCAGTACTA
677 EPHB2 NM_017449 17975764 2048 D-003122-06 GTACAACGCCACAGCCATA
678 EPHB2 NM_017449 17975764 2048 D-003122-07 GGAAAGCAATGACTGTTCT
679 EPHB2 NM_017449 17975764 2048 D-003122-08 CGGACAAGCTGCAACACTA
680 EPHB3 EPHB3 NM_004443 17975767 2049 D-003123-05
GGTGTGATCTCCAATGTGA 681 EPHB3 NM_004443 17975767 2049 D-003123-06
GGGATGACCTCCTGTACAA 682 EPHB3 NM_004443 17975767 2049 D-003123-07
CAGAAGACCTGCTCCGTAT 683 EPHB3 NM_004443 17975767 2049 D-003123-08
GAGATGAAGTACTTTGAGA 684 EPHB4 EPHB4 NM_004444 17975769 2050
D-003124-05 GGACAAACACGGACAGTAT 685 EPHB4 NM_004444 17975769 2050
D-003124-06 GTACTAAGGTCTACATCGA 686 EPHB4 NM_004444 17975769 2050
D-003124-07 GGAGAGAAGCAGAATATTC 687 EPHB4 NM_004444 17975769 2050
D-003124-08 GCCAATAGCCACTCTAACA 688 EPHB6 EPHB6 NM_004445 4758291
2051 D-003125-05 GGAAGTCGATCCTGCTTAT 689 EPHB6 NM_004445 4758291
2051 D-003125-06 GGACCAAGGTGGACACAAT 690 EPHB6 NM_004445 4758291
2051 D-003125-07 TGTGGGAAGTGATGAGTTA 691 EPHB6 NM_004445 4758291
2051 D-003125-08 CGGGAGACCTTCACCCTTT 692 ERBB2 ERBB2 NM_004448
4758297 2064 D-003126-05 GGACGAATTCTGCACAATG 693 ERBB2 NM_004448
4758297 2064 D-003126-06 GACGAATTCTGCACAATGG 694 ERBB2 NM_004448
4758297 2064 D-003126-07 CTACAACACAGACACGTTT 695 ERBB2 NM_004448
4758297 2064 D-003126-08 AGACGAAGCATACGTGATG 696 ERBB3 ERBB3
NM_001982 4503596 2065 D-003127-05 AAGAGGATGTCAACGGTTA 697 ERBB3
NM_001982 4503596 2065 D-003127-06 GAAGACTGCCAGACATTGA 698 ERBB3
NM_001982 4503596 2065 D-003127-07 GACAAACACTGGTGCTGAT 699 ERBB3
NM_001982 4503596 2065 D-003127-08 GCAGTGGATTCGAGAAGTG 700 ERBB4
ERBB4 NM_005235 4885214 2066 D-003128-05 GAGGAAAGATGCCAATTAA 701
ERBB4 NM_005235 4885214 2066 D-003128-06 GCAGGAAACATCTATATTA 702
ERBB4 NM_005235 4885214 2066 D-003128-07 GATCACAACTGCTGCTTAA 703
ERBB4 NM_005235 4885214 2066 D-003128-08 CCTCAAAGATACCTAGTTA 704
FER FER NM_005246 4885230 2241 D-003129-05 GGAGTGACCTGAAGAATTC 705
FER NM_005246 4885230 2241 D-003129-06 TAAAGCAGATTCCCATTAA 706 FER
NM_005246 4885230 2241 D-003129-07 GGAAAGTACTGTCCAAATG 707 FER
NM_005246 4885230 2241 D-003129-08 GAACAACGGCTGCTAAAGA 708 FES FES
NM_002005 13376997 2242 D-003130-05 CGAGGATCCTGAAGCAGTA 709 FES
NM_002005 13376997 2242 D-003130-06 AGGAATACCTGGAGATTAG 710 FES
NM_002005 13376997 2242 D-003130-07 CAACAGGAGCTCCGGAATG 711 FES
NM_002005 13376997 2242 D-003130-08 GGTGTTGGGTGAGCAGATT 712 FGFR1
FGFR1 NM_000604 13186232 2260 D-003131-05 TAAGAAATGTCTCCTTTGA 713
FGFR1 NM_000604 13186232 2260 D-003131-06 GAAGACTGCTGGAGTTAAT 714
FGFR1 NM_000604 13186232 2260 D-003131-07 GATGGTCCCTTGTATGTCA 715
FGFR1 NM_000604 13186232 2260 D-003131-08 CTTAAGAAATGTCTCCTTT 716
FGFR2 FGFR2 NM_000141 13186239 2263 D-003132-05 CCAAATCTCTCAACCAGAA
717 FGFR2 NM_000141 13186239 2263 D-003132-06 GAACAGTATTCACCTAGTT
718 FGFR2 NM_000141 13186239 2263 D-003132-07 GGCCAACACTGTCAAGTTT
719 FGFR2 NM_000141 13186239 2263 D-003132-08 GTGAAGATGTTGAAAGATG
720 FGFR3 FGFR3 NM_000142 13112046 2261 D-003133-05
TGTCGGACCTGGTGTCTGA 721 FGFR3 NM_000142 13112046 2261 D-003133-06
GCATCAAGCTGCGGCATCA 722 FGFR3 NM_000142 13112046 2261 D-003133-07
GGACGGCACACCCTACGTT 723 FGFR3 NM_000142 13112046 2261 D-003133-08
TGCACAACCTCGACTACTA 724 FGFR4 FGFR4 NM_002011 13112051 2264
D-003134-05 GCACTGGAGTCTCGTGATG 725 FGFR4 NM_002011 13112051 2264
D-003134-06 CATAGGGACCTCTCGAATA 726 FGFR4 NM_002011 13112051 2264
D-003134-07 ATACGGACATCATCCTGTA 727 FGFR4 NM_002011 13112051 2264
D-003134-08 ATAGGGACCTCTCGAATAG 728 FGR FGR NM_005248 4885234 2268
D-003135-05 GCGATCATGTGAAGCATTA 729 FGR NM_005248 4885234 2268
D-003135-06 TCACTGAGCTCATCACCAA 730 FGR NM_005248 4885234 2268
D-003135-07 GAAGAGTGGTACTTTGGAA 731 FGR NM_005248 4885234 2268
D-003135-08 CCCAGAAGCTGCCCTCTTT 732 FLT1 FLT1 NM_002019 4503748
2321 D-003136-05 GAGCAAACGTGACTTATTT 733 FLT1 NM_002019 4503748
2321 D-003136-06 CCAAATGGGTTTCATGTTA 734 FLT1 NM_002019 4503748
2321 D-003136-07 CAACAAGGATGCAGCACTA 735 FLT1 NM_002019 4503748
2321 D-003136-08 GGACGTAACTGAAGAGGAT 736 FLT3 FLT3 NM_004119
4758395 2322 D-003137-05 GAAGGCATCTACACCATTA 737 FLT3 NM_004119
4758395 2322 D-003137-06 GAAGGAGTCTGGAATAGAA 738 FLT3 NM_004119
4758395 2322 D-003137-07 GAATTTAAGTCGTGTGTTC 739 FLT3 NM_004119
4758395 2322 D-003137-08 GGAATTCATTTCACTCTGA 740 FLT4 FLT4
NM_002020 4503752 2324 D-003138-05 GCAAGAACGTGCATCTGTT 741 FLT4
NM_002020 4503752 2324 D-003138-06 GCGAATACCTGTCCTACGA 742 FLT4
NM_002020 4503752 2324 D-003138-07 GAAGACATTTGAGGAATTC 743 FLT4
NM_002020 4503752 2324 D-003138-08 GAGCAGCCATTCATCAACA 744 FRK FRK
NM_002031 4503786 2444 D-003139-05 GAAACAGACTCTTCATATT 745 FRK
NM_002031 4503786 2444 D-003139-06 GAACAATACCACTCCAGTA 746 FRK
NM_002031 4503786 2444 D-003139-07 CAAGACCGGTTCCTTTCTA 747 FRK
NM_002031 4503786 2444 D-003139-08 GCAAGAATATCTCCAAAAT 748 FYN FYN
NM_002037 23510344 2534 D-003140-05 GGAATGGACTCATATGCAA 749 FYN
NM_002037 23510344 2534 D-003140-06 GCAGAAGAGTGGTACTTTG 750 FYN
NM_002037 23510344 2534 D-003140-07 CAAAGGAAG1TTACTGGAT 751 FYN
NM_002037 23510344 2534 D-003140-08 GAAGAGTGGTACTTTGGAA 752 HCK HCK
NM_002110 4504356 3055 D-003141-05 GAGATACCGTGAAACATTA 753 HCK
NM_002110 4504356 3055 D-003141-06 GCAGGGAGATACCGTGAAA 754 HCK
NM_002110 4504356 3055 D-003141-07 CATCGTGGTTGCCCTGTAT 755 HCK
NM_002110 4504356 3055 D-003141-08 TGTGTAAGATTGCTGACTT 756 ITK ITK
NM_005546 21614549 3702 D-003144-05 CAAATAATCTGGAAACCTA 757 ITK
NM_005546 21614549 3702 D-003144-06 GAAGAAACGAGGAATAATA 758 ITK
NM_005546 21614549 3702 D-003144-07 GAAACTCTCTCATCCCAAA 759 ITK
NM_005546 21614549 3702 D-003144-08 GGAATGGGCATGAAGGATA 760 JAK1
JAK1 NM_002227 4504802 3716 D-003145-05 CCACATAGCTGATCTGAAA 761
JAK1 NM_002227 4504802 3716 D-003145-06 TGAAATCACTCACATTGTA 762
JAK1 NM_002227 4504802 3716 D-003145-07 TAAGGAACCTCTATCATGA 763
JAK1 NM_002227 4504802 3716 D-003145-08 GCAGGTGGCTGTTAAATCT 764
JAK2 JAK2 NM_004972 13325062 3717 D-003146-05 GCAAATAGATCCAGTTCTT
765 JAK2 NM_004972 13325062 3717 D-003146-06 GAGCAAAGATCCAAGACTA
766 JAK2 NM_004972 13325062 3717 D-003146-07 GCCAGAAACTTGAAACTTA
767 JAK2 NM_004972 13325062 3717 D-003146-08 GTACAGATTTCGCAGATTT
768 JAK3 JAK3 NM_000215 4557680 3718 D-003147-05
GCGCCTATCTTTCTCCTTT 769 JAK3 NM_000215 4557680 3718 D-003147-06
CCAGAAATCGTAGACATTA 770 JAK3 NM_000215 4557680 3718 D-003147-07
CCTCATCTCTTCAGACTAT 771 JAK3 NM_000215 4557680 3718 D-003147-08
TGTACGAGCTCTTCACCTA 772 KDR KDR NM_002253 11321596 3791 D-003148-05
GGAAATCTCTTGCAAGCTA 773 KDR NM_002253 11321596 3791 D-003148-06
GATTACAGATCTCCATTTA 774 KDR NM_002253 11321596 3791 D-003148-07
GCAGACAGATCTACGTTTG 775 KDR NM_002253 11321596 3791 D-003148-08
GCGATGGCCTCTTCTGTAA 776 KIAA1079 KIAA1079 NM_014916 7662475 22853
D-003149-05 GAAATTCTCTCAACTGATG 777 KIAA1079 NM_014916 7662475
22853 D-003149-06 GCAGAGGTCTTCACACTTT 778 KIAA1079 NM_014916
7662475 22853 D-003149-07 TAAATGATCTTCAGACAGA 779 KIAA1079
NM_014916 7662475 22853 D-003149-08 GAGCAGCCCTACTCTGATA 780 KIT KIT
NM_000222 4557694 3815 D-003150-05 AAACACGGCTTAAGCAATT 781 KIT
NM_000222 4557694 3815 D-003150-06 GAACAGAACCTTCACTGAT 782 KIT
NM_000222 4557694 3815 D-003150-07 GGGAAGCCCTCATGTCTGA 783 KIT
NM_000222 4557694 3815 D-003150-08 GCAATTCCATTTATGTGTT 784 LCK LCK
NM_005356 20428651 3932 D-003151-05 GAACTGCCATTATCCCATA 785 LCK
NM_005356 20428651 3932 D-003151-06 GAGAGGTGGTGAAACATTA 786 LCK
NM_005356 20428651 3932 D-003151-07 GGGCCAAGTTTCCCATTAA 787 LCK
NM_005356 20428651 3932 D-003151-08 GCACGCTGCTCATCCGAAA 788 LTK LTK
NM_002344 4505044 4058 D-003152-05 TGAATTCACTCCTGCCAAT
789 LTK NM_002344 4505044 4058 D-003152-06 GTGGCAACCTCAACACTGA 790
LTK NM_002344 4505044 4058 D-003152-07 GGAGCTAGCTGTGGATAAC 791 LTK
NM_002344 4505044 4058 D-003152-08 GCAAGTTTCGCCATCAGAA 792 LYN LYN
NM_002350 4505054 4067 D-003153-05 GCAGATGGCTTGTGCAGAA 793 LYN
NM_002350 4505054 4067 D-003153-06 GGAGAAGGCTTGTATTAGT 794 LYN
NM_002350 4505054 4067 D-003153-07 GATGAGCTCTATGACATTA 795 LYN
NM_002350 4505054 4067 D-003153-08 GGTGCTAAGTTCCCTATTA 796 MATK
MATK NM_002378 21450841 4145 D-003154-05 TGAAGAATATCAAGTGTGA 797
MATK NM_002378 21450841 4145 D-003154-06 CCGCTCAGCTCCTGCAGTT 798
MATK NM_002378 21450841 4145 D-003154-07 TACTGAACCTGCAGCATTT 799
MATK NM_002378 21450841 4145 D-003154-08 TGGGAGGTCTTCTCATATG 800
MERTK MERTK NM_006343 5453737 10461 D-003155-05 GAACTTACCTTACATAGCT
801 MERTK NM_006343 5453737 10461 D-003155-06 GGACCTGCATACTTACTTA
802 MERTK NM_006343 5453737 10461 D-003155-07 TGACAGGAATCTTCTAATT
803 MERTK NM_006343 5453737 10461 D-003155-08 GGTAATGGCTCAGTCATGA
804 MET MET NM_000245 4557746 4233 D-003156-05 GAAAGAACCTCTCAACATT
805 MET NM_000245 4557746 4233 D-003156-06 GGACAAGGCTGACCATATG 806
MET NM_000245 4557746 4233 D-003156-07 CCAATGACCTGCTGAAATT 807 MET
NM_000245 4557746 4233 D-003156-08 GAGCATACATTAAACCAAA 808 MST1R
MST1R NM_002447 4505264 4486 D-003157-05 GGATGGAGCTGCTGGCTTT 809
MST1R NM_002447 4505264 4486 D-003157-06 CTGCAGACCTATAGATTTA 810
MST1R NM_002447 4505264 4486 D-003157-07 GCACCTGTCTCACTCTTGA 811
MST1R NM_002447 4505264 4486 D-003157-08 GAAAGAGTCCATCCAGCTA 812
MUSK MUSK NM_005592 5031926 4593 D-003158-05 GAAGAAGCCTCGGCAGATA
813 MUSK NM_005592 5031926 4593 D-003158-06 GTAATAATCTCCATCATGT 814
MUSK NM_005592 5031926 4593 D-003158-07 GGAATGAACTGAAAGTAGT 815
MUSK NM_005592 5031926 4593 D-003158-08 GAGATTTCCTGGACTAGAA 816
NTRK1 NTRK1 NM_002529 4585711 4914 D-003159-05 GGACAACCCTTTCGAGTTC
817 NTRK1 NM_002529 4585711 4914 D-003159-06 CCAGTGACCTCAACAGGAA
818 NTRK1 NM_002529 4585711 4914 D-003159-07 CCACAATACTTCAGTGATG
819 NTRK1 NM_002529 4585711 4914 D-003159-08 GAAGAGTGGTCTCCGTTTC
820 NTRK2 NTRK2 NM_006180 21361305 4915 D-00316D-05
GAACAGAAGTAATGAAATC 821 NTRK2 NM_006180 21361305 4915 D-00316D-06
GTAATGCTGTTTCTGCTTA 822 NTRK2 NM_006180 21361305 4915 D-00316D-07
GCAAGACACTCCAAGTTTG 823 NTRK2 NM_006180 21361305 4915 D-00316D-08
GAAAGTCTATCACATTATC 824 NTRK3 NTRK3 NM_002530 4505474 4916
D-003161-05 GAGCGAATCTGCTAGTGAA 825 NTRK3 NM_002530 4505474 4916
D-003161-06 GAAGTTCACTACAGAGAGT 826 NTRK3 NM_002530 4505474 4916
D-003161-07 GGTCGACGGTCCAAATTTG 827 NTRK3 NM_002530 4505474 4916
D-003161-08 GAATATCACTTCCATACAC 828 PDGFRA PDGFRA NM_006206
15451787 5156 D-003162-05 GAAACTTCCTGGACTATTT 829 PDGFRA NM_006206
15451787 5156 D-003162-06 GAGATTTGGTCAACTATTT 830 PDGFRA NM_006206
15451787 5156 D-003162-07 GCACGCCGCTTCCTGATAT 831 PDGFRA NM_006206
15451787 5156 D-003162-08 CATCAGAGCTGGATCTAGA 832 PDGFRB PDGFRB
NM_002609 15451788 5159 D-003163-05 GAAAGGAGACGTCAAATAT 833 PDGFRB
NM_002609 15451788 5159 D-003163-06 GGAATGAGGTGGTCAACTT 834 PDGFRB
NM_002609 15451788 5159 D-003163-07 CAACGAGTCTCCAGTGCTA 835 PDGFRB
NM_002609 15451788 5159 D-003163-08 GAGAGGACCTGCCGAGCAA 836 PTK2
PTK2 NM_005607 27886592 5747 D-003164-05 GAAGTTGGGTTGTCTAGAA 837
PTK2 NM_005607 27886592 5747 D-003164-06 GAAGAACAATGATGTAATC 838
PTK2 NM_005607 27886592 5747 D-003164-07 GGAAATTGCTTTGAAGTTG 839
PTK2 NM_005607 27886592 5747 D-003164-08 GGTTCAAGCTGGATTATTT 840
PTK2B PTK2B NM_004103 27886583 2185 D-003165-05 GAACATGGCTGACCTCATA
841 PTK2B NM_004103 27886583 2185 D-003165-06 GGACCACGCTGCTCTATTT
842 PTK2B NM_004103 27886583 2185 D-003165-07 GGACGAGGACTATTACAAA
843 PTK2B NM_004103 27886583 2185 D-003165-08 TGGCAGAGCTCATCAACAA
844 PTK6 PTK6 NM_005975 27886594 5753 D-003166-05
GAGAAAGTCCTGCCCGTTT 845 PTK6 NM_005975 27886594 5753 D-003166-06
TGAAGAAGCTGCGGCACAA 846 PTK6 NM_005975 27886594 5753 D-003166-07
CCGCGACTCTGATGAGAAA 847 PTK6 NM_005975 27886594 5753 D-003166-08
TGCCCGAGCTTGTGAACTA 848 PTK7 PTK7 NM_002821 27886610 5754
D-003167-05 GAGAGAAGCCCACTATTAA 849 PTK7 NM_002821 27886610 5754
D-003167-06 CGAGAGAAGCCCACTATTA 850 PTK7 NM_002821 27886610 5754
D-003167-07 GGAGGGAGTTGGAGATGTT 851 PTK7 NM_002821 27886610 5754
D-003167-08 GAAGACATGCCGCTATTTG 852 PTK9 PTK9 NM_002822 4506274
5756 D-003168-05 GAAGAACTACGACAGATTA 853 PTK9 NM_002822 4506274
5756 D-003168-09 GAAGGAGACTATTTAGAGT 854 PTK9 NM_002822 4506274
5756 D-003168-10 GAGCGGATGCTGTATTCTA 855 PTK9 NM_002822 4506274
5756 D-003168-11 CTGCAGACTTCCTTTATGA 856 PTK9L PTK9L NM_007284
31543446 11344 D-003169-05 AGAGAGAGCTCCAGCAGAT 857 PTK9L NM_007284
31543446 11344 D-003169-06 TTAACGAGGTGAAGACAGA 858 PTK9L NM_007284
31543446 11344 D-003169-07 ACACAGAGCCCACGGATGT 859 PTK9L NM_007284
31543446 11344 D-003169-08 GCTGGGATCAGGACTATGA 860 RET RET
NM_000323 21536316 5979 D-003170-05 GCAAAGACCTGGAGAAGAT 861 RET
NM_000323 21536316 5979 D-003170-06 GCACACGGCTGCATGAGAA 862 RET
NM_000323 21536316 5979 D-003170-07 GAACTGGCCTGGAGAGAGT 863 RET
NM_000323 21536316 5979 D-003170-08 TTAAATGGATGGCAATTGA 864 ROR1
ROR1 NM_005012 4826867 4919 D-003171-05 GCAAGCATCTTTACTAGGA 865
ROR1 NM_005012 4826867 4919 D-003171-06 GAGCAAGGCTAAAGAGCTA 866
ROR1 NM_005012 4826867 4919 D-003171-07 GAGAGCAACTTCATGTAAA 867
ROR1 NM_005012 4826867 4919 D-003171-08 GAGAATGTCCTGTGTCAAA 868
ROR2 ROR2 NM_004560 19743897 4920 D-003172-05 GGAACTCGCTGCTGCCTAT
869 ROR2 NM_004560 19743897 4920 D-003172-06 GCAGGTGCCTCCTCAGATG
870 ROR2 NM_004560 19743897 4920 D-003172-07 GCAATGTGCTAGTGTACGA
871 ROR2 NM_004560 19743897 4920 D-003172-08 GAAGACAGAATATGGTTCA
872 ROS1 ROS1 NM_002944 19924164 6098 D-003173-05
GAGGAGACCTTCTTACTTA 873 ROS1 NM_002944 19924164 6098 D-003173-06
TTACAGAGGTTCAGGATTA 874 ROS1 NM_002944 19924164 6098 D-003173-07
GAACAAACCTAAGCATGAA 875 ROS1 NM_002944 19924164 6098 D-003173-08
GAAAGAGCACTTCAAATAA 876 RYK RYK NM_002958 11863158 6259 D-003174-05
GAAAGATGGTTACCGAATA 877 RYK NM_002958 11863158 6259 D-003174-06
CAAAGTAGATTCTGAAGTT 878 RYK NM_002958 11863158 6259 D-003174-07
TCACTACGCTCTATCCTTT 879 RYK NM_002958 11863158 6259 D-003174-08
GGTGAAGGATATAGCAATA 880 SRC SRC NM_005417 21361210 6714 D-003175-05
GAGAACCTGGTGTGCAAAG 881 SRC NM_005417 21361210 6714 D-003175-09
GAGAGAACCTGGTGTGCAA 882 SRC NM_005417 21361210 6714 D-003175-10
GGAGTTTGCTGGACTTTCT 883 SRC NM_005417 21361210 6714 D-003175-11
GAAAGTGAGACCACGAAAG 884 SYK SYK NM_003177 21361552 6850 D-003176-05
GGAATAATCTCAAGAATCA 885 SYK NM_003177 21361552 6850 D-003176-06
GAACTGGGCTCTGGTAATT 886 SYK NM_003177 21361552 6850 D-003176-07
GGAAGAATCTGAGCAAATT 887 SYK NM_003177 21361552 6850 D-003176-08
GAACAGACATGTCAAGGAT 888 TEC TEC NM_003215 4507428 7006 D-003177-05
GAAATTGTCTAGTAAGTGA 889 TEC NM_003215 4507428 7006 D-003177-06
CACCTGAAGTGTTTAATTA 890 TEC NM_003215 4507428 7006 D-003177-07
GTACAAAGTCGCAATCAAA 891 TEC NM_003215 4507428 7006 D-003177-08
TGGAGGAGATTCTTATTAA 892 TEK TEK NM_000459 4557868 7010 D-003178-05
GAAAGAATATGCCTCCAAA 893 TEK NM_000459 4557868 7010 D-003178-06
GGAATGACATCAAATTTCA 894 TEK NM_000459 4557868 7010 D-003178-07
TGAAGTACCTGATATTCTA 895 TEK NM_000459 4557868 7010 D-003178-08
CGAAAGACCTACGTGAATA 896 TIE TIE NM_005424 4885630 7075 D-003179-05
GAGAGGAGGTTTATGTGAA 897 TIE NM_005424 4885630 7075 D-003179-06
GGGACAGCCTCTACCCTTA 898 TIE NM_005424 4885630 7075 D-003179-07
GAAGTTCTGTGCAAATTGG 899 TIE NM_005424 4885630 7075 D-003179-08
CAACATGGCCTCAGAACTG 900 TNK1 TNK1 NM_003985 4507610 8711
D-003180-05 GTTCTGGGCCTAAGTCTAA 901 TNK1 NM_003985 4507610 8711
D-003180-06 GAACTGGGTCTACAAGATC 902 TNK1 NM_003985 4507610 8711
D-003180-07 CGAGAGGTATCGGTCATGA 903 TNK1 NM_003985 4507610 8711
D-003180-08 GGCGCATCCTGGAGCATTA 904 TXK TXK NM_003328 4507742 7294
D-003181-05 GAACATCTATTGAGACAAG 905 TXK NM_003328 4507742 7294
D-003181-06 TCAAGGCACTTTATGATTT 906 TXK NM_003328 4507742 7294
D-003181-07 GGAGAGGAATGGCTATATT 907 TXK NM_003328 4507742 7294
D-003181-08 GGATATATGTGAAGGAATG 908 TYK2 TYK2 NM_003331 4507748
7297 D-003182-05 GAGGAGATCCACCACTTTA 909 TYK2 NM_003331 4507748
7297 D-003182-06 GCATCCACATTGCACATAA 910 TYK2 NM_003331 4507748
7297 D-003182-07 TCAAATACCTAGCCACACT 911 TYK2 NM_003331 4507748
7297 D-003182-08 CAATCTTGCTGACGTCTTG 912 TYRO3 TYRO3 NM_006293
27597077 7301 D-003183-05 GGTAGAAGGTGTGCCATTT 913 TYRO3 NM_006293
27597077 7301 D-003183-06 ACGCTGAGATTTACAACTA 914 TYRO3 NM_006293
27597077 7301 D-003183-07 GGATGGCTCCTTTGTGAAA 915 TYRO3 NM_006293
27597077 7301 D-003183-08 GAGAGGAACTACGAAGATC 916 YES1 YES1
NM_005433 21071041 7525 D-003184-05 GAAGGACCCTGATGAAAGA 917 YES1
NM_005433 21071041 7525 D-003184-06 TAAGAAGGGTGAAAGATTT 918 YES1
NM_005433 21071041 7525 D-003184-07 TCAAGAAGCTCAGATAATG 919 YES1
NM_005433 21071041 7525 D-003184-08 CAGAATCCCTCCATGAATT 920
[0614]
13TABLE VIII Gene Locus SEQ. ID Name Acc# Gl Link Duplex # Full
Sequence NO. APC2 APC2 NM_013366 7549800 29882 D-003200-05
GCAAGGACCTCTTCATCAA 921 APC2 NM_013366 7549800 29882 D-003200-06
GAGAAGAAGTCCACACTAT 922 APC2 NM_013366 7549800 29882 D-003200-07
GGAATGCCATCTCCCAATG 923 APC2 NM_013366 7549800 29882 D-003200-09
CAACACGTGTGACATCATC 924 ATM ATM NM_000051 20336202 472 D-003201-05
GCAAGCAGCTGAAACAAAT 925 ATM NM_000051 20336202 472 D-003201-06
GAATGTTGCTTTCTGAATT 926 ATM NM_000051 20336202 472 D-003201-07
GACCTGAAGTCTTATTTAA 927 ATM NM_000051 20336202 472 D-003201-08
AGACAGAATTCCCAAATAA 928 ATR ATR NM_001184 20143978 545 D-003202-05
GAACAACACTGCTGGTTTG 929 ATR NM_001184 20143978 545 D-003202-06
GAAGTCATCTGTTCATTAT 930 ATR NM_001184 20143978 545 D-003202-07
GAAATAAGGTAGACTCAAT 931 ATR NM_001184 20143978 545 D-003202-08
CAACATAAATCCAAGAAGA 932 BTAK BTAK NM_003600 3213196 6790
D-003545-04 CAAAGAATCAGCTAGCAAA 933 BTAK NM_003600 3213196 6790
D-003545-05 GAAGAGAGTTATTCATAGA 934 BTAK NM_003600 3213196 6790
D-003545-07 CAAATGCCCTGTCTTACTG 935 BTAK NM_003600 3213196 6790
D-003545-09 TCTCGTGACTCAGCAAATT 936 CCNA1 CCNA1 NM_003914 16306528
890 D-003204-05 GAACCTGGCTAAGTACGTA 937 CCNA1 NM_003914 16306528
890 D-003204-06 GCAGATCCATTCTTGAAAT 938 CCNA1 NM_003914 16306528
890 D-003204-07 TCACAAGAATCAGGTGTTA 939 CCNA1 NM_003914 16306528
890 D-003204-08 CATAAAGCGTACCTTGATA 940 CCNA2 CCNA2 NM_001237
16950653 890 D-003205-05 GCTGTGAACTACATTGATA 941 CCNA2 NM_001237
16950653 890 D-003205-06 GATGATACCTACACCAAGA 942 CCNA2 NM_001237
16950653 890 D-003205-07 GCTGTTAGCCTCAAAGTTT 943 CCNA2 NM_001237
16950653 890 D-003205-08 AAGCTGGCCTGAATCATTA 944 CCNB1 CCNB1
NM_031966 14327895 891 D-003206-05 CAACATTACCTGTCATATA 945 CCNB1
NM_031966 14327895 891 D-003206-06 CCAAATACCTGATGGAACT 946 CCNB1
NM_031966 14327895 891 D-003206-07 GAAATGTACCCTCCAGAAA 947 CCNB1
NM_031966 14327895 891 D-003206-08 GCACCTGGCTAAGAATGTA 948 CCNB2
CCNB2 NM_004701 10938017 9133 D-003207-05 CAACAAATGTCAACAAACA 949
CCNB2 NM_004701 10938017 9133 D-003207-06 GCAGCAAACTCCTGAAGAT 950
CCNB2 NM_004701 10938017 9133 D-003207-07 CCAGTGATTTGGAGAATAT 951
CCNB2 NM_004701 10938017 9133 D-003207-08 GTGACTACGTTAAGGATAT 952
CCNB3 CCNB3 NM_033031 14719419 85417 D-003208-05
TGAACAAACTGCTGACTTT 953 CCNB3 NM_033031 14719419 85417 D-003208-06
GCTAGCTGCTGCCTCCTTA 954 CCNB3 NM_033031 14719419 85417 D-003208-07
CAACTCACCTCGTGTGGAT 955 CCNB3 NM_033031 14719419 85417 D-003208-08
GTGGATCTCTACCTAATGA 956 CCNC CCNC NM_005190 7382485 892 D-003209-05
GCAGAGCTCCCACTATTTG 957 CCNC NM_005190 7382485 892 D-003209-06
GGAGTAGTTTCAAATACAA 958 CCNC NM_005190 7382485 892 D-003209-07
GACCTTTGCTCCAGTATGT 959 CCNC NM_005190 7382485 892 D-003209-08
GAGATTCTATGCCAGGTAT 960 CCND1 CCND1 NM_053056 16950654 595
D-003210-05 TGAACAAGCTCAAGTGGAA 961 CCND1 NM_053056 16950654 595
D-003210-06 CCAGAGTGATCAAGTGTGA 962 CCND1 NM_053056 16950654 595
D-003210-07 GTTCGTGGCCTCTAAGATG 963 CCND1 NM_053056 16950654 595
D-003210-08 CCGAGAAGCTGTGCATCTA 964 CCND2 CCND2 NM_001759 16950656
894 D-003211-06 TGAATTACCTGGACCGTTT 965 CCND2 NM_001759 16950656
894 D-003211-07 CGGAGAAGCTGTGCATTTA 966 CCND2 NM_001759 16950656
894 D-003211-08 CTACAGACGTGCGGGATAT 967 CCND2 NM_001759 16950656
894 D-003211-09 CAACACAGACGTGGATTGT 968 CCND3 CCND3 NM_001760
16950657 896 D-003212-05 GGACCTGGCTGCTGTGATT 969 CCND3 NM_001760
16950657 896 D-003212-06 GATTATACCTTTGCCATGT 970 CCND3 NM_001760
16950657 896 D-003212-07 GACCAGCACTCCTACAGAT 971 CCND3 NM_001760
16950657 896 D-003212-08 TGCGGAAGATGCTGGCTTA 972 CCNE1 CCNE1
NM_001238 17318558 898 D-003213-05 GTACTGAGCTGGGCAAATA 973 CCNE1
NM_001238 17318558 898 D-003213-06 GGAAATCTATCCTCCAAAG 974 CCNE1
NM_001238 17318558 898 D-003213-07 GGAGGTGTGTGAAGTCTAT 975 CCNE1
NM_001238 17318558 898 D-003213-08 CTAAATGACTTACATGAAG 976 CCNE2
CCNE2 NM_057749 17318564 9134 D-003214-05 GGATGGAACTCATTATATT 977
CCNE2 NM_057749 17318564 9134 D-003214-06 GCAGATATGTTCATGACAA 978
CCNE2 NM_057749 17318564 9134 D-003214-07 CATAATATCCAGACACATA 979
CCNE2 NM_057749 17318564 9134 D-003214-08 TAAGAAAGCCTCAGGTTTG 980
CCNF CCNF NM_001761 4502620 899 D-003215-05 TCACAAAGCATCCATATTG 981
CCNF NM_001761 4502620 899 D-003215-06 GAAGTCATGTTTACAGTGT 982 CCNF
NM_001761 4502620 899 D-003215-07 TAGCCTACCTCTACAATGA 983 CCNF
NM_001761 4502620 899 D-003215-08 GCACCCGGTTTATCAGTAA 984 CCNG1
CCNG1 NM_004060 8670528 900 D-003216-05 GATAATGGCCTCAGAATGA 985
CCNG1 NM_004060 8670528 900 D-003216-06 GCACGGCAATTGAAGCATA 986
CCNG1 NM_004060 8670528 900 D-003216-07 GGAATAGAATGTCTTCAGA 987
CCNG1 NM_004060 8670528 900 D-003216-08 TAACTCACCTTCCAACAAT 988
CCNG2 CCNG2 NM_004354 4757935 901 D-003217-05 GGAGAGAGTTGGTTTCTAA
989 CCNG2 NM_004354 4757935 901 D-003217-06 GGTGAAACCTAAACATTTG 990
CCNG2 NM_004354 4757935 901 D-003217-07 GAAATACTGAGCCTTGATA 991
CCNG2 NM_004354 4757935 901 D-003217-08 TGCCAAAGTTGAAGATTTA 992
CCNH CCNH NM_001239 17738313 902 D-003218-05 GCTGATGACTTTCTTAATA
993 CCNH NM_001239 17738313 902 D-003218-06 CAACTTAATTTCCACCTTA 994
CCNH NM_001239 17738313 902 D-003218-07 ATACACACCTTCCCAAATT 995
CCNH NM_001239 17738313 902 D-003218-08 GCTATGAAGATGATGATTA 996
CCNI CCNI NM_006835 17738314 10983 D-003219-05 GCAAGCAGACCTCTACTAA
997 CCNI NM_006835 17738314 10983 D-003219-07 TGAGAGAATTCCAGTACTA
998 CCNI NM_006835 17738314 10983 D-003219-08 GGAATCAAACGGCTCTATA
999 CCNI NM_006835 17738314 10983 D-003219-09 GAATTGGGATCTTCACACA
1000 CCNT1 CCNT1 NM_001240 17978465 904 D-003220-05
TATCAACACTGCTATAGTA 1001 CCNT1 NM_001240 17978465 904 D-003220-06
GAACAAACGTCCTGGTGAT 1002 CCNT1 NM_001240 17978465 904 D-003220-07
GCACAAGACTCACCCATCT 1003 CCNT1 NM_001240 17978465 904 D-003220-08
GCACAGACTTCTTACTTCA 1004 CCNT2A CCNT2A NM_001241 17978467 905
D-003221-05 GCACAGACATCCTATTTCA 1005 CCNT2A NM_001241 17978467 905
D-003221-06 GCAGGGACCTTCTATATCA 1006 CCNT2A NM_001241 17978467 905
D-003221-07 GAACAGCTATATTCACAGA 1007 CCNT2A NM_001241 17978467 905
D-003221-09 TTATATAGCTGCCCAGGTA 1008 CCNT2B CCNT2B NM_058241
17978468 905 D-003222-05 GCACAGACATCCTATTTCA 1009 CCNT2B NM_058241
17978468 905 D-003222-06 GCAGGGACCTTCTATATCA 1010 CCNT2B NM_058241
17978468 905 D-003222-07 GAACAGCTATATTCACAGA 1011 CCNT2B NM_058241
17978468 905 D-003222-08 GGTGAAATGTACCCAGTTA 1012 CDC16 CDC16
NM_003903 14110370 8881 D-003223-05 GTAGATGGCTTGCAAGAGA 1013 CDC16
NM_003903 14110370 8881 D-003223-06 TAAAGTAGCTTCACTCTCT 1014 CDC16
NM_003903 14110370 8881 D-003223-07 GCTACAAGCTTACTTCTGT 1015 CDC16
NM_003903 14110370 8881 D-003223-08 TGGAAGAGCCCATCAATAA 1016 CDC2
CDC2 NM_033379 27886643 983 D-003552-01 GTACAGATCTCCAGAAGTA 1017
CDC2 NM_033379 27886643 983 D-003552-02 GATCAACTCTTCAGGATTT 1018
CDC2 NM_033379 27886643 983 D-003552-03 GGTTATATCTCATCTTTGA 1019
CDC2 NM_033379 27886643 983 D-003552-04 GAACTTCGTCATCCAAATA 1020
CDC20 CDC20 NM_001255 4557436 991 D-003225-05 GGGAATATATATCCTCTGT
1021 CDC20 NM_001255 4557436 991 D-003225-06 GAAACGGCTTCGAAATATG
1022 CDC20 NM_001255 4557436 991 D-003225-07 GAAGACCTGCCGTTACATT
1023 CDC20 NM_001255 4557436 991 D-003225-08 CACCAGTGATCGACACATT
1024 CDC25A CDC25A NM_001789 4502704 993 D-003226-05
GAAATTATGGCATCTGTTT 1025 CDC25A NM_001789 4502704 993 D-003226-06
TACAAGGAGTTCTTTATGA 1026 CDC25A NM_001789 4502704 993 D-003226-07
CCACGAGGACTTTAAAGAA 1027 CDC25A NM_001789 4502704 993 D-003226-08
TGGGAAACATCAGGATTTA 1028 CDC25B CDC25B NM_004358 11641416 994
D-003227-05 GCAGATACCCCTATGAATA 1029 CDC25B NM_004358 11641416 994
D-003227-06 CTAGGTCGCTTCTCTCTGA 1030 CDC25B NM_004358 11641416 994
D-003227-07 GAGAGCTGATTGGAGATTA 1031 CDC25B NM_004358 11641416 994
D-003227-08 AAAAGGACCTCGTCATGTA 1032 CDC25C CDC25C NM_001790
12408659 995 D-003228-05 GAGCAGAAGTGGCCTATAT 1033 CDC25C NM_001790
12408659 995 D-003228-06 CAGAAGAGATTTCAGATGA 1034 CDC25C NM_001790
12408659 995 D-003228-07 CCAGGGAGCCTTAAACTTA 1035 CDC25C NM_001790
12408659 995 D-003228-08 GAAACTTGGTGGACAGTGA 1036 CDC27 CDC27
NM_001256 16554576 996 D-003229-06 CATGCAAGCTGAAAGAATA 1037 CDC27
NM_001256 16554576 996 D-003229-07 CAACACAAGTACCTAATCA 1038 CDC27
NM_001256 16554576 996 D-003229-08 GGAGATGGATCCTAGTTAC 1039 CDC27
NM_001256 16554576 996 D-003229-09 GAAAAGCCATGATGATATT 1040 CDC34
CDC34 NM_004359 16357476 997 D-003230-05 GCTCAGACCTCTTCTACGA 1041
CDC34 NM_004359 16357476 997 D-003230-06 GGACGAGGGCGATCTATAC 1042
CDC34 NM_004359 16357476 997 D-003230-07 GATCGGGAGTACACAGACA 1043
CDC34 NM_004359 16357476 997 D-003230-08 TGAACGAGCCCAACACCTT 1044
CDC37 CDC37 NM_007065 16357478 11140 D-003231-05
GCGAGGAGACAGCCAATTA 1045 CDC37 NM_007065 16357478 11140 D-003231-06
CACAAGACCTTCGTGGAAA 1046 CDC37 NM_007065 16357478 11140 D-003231-07
ACAATCGTCATGCAATTTA 1047 CDC37 NM_007065 16357478 11140 D-003231-08
GAGGAGAAATGTGCACTCA 1048 CDC45L CDC45L NM_003504 34335230 8318
D-003232-05 GCACACGGATCTCCTTTGA 1049 CDC45L NM_003504 34335230 8318
D-003232-06 GCAAACACCTGCTCAAGTC 1050 CDC45L NM_003504 34335230 8318
D-003232-07 TGAAGAGTCTGCAAATAAA 1051 CDC45L NM_003504 34335230 8318
D-003232-08 GGACGTGGATGCTCTGTGT 1052 CDC6 CDC6 NM_001254 16357469
990 D-003233-05 GAACACAGCTGTCCCAGAT 1053 CDC6 NM_001254 16357469
990 D-003233-06 GAGCAGAGATGTCCACTGA 1054 CDC6 NM_001254 16357469
990 D-003233-07 GGAAATATCTTAGCTACTG 1055 CDC6 NM_001254 16357469
990 D-003233-08 GGACGAAGATTGGTATTTG 1056 CDC7 CDC7 NM_003503
11038647 8317 D-003234-05 GGAATGAGGTACCTGATGA 1057 CDC7 NM_003503
11038647 8317 D-003234-06 CAGGAAAGGTGTTCACAAA 1058 CDC7 NM_003503
11038647 8317 D-003234-07 CTACACAAATGCACAAATT 1059 CDC7 NM_003503
11038647 8317 D-003234-08 GTACGGGAATATATGCTTA 1060 CDK10 CDK10
NM_003674 32528262 8558 D-003235-05 GAACTGCTGTTGGGAACCA 1061 CDK10
NM_003674 32528262 8558 D-003235-06 GGAAGCAGCCCTACAACAA 1062 CDK10
NM_003674 32528262 8558 D-003235-07 GCACGCCCAGTGAGAACAT 1063 CDK10
NM_003674 32528262 8558 D-003235-08 GGAAGCAGCCCTACAACAA 1064 CDK2
CDK2 NM_001798 16936527 1017 D-003236-05 GAGCTTAACCATCCTAATA 1065
CDK2 NM_001798 16936527 1017 D-003236-06 GAGCTTAACCATCCTAATA 1066
CDK2 NM_001798 16936527 1017 D-003236-07 GTACCGAGCTCCTGAAATC 1067
CDK2 NM_001798 16936527 1017 D-003236-08 GAGAGGTGGTGGCGCTTAA 1068
CDK3 CDK3 NM_001258 4557438 1018 D-003237-05 GAGCATTGGTTGCATCTTT
1069 CDK3 NM_001258 4557438 1018 D-003237-06 GATCGGAGAGGGCACCTAT
1070 CDK3 NM_001258 4557438 1018 D-003237-07 GAAGCTCTATCTGGTGTTT
1071 CDK3 NM_001258 4557438 1018 D-003237-08 GCAGAGATGGTGACTCGAA
1072 CDK4 CDK4 NM_000075 456426 1019 D-003238-05
GCAGCACTCTTATCTACAT 1073 CDK4 NM_000075 456426 1019 D-003238-06
GGAGGAGGCCTTCCCATCA 1074 CDK4 NM_000075 456426 1019 D-003238-07
TCGAAAGCCTCTCTTCTGT 1075 CDK4 NM_000075 456426 1019 D-003238-08
GTACCGAGCTCCCGAAGTT 1076 CDK5 CDK5 NM_004935 4826674 1020
D-003239-05 TGACCAAGCTGCCAGACTA 1077 CDK5 NM_004935 4826674 1020
D-003239-06 GAGCTGAAATTGGCTGATT 1078 CDK5 NM_004935 4826674 1020
D-003239-07 CAACATCCCTGGTGAACGT 1079 CDK5 NM_004935 4826674 1020
D-003239-08 GGATTCCCGTCCGCTGTTA 1080 CDK6 CDK6 NM_001259 16950658
1021 D-003240-05 GCAAAGACCTACTTCTGAA 1081 CDK6 NM_001259 16950658
1021 D-003240-06 GAAGAAGACTGGCCTAGAG 1082 CDK6 NM_001259 16950658
1021 D-003240-07 GGTCTGGACTTTCTTCATT 1083 CDK6 NM_001259 16950658
1021 D-003240-08 TAACAGATATCGATGAACT 1084 CDK7 CDK7 NM_001799
16950659 1022 D-003241-05 GGACATAGATCAGAAGCTA 1085 CDK7 NM_001799
16950659 1022 D-003241-06 CAATAGAGCTTATACACAT 1086 CDK7 NM_001799
16950659 1022 D-003241-07 CATACAAGGCTTATTCTTA 1087 CDK7 NM_001799
16950659 1022 D-003241-08 GGAGACGACTTACTAGATC 1088 CDK8 CDK8
NM_001260 4502744 1024 D-003242-05 CCACAGTACTCACATCAGA 1089 CDK8
NM_001260 4502744 1024 D-003242-06 GCAATAACCACACTAATGG 1090 CDK8
NM_001260 4502744 1024 D-003242-07 GAAGAAAGTGAGAGTTGTT 1091 CDK8
NM_001260 4502744 1024 D-003242-08 GAACATGACCTCTGGCATA 1092 CDK9
CDK9 NM_0012611 7017983 1025 D-003243-05 GGCCAAACGTGGACAACTA 1093
CDK9 NM_0012611 7017983 1025 D-003243-06 TGACGTCCATGTTCGAGTA 1094
CDK9 NM_0012611 7017983 1025 D-003243-07 CCAACCAGACGGAGTTTGA 1095
CDK9 NM_0012611 7017983 1025 D-003243-08 GAAGGTGGCTCTGAAGAAG 1096
CDKN1C CDKN1C NM_000076 4557440 1028 D-003244-05
GACCAGAACCGCTGGGATT 1097 CDKN1C NM_000076 4557440 1028 D-003244-06
GGACCGAAGTGGACAGCGA 1098 CDKN1C NM_000076 4557440 1028 D-003244-08
GCAAGAGATCAGCGCCTGA 1099 CDKN1C NM_000076 4557440 1028 D-003244-09
CCGCTGGGATTACGACTTC 1100 CDKN2B CDKN2B NM_004936 17981693 1030
D-003245-05 GCGAGGAGAACAAGGGCAT 1101 CDKN2B NM_004936 17981693 1030
D-003245-06 CCAACGGAGTCAACCGTTT 1102 CDKN2B NM_004936 17981693 1030
D-003245-07 CGATCCAGGTCATGATGAT 1103 CDKN2B NM_004936 17981693 1030
D-003245-08 CCTGGAAGCCGGCGCGGAT 1104 CDKN2C CDKN2C NM_001262
17981697 1031 D-003246-05 GGACACCGCCTGTGATTTG 1105 CDKN2C NM_001262
17981697 1031 D-003246-06 GCCAGGAGACTGCTACTTA 1106 CDKN2C NM_001262
17981697 1031 D-003246-07 TGAAAGACCGAACTGGTTT 1107 CDKN2C NM_001262
17981697 1031 D-003246-08 GAACCTGCCCTTGCACTTG 1108 CDKN2D CDKN2D
NM_001800 17981700 1032 D-003247-05 TGGCAGTTCAAGAGGGTCA 1109 CDKN2D
NM_001800 17981700 1032 D-003247-06 CTCAGGACCTCGTGGACAT 1110 CDKN2D
NM_001800 17981700 1032 D-003247-07 TGAAGGTCCTAGTGGAGCA 1111 CDKN2D
NM_001800 17981700 1032 D-003247-08 AGACGGCGCTGCAGGTCAT 1112 CDT1
CDT1 NM_030928 19923847 81620 D-003248-05 CCAAGGAGGCACAGAAGCA 1113
CDT1 NM_030928 19923847 81620 D-003248-06 GCTTCAACGTGGATGAAGT 1114
CDT1 NM_030928 19923847 81620 D-003248-07 TCTCCGGGCCAGAAGATAA 1115
CDT1 NM_030928 19923847 81620 D-003248-08 GCGCAATGTTGGCCAGATC 1116
CENPA CENPA NM_001809 4585861 1058 D-003249-05 GCACACACCTCTTGATAAG
1117 CENPA NM_001809 4585861 1058 D-003249-06 GCAAGAGAAATATGTGTTA
1118 CENPA NM_001809 4585861 1058 D-003249-07 TTACATGCAGGCCGAGTTA
1119 CENPA NM_001809 4585861 1058 D-003249-08 GAGACAAGGTTGGCTAAAG
1120 CENPB CENPB NM_001810 26105977 1059 D-003250-05
GGACATAGCCGCCTGCTTT 1121 CENPB NM_001810 26105977 1059 D-003250-06
GCACGATCCTGAAGAACAA 1122 CENPB NM_001810 26105977 1059 D-003250-07
GGAGGAGGGTGATGTTGAT 1123 CENPB NM_001810 26105977 1059 D-003250-08
CCGAATGGCTGCAGAGTCT 1124 CENPC1 CENPC1 NM_001812 4502778 1060
D-003251-05 GCGAATAGATTATCAAGGA 1125 CENPC1 NM_001812 4502778 1060
D-003251-06 GAACAGAATCCATCACAAA 1126 CENPC1 NM_001812 4502778 1060
D-003251-07 CCATAAACCTCACCCAGTA 1127 CENPC1 NM_001812 4502778 1060
D-003251-08 CAAGAGAACACGTTTGAAA 1128 CENPE CENPE NM_001813 4502780
1062 D-003252-05 GAAGACAGCTCAAATAATA 1129 CENPE NM_001813 4502780
1062 D-003252-06 CAACAAAGCTACTAAATCA 1130 CENPE NM_001813 4502780
1062 D-003252-07 GGAAAGAAGTGCTACCATA 1131 CENPE NM_001813 4502780
1062 D-003252-08 GGAAAGAAATGACACAGTT 1132 CENPF CENPF NM_016343
14670380 1063 D-003253-05 GCGAATATCTGAATTAGAA 1133 CENPF NM_016343
14670380 1063 D-003253-06 GGAAATTAATGCATCCTTA 1134 CENPF NM_016343
14670380 1063 D-003253-07 GAGCGAGGCTGGTGGTTTA 1135 CENPF NM_016343
14670380 1063 D-003253-08 CAAGTCATCTTTCATCTAA 1136 CENPH CENPH
NM_022909 21264590 64946 D-003254-05 GAAAGAAGAGATTGCAATT 1137 CENPH
NM_022909 21264590 64946 D-003254-06 CAGAACAAATTATGCAAGA 1138 CENPH
NM_022909 21264590 64946 D-003254-07 CTAGTGTGCTCATGGATAA 1139 CENPH
NM_022909 21264590 64946 D-003254-08 GAAACACCTATTAGAGCTA 1140 CHEK1
CHEK1 NM_001274 20127419 1111 D-003255-05 CAAATTGGATGCAGACAAA 1141
CHEK1 NM_001274 20127419 1111 D-003255-06 GCAACAGTATTTCGGTATA 1142
CHEK1 NM_001274 20127419 1111 D-003255-07 GGACTTCTCTCCAGTAAAC 1143
CHEK1 NM_001274 20127419 1111 D-003255-08 AAAGATAGATGGTACAACA 1144
CHEK2 CHEK2 NM_007194 22209010 11200 D-003256-02
CTCTTACATTGCATACATA 1145 CHEK2 NM_007194 22209010 11200 D-003256-03
TAAACGCCTGAAAGAAGCT 1146 CHEK2 NM_007194 22209010 11200 D-003256-04
GCATAGGACTCAAGTGTCA 1147 CHEK2 NM_007194
22209010 11200 D-003256-05 GAAATTGCACTGTCACTAA 1148 CNK CNK
NM_004073 4758015 1263 D-003257-05 GCGAGAAGATCCTAAATGA 1149 CNK
NM_004073 4758015 1263 D-003257-07 GCAAGTGGGTTGACTACTC 1150 CNK
NM_004073 4758015 1263 D-003257-08 GCACATCCGTTGGCCATCA 1151 CNK
NM_004073 4758015 1263 D-003257-09 GACCTCAAGTTGGGAAATT 1152 CRI1
CRI1 NM_014335 7656937 23741 D-003258-05 GTGATGAGATTATTGATAG 1153
CRI1 NM_014335 7656937 23741 D-003258-06 GGACGAGGGCGAGGAATTT 1154
CRI1 NM_014335 7656937 23741 D-003258-07 GGAAACGGAGCCTTGCTAA 1155
CRI1 NM_014335 7656937 23741 D-003258-08 TCAATCGTCTGACCGAAGA 1156
E2F1 E2F1 NM_005225 12669910 1869 D-003259-05 GAACAGGGCCACTGACTCT
1157 E2F1 NM_005225 12669910 1869 D-003259-06 TGGACCACCTGATGAATAT
1158 E2F1 NM_005225 12669910 1869 D-003259-07 CCCAGGAGGTCACTTCTGA
1159 E2F1 NM_005225 12669910 1869 D-003259-08 GGCTGGACCTGGAAACTGA
1160 E2F2 E2F2 NM_004091 34485718 1870 D-003260-05
GGGAGAAGACTCGGTATGA 1161 E2F2 NM_004091 34485718 1870 D-003260-06
GAGGACAACCTGCAGATAT 1162 E2F2 NM_004091 34485718 1870 D-003260-07
TGAAGGAGCTGATGAACAC 1163 E2F2 NM_004091 34485718 1870 D-003260-08
CCAAGAAGTTCA1TTACCT 1164 E2F3 E2F3 NM_001949 12669913 1871
D-003261-05 GAAATTAGATGAACTGATC 1165 E2F3 NM_001949 12669913 1871
D-003261-06 TGAAGTGCCTGACTCAATA 1166 E2F3 NM_001949 12669913 1871
D-003261-07 GAACAAGGCAGCAGAAGTG 1167 E2F3 NM_001949 12669913 1871
D-003261-08 GAAACACACAGTCCAATGA 1168 E2F4 E2F4 NM_001950 12669914
1874 D-003262-05 GGAGATTGCTGACAAACTG 1169 E2F4 NM_001950 12669914
1874 D-003262-06 GAAGGTATCGGGCTAATCG 1170 E2F4 NM_001950 12669914
1874 D-003262-07 GTGCAGAAGTCCAGGGAAT 1171 E2F4 NM_001950 12669914
1874 D-003262-08 GGACAGTGGTGAGCTCAGT 1172 E2F5 E2F5 NM_001951
12669916 1875 D-003263-05 GCAGATGACTACAACTTTA 1173 E2F5 NM_001951
12669916 1875 D-003263-06 GACATCAGCTACAGATATA 1174 E2F5 NM_001951
12669916 1875 D-003263-07 CAACATGTCTCTGAAAGAA 1175 E2F5 NM_001951
12669916 1875 D-003263-08 GAAGACATCTGTAATTGCT 1176 E2F6 E2F6
NM_001952 12669917 1876 D-003264-05 TAAACAAGGTTGCAACGAA 1177 E2F6
NM_001952 12669917 1876 D-003264-06 TAGCATATGTGACCTATCA 1178 E2F6
NM_001952 12669917 1876 D-003264-07 GAAACCAGATTGGATGTTC 1179 E2F6
NM_001952 12669917 1876 D-003264-09 GGAACTTTCTGACTTATCA 1180 FOS
FOS NM_005252 6552332 2353 D-003265-05 GGGATAGCCTCTCTTACTA 1181 FOS
NM_005252 6552332 2353 D-003265-06 GAACAGTTATCTCCAGAAG 1182 FOS
NM_005252 6552332 2353 D-003265-07 GGAGACAGACCAACTAGAA 1183 FOS
NM_005252 6552332 2353 D-003265-08 AGACCGAGCCCTTTGATGA 1184 HIPK2
HIPK2 NM_022740 13430859 28996 D-003266-06 GAGAATCACTCCAATCGAA 1185
HIPK2 NM_022740 13430859 28996 D-003266-07 AGACAGGGATTAAGTCAAA 1186
HIPK2 NM_022740 13430859 28996 D-003266-08 GGACAAAGACAACTAGGTT 1187
HIPK2 NM_022740 13430859 28996 D-003266-09 GCACACACGTCAAATCATG 1188
HUS1 HUS1 NM_004507 31077213 3364 D-003267-05 ACAAAGGCCTTATGCAATA
1189 HUS1 NM_004507 31077213 3364 D-003267-06 GAAGTGCACATAGATATTA
1190 HUS1 NM_004507 31077213 3364 D-003267-07 AAGCTTAACTTCATCCTTT
1191 HUS1 NM_004507 31077213 3364 D-003267-08 GAACTTCTTCAACGAATTT
1192 JUN JUN NM_002228 7710122 3725 D-003268-05 TGGAAACGACCTTCTATGA
1193 JUN NM_002228 7710122 3725 D-003268-06 GAACTGCACAGCCAGAACA
1194 JUN NM_002228 7710122 3725 D-003268-07 GAGCTGGAGCGCCTGATAA
1195 JUN NM_002228 7710122 3725 D-003268-08 TAACGCAGCAGTTGCAAAC
1196 JUNB JUNB NM_002229 4504808 3726 D-003269-05
GCATCAAAGTGGAGCGCAA 1197 JUNB NM_002229 4504808 3726 D-003269-06
TGGAAGACCAAGAGCGCAT 1198 JUNB NM_002229 4504808 3726 D-003269-07
CATACACAGCTACGGGATA 1199 JUNB NM_002229 4504808 3726 D-003269-08
CCATCAACATGGAAGACCA 1200 LOC51053 LOC51053 NM_015895 20127542 51053
D-003270-05 GGAGAAAGGCGCTGTATGA 1201 LOC51053 NM_015895 20127542
51053 D-003270-06 GAATAGTTCTGTCCCAAGA 1202 LOC51053 NM_015895
20127542 51053 D-003270-07 GAACATGTACAGTATATGG 1203 LOC51053
NM_015895 20127542 51053 D-003270-08 GCAGAAACAAGAAGAAATC 1204
MAD2L1 MAD2L1 NM_002358 6466452 4085 D-003271-05
GAAAGATGGCAGTTTGATA 1205 MAD2L1 NM_002358 6466452 4085 D-003271-06
TAAATAATGTGGTGGAACA 1206 MAD2L1 NM_002358 6466452 4085 D-003271-07
GAAATCCGTTCAGTGATCA 1207 MAD2L1 NM_002358 6466452 4085 D-003271-08
TTACTCGAGTGCAGAAATA 1208 MAD2L2 MAD2L2 NM_006341 6006019 10459
D-003272-05 GGAAGAGCGCGCTCATAAA 1209 MAD2L2 NM_006341 6006019 10459
D-003272-06 TGGAAGAGCGCGCTCATAA 1210 MAD2L2 NM_006341 6006019 10459
D-003272-07 AGCCACTCCTGGAGAAGAA 1211 MAD2L2 NM_006341 6006019 10459
D-003272-08 TGGAGAAATTCGTCTTTGA 1212 MCM2 MCM2 NM_004526 33356546
4171 D-003273-05 GAAGATCTTTGCCAGCATT 1213 MCM2 NM_004526 33356546
4171 D-003273-06 GGATAAGGCTCGTCAGATC 1214 MCM2 NM_004526 33356546
4171 D-003273-07 CAGAGCAGGTGACATATCA 1215 MCM2 NM_004526 33356546
4171 D-003273-08 GCCGTGGGCTCCTGTATGA 1216 MCM3 MCM3 NM_002388
33356548 4172 D-003274-05 GGACATCAATATTCTTCTA 1217 MCM3 NM_002388
33356548 4172 D-003274-06 GCCAGGACATCTCCAGTTA 1218 MCM3 NM_002388
33356548 4172 D-003274-07 GCAGGTATGACCAGTATAA 1219 MCM3 NM_002388
33356548 4172 D-003274-08 GGAAATGCCTCAAGTACAC 1220 MCM4 MCM4
XM_030274 22047061 4173 D-003275-05 GGACATATCTATTCTTACT 1221 MCM4
XM_030274 22047061 4173 D-003275-06 GATGTTAGTTCACCACTGA 1222 MCM4
XM_030274 22047061 4173 D-003275-07 CCAGCTGCCTCATACTTTA 1223 MCM4
XM_030274 22047061 4173 D-003275-08 GAAAGTACAAGATCGGTAT 1224 MCM5
MCM5 NM_006739 23510447 4174 D-003276-05 GAAGATCCCTGGCATCATC 1225
MCM5 NM_006739 23510447 4174 D-003276-06 GAACAGGGTTACCATCATG 1226
MCM5 NM_006739 23510447 4174 D-003276-07 GGACAACATTGACTTCATG 1227
MCM5 NM_006739 23510447 4174 D-003276-08 CCAAGGAGGTAGCTGATGA 1228
MCM6 MCM6 NM_005915 33469920 4175 D-003277-05 GGAAAGAGCTCAGAGATGA
1229 MCM6 NM_005915 33469920 4175 D-003277-06 GAGCAGCGATGGAGAAATT
1230 MCM6 NM_005915 33469920 4175 D-003277-07 GGAAACACCTGATGTCAAT
1231 MCM6 NM_005915 33469920 4175 D-003277-08 CCAAACATCTGCCGAAATC
1232 MCM7 MCM7 NM_005916 33469967 4176 D-003278-05
GGAAATATCCCTCGTAGTA 1233 MCM7 NM_005916 33469967 4176 D-003278-06
GGAAGAAGCAGTTCAAGTA 1234 MCM7 NM_005916 33469967 4176 D-003278-07
CAACAAGCCTCGTGTGATC 1235 MCM7 NM_005916 33469967 4176 D-003278-08
GGAGAGAACACAAGGATTG 1236 MDM2 MDM2 NM_002392 4505136 4193
D-003279-05 GGAGATATGTTGTGAAAGA 1237 MDM2 NM_002392 4505136 4193
D-003279-06 CCACAAATCTGATAGTATT 1238 MDM2 NM_002392 4505136 4193
D-003279-07 GATGAGGTATATCAAGTTA 1239 MDM2 NM_002392 4505136 4193
D-003279-08 GGAAGAAACCCAAGACAAA 1240 MK167 MK167 NM_002417 19923216
4288 D-003280-05 GCACAAAGCTTGGTTATAA 1241 MK167 NM_002417 19923216
4288 D-003280-06 CCTAAGACCTGAACTATTT 1242 MK167 NM_002417 19923216
4288 D-003280-07 CAAAGAGGAACACAAATTA 1243 MK167 NM_002417 19923216
4288 D-003280-08 GTAAATGGGTCTGTTATTG 1244 MNAT1 MNAT1 NM_002431
4505224 4331 D-003281-05 GGAAGAAGCTTTAGAAGTG 1245 MNAT1 NM_002431
4505224 4331 D-003281-06 TAGATGAGCTGGAGAGTTC 1246 MNAT1 NM_002431
4505224 4331 D-003281-07 GGACCTTGCTGGAGGCTAT 1247 MNAT1 NM_002431
4505224 4331 D-003281 -08 GCAGATAGAGACATATGGA 1248 MYC MYC
NM_002467 31543215 4609 D-003282-05 CAGAGAAGCTGGCCTCCTA 1249 MYC
NM_002467 31543215 4609 D-003282-06 GAAACGACGAGAACAGTTG 1250 MYC
NM_002467 31543215 4609 D-003282-07 CGACGAGACCTTCATCAAA 1251 MYC
NM_002467 31543215 4609 D-003282-08 CCACACATCAGCACAACTA 1252 ORC1L
ORC1L NM_004153 31795543 4998 D-003283-05 GAACAGGAATTCCAAGACA 1253
ORC1L NM_004153 31795543 4998 D-003283-06 TAAGAAACGTGCTCGAGTA 1254
ORC1L NM_004153 31795543 4998 D-003283-07 GAGATCACCTCACCTTCTA 1255
ORC1L NM_004153 31795543 4998 D-003283-08 GCAGAGAGCCCTTCTTGGA 1256
ORC2L ORC2L NM_006190 32454751 4999 D-003284-05 GAAGAAACCTCCTATGAGA
1257 ORC2L NM_006190 32454751 4999 D-003284-06 GAAGGGAACTGATGGAGTA
1258 ORC2L NM_006190 32454751 4999 D-003284-07 GAAGAATGATCCTGAGATT
1259 ORC2L NM_006190 32454751 4999 D-003284-08 GAAGAGATGTTCAAGAATC
1260 ORC3L ORC3L NM_012381 32483366 23595 D-003285-05
GGACTGCTGTGTAGATATA 1261 ORC3L NM_012381 32483366 23595 D-003285-06
GAACTGATGACCATACTTG 1262 ORC3L NM_012381 32483366 23595 D-003285-07
AAAGATCTCTCTGCCAATA 1263 ORC3L NM_012381 32483366 23595 D-003285-08
CAGCACAGCTAAGAGAATA 1264 ORC4L ORC4L NM_002552 32454749 5000
D-003286-06 GAAAGCACATTCCGTTTAT 1265 ORC4L NM_002552 32454749 5000
D-003286-07 TGAAAGAACTCATGGAAAT 1266 ORC4L NM_002552 32454749 5000
D-003286-08 GCTGAGAAGTGGAATGAAA 1267 ORC4L NM_002552 32454749 5000
D-003286-09 CCAGTGATCTTCATATTAG 1268 ORC5L ORC5L NM_002553 32454752
5001 D-003287-05 GAAATAACCTGTGAAACAT 1269 ORC5L NM_002553 32454752
5001 D-003287-06 CAGATTACCTCTCTAGTGA 1270 ORC5L NM_002553 32454752
5001 D-003287-07 GAACTTCCATATTACTCTA 1271 ORC5L NM_002553 32454752
5001 D-003287-08 GTATTCAGCTGATTTCTAT 1272 ORC6L ORC6L NM_014321
32454755 23594 D-003288-05 GAACATGGCTTCAAAGATA 1273 ORC6L NM_014321
32454755 23594 D-003288-06 GGACAGGGCTTATTTAATT 1274 ORC6L NM_014321
32454755 23594 D-003288-07 GAAAGAAGATAGTGGTTGA 1275 ORC6L NM_014321
32454755 23594 D-003288-08 TATCAGAGCTGTCTTAAAT 1276 PCNA PCNA
NM_002592 33239449 5111 D-003289-05 GATCGAGGATGAAGAAGGA 1277 PCNA
NM_002592 33239449 5111 D-003289-07 GCCGAGATCTCAGCCATAT 1278 PCNA
NM_002592 33239449 5111 D-003289-09 GAGGCCTGCTGGGATATTA 1279 PCNA
NM_002592 33239449 5111 D-003289-10 GTGGAGAACTTGGAAATGG 1280 PLK
PLK NM_005030 21359872 5347 D-003290-05 CAACCAAAGTCGAATATGA 1281
PLK NM_005030 21359872 5347 D-003290-06 CAAGAAGAATGAATACAGT 1282
PLK NM_005030 21359872 5347 D-003290-07 GAAGATGTCCATGGAAATA 1283
PLK NM_005030 21359872 5347 D-003290-08 CAACACGCCTCATCCTCTA 1284
PIN1 PIN1 NM_006221 5453897 5300 D-003291-05 GGACCAAGGAGGAGGCCCT
1285 PIN1 NM_006221 5453897 5300 D-003291-06 CGTCCTGGCGGCAGGAGAA
1286 PIN1 NM_006221 5453897 5300 D-003291-07 CGGGAGAGGAGGACTTTGA
1287 PIN1 NM_006221 5453897 5300 D-003291-08 AGTCGGGAGAGGAGGACTT
1288 PIN1L PIN1L NM_006222 5453899 5301 D-003292-06
CGACCTGGCGGCAGGAAAT 1289 PIN1L NM_006222 5453899 5301 D-003292-07
AGGCAGGAGAGAAGGACTT 1290 PIN1L NM_006222 5453899 5301 D-003292-08
GCTACATCCAGAAGATCAA 1291 PIN1L NM_006222 5453899 5301 D-003292-09
GGACAGTGTTCACGGATTC 1292 RAD1 RAD1 NM_002853 19718797 5810
D-003293-05 GAAGATGGACAAATATGTT 1293 RAD1 NM_002853 19718797 5810
D-003293-06 GGAAGAGTCTGTTACTTTT 1294 RAD1 NM_002853 19718797 5810
D-003293-07 GATAACAGAGGCTTCCTTT 1295 RAD1 NM_002853 19718797 5810
D-003293-08 GCATTAGTCCTATCTTGTA 1296 RAD17 RAD17 NM_133338 19718783
5884 D-003294-05 GAATCAAGCTTCCATATGT 1297 RAD17 NM_133338 19718783
5884 D-003294-06 CAACAAAGCCCGAGGATAT 1298 RAD17 NM_133338 19718783
5884 D-003294-07 ACACATGCCTGGAGACTTA 1299 RAD17 NM_133338 19718783
5884 D-003294-08 CTACATAGATTTCTTCATG 1300 RAD9A RAD9A NM_004584
19924112 5883 D-003295-05 TCAGCAAACTTGAATCTTA 1301 RAD9A NM_004584
19924112 5883 D-003295-06 GACATTGACTCTTACATGA 1302 RAD9A NM_004584
19924112 5883 D-003295-08 GGAAACCACTATAGGCAAT 1303 RAD9A NM_004584
19924112 5883 D-003295-09 CGGACGACTTTGCCAATGA 1304 RB1 RB1
NM_000321 19924112 5925 D-003296-05 GAAAGGACATGTGAACTTA 1305 RB1
NM_000321 19924112 5925 D-003296-06 GAAGAAGTATGATGTATTG 1306 RB1
NM_000321 4506434 5925 D-003296-07 GAAATGACTTCTACTCGAA 1307 RB1
NM_000321 4506434 5925 D-003296-08 GGAGGGAACATCTATATTT 1308 RBBP2
RBBP2 NM_005056 4826967 5927 D-003297-05 CAAAGAAGCTGAATAAACT 1309
RBBP2 NM_005056 4826967 5927 D-003297-06 CAACACATATGGCGGATTT 1310
RBBP2 NM_005056 4826967 5927 D-003297-07 GGACAAACCTAGAAAGAAG 1311
RBBP2 NM_005056 4826967 5927 D-003297-08 GAAAGGCACTCTCTCTGTT 1312
RBL1 RBL1 NM_002895 34577078 5933 D-003298-05 CAAGAGAAGTTGTGGCATA
1313 RBL1 NM_002895 34577078 5933 D-003298-06 CAGCAGCACTCCATTTATA
1314 RBL1 NM_002895 34577078 5933 D-003298-07 ACAGAAAGGTCTATCATTT
1315 RBL1 NM_002895 34577078 5933 D-003298-08 GGACATAAAGTTACAATTC
1316 RBL2 RBL2 NM_005611 21361291 5934 D-003299-05
GAGCAGAGCTTAATCGAAT 1317 RBL2 NM_005611 21361291 5934 D-003299-06
GAGAATAGCCCTTGTGTGA 1318 RBL2 NM_005611 21361291 5934 D-003299-07
GGACTTAGTTTATGGAAAT 1319 RBL2 NM_005611 21361291 5934 D-003299-08
GAATTTAGATGAGCGGATA 1320 RBP1 RBP1 NM_002899 8400726 5947
D-003300-05 GAGACAAGCTCCAGTGTGT 1321 RBP1 NM_002899 8400726 5947
D-003300-06 GCAAGCAAGTATTCAAGAA 1322 RBP1 NM_002899 8400726 5947
D-003300-07 GCAGGACGGTGACCATATG 1323 RBP1 NM_002899 8400726 5947
D-003300-08 GCAAGTGCATGACAACAGT 1324 RPA3 RPA3 NM_002947 19923751
6119 D-003322-05 GGAAGTGGTTGGAAGAGTA 1325 RPA3 NM_002947 19923751
6119 D-003322-06 GAAGATAGCCATCCTTTTG 1326 RPA3 NM_002947 19923751
6119 D-003322-07 CATGCTAGCTCAATTCATC 1327 RPA3 NM_002947 19923751
6119 D-003322-08 GATCTTGGACTTTACAATG 1328 SKP1A SKP1A NM_006930
25777710 6500 D-003323-05 GGAGAGATATTTGAAGTTG 1329 SKP1A NM_006930
25777710 6500 D-003323-06 GGGAATGGATGATGAAGGA 1330 SKP1A NM_006930
25777710 6500 D-003323-07 CAAACAATCTGTGACTATT 1331 SKP1A NM_006930
25777710 6500 D-003323-08 TCAATTAAGTTGCAGAGTT 1332 SKP2 SKP2
NM_005983 16306594 6502 D-003324-05 CATCTAGACTTAAGTGATA 1333 SKP2
NM_005983 16306594 6502 D-003324-06 GAAATCAGATCTCTCTACT 1334 SKP2
NM_005983 16306594 6502 D-003324-07 CTAAAGGTCTCTGGTGTTT 1335 SKP2
NM_005983 16306594 6502 D-003324-08 GATGGTACCC1TCAACTGT 1336 SNK
SNK NM_006622 5730054 10769 D-003325-05 GAAGACATCTACAAGCTTA 1337
SNK NM_006622 5730054 10769 D-003325-06 GAAATACCTTCATGAACAA 1338
SNK NM_006622 5730054 10769 D-003325-07 GAAGGTCAATGGCTCATAT 1339
SNK NM_006622 5730054 10769 D-003325-08 CCGGAGATCTCGCGGATTA 1340
STK12 STK12 NM_004217 4759177 9212 D-003326-07 CAGAAGAGCTGCACATTTG
1341 STK12 NM_004217 4759177 9212 D-003326-08 CCAAACTGCTCAGGCATAA
1342 STK12 NM_004217 4759177 9212 D-003326-09 ACGCGGCACTTCACAATTG
1343 STK12 NM_004217 4759177 9212 D-003326-10 TGGGACACCCGACATCTTA
1344 TFDP1 TFDP1 NM_007111 34147667 7027 D-003327-05
GGAAGCAGCTCTTGCCAAA 1345 TFDP1 NM_007111 34147667 7027 D-003327-06
GAGGAGACTTGAAAGAATA 1346 TFDP1 NM_007111 34147667 7027 D-003327-07
GAACTTAGAGGTGGAAAGA 1347 TFDP1 NM_007111 34147667 7027 D-003327-08
GCGAGAAGGTGCAGAGGAA 1348 TFDP2 TFDP2 NM_006286 5454111 7029
D-003328-05 GAAAGTGTGTGAGAAAGTT 1349 TFDP2 NM_006286 5454111 7029
D-003328-06 CACAGGACCTTCTTGGTTA 1350 TFDP2 NM_006286 5454111 7029
D-003328-07 CGAAATCCCTGGTGCCAAA 1351 TFDP2 NM_006286 5454111 7029
D-003328-08 TGAGATCCATGATGACATA 1352 TP53 TP53 NM_000546 8400737
7157 D-003329-05 GAGGTTGGCTCTGACTGTA 1353 TP53 NM_000546 8400737
7157 D-003329-06 CAGTCTACCTCCCGCCATA 1354 TP53 NM_000546 8400737
7157 D-003329-07 GCACAGAGGAAGAGAATCT 1355 TP53 NM_000546 8400737
7157 D-003329-08 GAAGAAACCACTGGATGGA 1356 TP63 TP63 NM_003722
31543817 8626 D-003330-05 CATCATGTCTGGACTATTT 1357 TP63 NM_003722
31543817 8626 D-003330-06 CAAACAAGATTGAGATTAG 1358 TP63 NM_003722
31543817 8626 D-003330-07 GCACACAGACAAATGAATT 1359 TP63 NM_003722
31543817 8626 D-003330-08 CGACAGTCTTGTACAATTT 1360 TP73 TP73
NM_005427 4885644 7161 D-003331-05 GCAAGCAGCCCATCAAGGA 1361 TP73
NM_005427 4885644 7161 D-003331-06 GAGACGAGGACACGTACTA 1362 TP73
NM_005427 4885644 7161 D-003331-07 CTGCAGAACCTGACCATTG 1363 TP73
NM_005427 4885644 7161 D-003331-08 GGCCATGCCTGTTTACAAG 1364 YWHAZ
YWHAZ NM_003406 21735623 7534 D-003332-05 GCAAGGAGCTGAATTATCC 1365
YWHAZ NM_003406 21735623 7534 D-003332-06 TAAGAGATATCTGCAATGA 1366
YWHAZ NM_003406 21735623 7534 D-003332-07 GACGGAAGGTGCTGAGAAA 1367
YWHAZ NM_003406 21735623 7534 D-003332-08 AGAGCAAAGTCTTCTATTT
1368
[0615]
14TABLE IX Gene SEQ. ID Name Accession # GI# Duplex # Sequence NO.
AR NM_000044 21322251 D-003400-01 GGAACTCGATCGTATCATT 1369 AR
NM_000044 21322251 D-003400-02 CAAGGGAGGTTACACCAAA 1370 AR
NM_000044 21322251 D-003400-03 TCAAGGAACTCGATCGTAT 1371 AR
NM_000044 21322251 D-003400-04 GAAATGATTGCACTATTGA 1372 ESR1
NM_000125 4503602 D-003401-01 GAATGTGCCTGGCTAGAGA 1373 ESR1
NM_000125 4503602 D-003401-02 CATGAGAGCTGCCAACCTT 1374 ESR1
NM_000125 4503602 D-003401-03 AGAGAAAGATTGGCCAGTA 1375 ESR1
NM_000125 4503602 D-003401-04 CAAGGAGACTCGCTACTGT 1376 ESR2
NM_001437 10835012 D-003402-01 GAACATCTGCTCAACATGA 1377 ESR2
NM_001437 10835012 D-003402-02 GCACGGCTCCATATACATA 1378 ESR2
NM_001437 10835012 D-003402-03 CAAGAAGATTCCCGGCTTT 1379 ESR2
NM_001437 10835012 D-003402-04 GGAAATGCGTAGAAGGAAT 1380 ESRRA
NM_004451 18860919 D-003403-01 GGCCTTCGCTGAGGACTTA 1381 ESRRA
NM_004451 18860919 D-003403-02 TGAATGCACTGGTGTCTCA 1382 ESRRA
NM_004451 18860919 D-003403-03 GCATTGAGCCTCTCTACAT 1383 ESRRA
NM_004451 18860919 D-003403-04 CCAGACAGCGGGCAAAGTG 1384 ESRRB
NM_004452 22035686 D-003404-01 TACCTGAGCTTACAAATTT 1385 ESRRB
NM_004452 22035686 D-003404-02 GCACTTCTATAGCGTCAAA 1386 ESRRB
NM_004452 22035686 D-003404-03 CAACTCCGATTCCATGTAC 1387 ESRRB
NM_004452 22035686 D-003404-04 GGACTCGCCACCCATGTTT 1388 ESRRG
NM_001438 4503604 D-003405-01 AAACAAAGATCGACACATT 1389 ESRRG
NM_001438 4503604 D-003405-02 TCAGGAAACTGTATGATGA 1390 ESRRG
NM_001438 4503604 D-003405-03 GAAGACCAGTCCAAATTAG 1391 ESRRG
NM_001438 4503604 D-003405-04 ATGAAGCGCTGCAGGATTA 1392 HNF4A
NM_000457 21361184 D-003406-01 CGACATCACTGGAGCATAT 1393 HNF4A
NM_000457 21361184 D-003406-02 GAAGGAAGCCGTCCAGAAT 1394 HNF4A
NM_000457 21361184 D-003406-03 CCAAGTACATCCCAGCTTT 1395 HNF4A
NM_000457 21361184 D-003406-04 GGACATGGCCGACTACAGT 1396 HNF4G
NM_004133 6631087 D-003407-01 GCACTGACATAAACGTTAA 1397 HNF4G
NM_004133 6631087 D-003407-02 ACAAAGAGATCCATGATGT 1398 HNF4G
NM_004133 6631087 D-003407-03 AGAGATCCATGATGTATAA 1399 HNF4G
NM_004133 6631087 D-003407-04 AAATGAACGTGACAGAATA 1400 H5AJ2425
NM_017532 8923776 D-003408-01 GAATGAATCTACACCTTTG 1401 H5AJ2425
NM_017532 8923776 D-003408-02 GGAAATACGTGGAGACACT 1402 H5AJ2425
NM_017532 8923776 D-003408-03 CCAGATAACTACGGCGATA 1403 H5AJ2425
NM_017532 8923776 D-003408-04 TGGCGTACCTTCTCATTGA 1404 NROB1
NM_000475 5016089 D-003409-01 CAGCATGGATGATATGATG 1405 NROB1
NM_000475 5016089 D-003409-02 CTGCTGAGATTCATCAATG 1406 NROB1
NM_000475 5016089 D-003409-03 ACAGATTCATCGAACTTAA 1407 NROB1
NM_000475 5016089 D-003409-04 GAACGTGGCGCTCCTGTAC 1408 NROB2
NM_021969 13259502 D-003410-01 GAATATGCCTGCCTGAAAG 1409 NROB2
NM_021969 13259502 D-003410-02 GGAATATGCCTGCCTGAAA 1410 NROB2
NM_021969 13259502 D-003410-03 CGTAGCCGCTGCCTATGTA 1411 NROB2
NM_021969 13259502 D-003410-04 GCCATTCTCTACGCACTTC 1412 NR1D1
NM_021724 13430847 D-003411-01 CAACACAGGTGGCGTCATC 1413 NR1D1
NM_021724 13430847 D-003411-02 GGCATGGTGTTACTGTGTA 1414 NR1D1
NM_021724 13430847 D-003411-03 CAACATGCATTCCGAGAAG 1415 NR1D1
NM_021724 13430847 D-003411-04 GCGCTTTGCTTCGTTGTTC 1416 NR1H2
NM_007121 11321629 D-003412-01 GAACAGATCCGGAAGAAGA 1417 NR1H2
NM_007121 11321629 D-003412-02 GAAGAACAGATCCGGAAGA 1418 NR1H2
NM_007121 11321629 D-003412-03 CTAAGCAAGTGCCTGGTTT 1419 NR1H2
NM_007121 11321629 D-003412-04 GCTAACAGCGGCTCAAGAA 1420 NR1H3
NM_005693 5031892 D-003413-01 GAACAGATCCGCCTGAAGA 1421 NR1H3
NM_005693 5031892 D-003413-02 GGAGATAGTTGACTTTGCT 1422 NR1H3
NM_005693 5031892 D-003413-03 GAGTTTGCCTTGCTCATTG 1423 NR1H3
NM_005693 5031892 D-003413-04 TGACT1TGCTAAACAGCTA 1424 NR1H4
NM_005123 4826979 D-003414-01 CAAGTGACCTCGACAACAA 1425 NR1H4
NM_005123 4826979 D-003414-02 GAAAGAATTCGAAATAGTG 1426 NR1H4
NM_005123 4826979 D-003414-03 CAACAGACTCTTCTACATT 1427 NR1H4
NM_005123 4826979 D-003414-04 GAACCATACTCGCAATACA 1428 NR1I2
NM_003889 11863133 D-003415-01 GAACCATGCTGACTTTGTA 1429 NR1I2
NM_003889 11863133 D-003415-02 GATGGACGCTCAGATGAAA 1430 NR1I2
NM_003889 11863133 D-003415-03 CAACCTACATGTTCAAAGG 1431 NR1I2
NM_003889 11863133 D-003415-04 CAGGAGCAATTCGCCATTA 1432 NR1I3
NM_005122 4826660 D-003416-01 GGAAATCTGTCACATCGTA 1433 NR1I3
NM_005122 4826660 D-003416-02 TCGCAGACATCAACACTTT 1434 NR1I3
NM_005122 4826660 D-003416-03 CCTCTTCGCTACACAATTG 1435 NR1I3
NM_005122 4826660 D-003416-04 GAACAGTTTGTGCAGTTTA 1436 NR2C1
NM_003297 4507672 D-003417-01 TGACAGCACTTGATCATAA 1437 NR2C1
NM_003297 4507672 D-003417-02 GGAAGGAAGTGTACACCTA 1438 NR2C1
NM_003297 4507672 D-003417-03 GAGCACATCTTCAAACTAC 1439 NR2C1
NM_003297 4507672 D-003417-04 GAAGAAATTGCACATCAAA 1440 NR2C2
NM_003298 4507674 D-003418-01 GAACAACGGTGACACTTCA 1441 NR2C2
NM_003298 4507674 D-003418-02 CTGATGAGCTCCAACATAA 1442 NR2C2
NM_003298 4507674 D-003418-03 CAACCTAAGTGAATCTTTG 1443 NR2C2
NM_003298 4507674 D-003418-04 GAAGACACCTACCGATTGG 1444 NR2E1
NM_003269 21361108 D-003419-01 GATCATATCTGAAATACAG 1445 NR2E1
NM_003269 21361108 D-003419-02 CAAGACTGCTTTCAGATAT 1446 NR2E1
NM_003269 21361108 D-003419-03 GTTAGATGCTACTGAATTT 1447 NR2E1
NM_003269 21361108 D-003419-04 CAATGTATCTCTATGAAGT 1448 NR2E3
NM_014249 7657394 D-003420-01 GAGAAGCTCCTTTGTGATA 1449 NR2E3
NM_014249 7657394 D-003420-02 GAAGCACTATGGCATCTAT 1450 NR2E3
NM_014249 7657394 D-003420-03 GAAGGATCCTGAGCACGTA 1451 NR2E3
NM_014249 7657394 D-003420-04 GAAGCTCCTTTGTGATATG 1452 NR2F1
NM_005654 20127484 D-003421-01 GAAACTCTCATCCGCGATA 1453 NR2F1
NM_005654 20127484 D-003421-02 TCTCATCCGCGATATGTTA 1454 NR2F1
NM_005654 20127484 D-003421-03 CAAGAAGTGCCTCAAAGTG 1455 NR2F1
NM_005654 20127484 D-003421-04 GGAACTTAACTTACACATG 1456 NR2F2
NM_021005 14149745 D-003422-01 GTACCTGTCCGGATATATT 1457 NR2F2
NM_021005 14149745 D-003422-02 CCAACCAGCCGACGAGATT 1458 NR2F2
NM_021005 14149745 D-003422-03 ACTCGTACCTGTCCGGATA 1459 NR2F2
NM_021005 14149745 D-003422-04 GGCCGTATATGGCAATTCA 1460 NR2F6
NM_005234 20070198 D-003423-01 CGACGCCTGTGGCCTCTCA 1461 NR2F6
NM_005234 20070198 D-003423-02 CAGCCGGTGTCCGAACTGA 1462 NR2F6
NM_005234 20070198 D-003423-03 CAACCGTGACTGCCAGATC 1463 NR2F6
NM_005234 20070198 D-003423-04 GTACTGCCGTCTCAAGAAG 1464 NR3C1
NM_000176 4504132 D-003424-01 GAGGACAGATGTACCACTA 1465 NR3C1
NM_000176 4504132 D-003424-02 GATAAGACCATGAGTATTG 1466 NR3C1
NM_000176 4504132 D-003424-03 GAAGACGATTCATTCCTTT 1467 NR3C1
NM_000176 4504132 D-003424-04 GGACAGATGTACCACTATG 1468 NR3C2
NM_000901 4505198 D-003425-01 GCAAACAGATGATCCAAGT 1469 NR3C2
NM_000901 4505198 D-003425-02 CAGCTAAGATTTATCAGAA 1470 NR3C2
NM_000901 4505198 D-003425-03 GCACGAAAGTCAAAGAAGT 1471 NR3C2
NM_000901 4505198 D-003425-04 GGTATCCGGTCTTAGAATA 1472 NR4A1
NM_002135 21361341 D-003426-01 GAAGGAAGTTGTCCGAACA 1473 NR4A1
NM_002135 21361341 D-003426-02 CAGGAGAGTTTGACACCTT 1474 NR4A1
NM_002135 21361341 D-003426-03 CAGTGGCTCTGACTACTAT 1475 NR4A1
NM_002135 21361341 D-003426-04 GAAGGCCGCTGTGCTGTGT 1476 NR4A2
NM_006186 5453821 D-003427-01 GCAATGCGTTCGTGGCTTT 1477 NR4A2
NM_006186 5453821 D-003427-02 CGGCTACACAGGAGAGTTT 1478 NR4A2
NM_006186 5453821 D-003427-03 CCACGTGACTTTCAACAAT 1479 NR4A2
NM_006186 5453821 D-003427-04 GAATACAGCTCCGATTTCT 1480 NR4A3
NM_006981 11276070 D-003428-01 CAAAGAAGATCAGACATTA 1481 NR4A3
NM_006981 11276070 D-003428-02 GATCAGACATTACTTATTG 1482 NR4A3
NM_006981 11276070 D-003428-03 CCAGAGATCTTGATTATTC 1483 NR4A3
NM_006981 11276070 D-003428-04 GAAGTTGTCCGTACAGATA 1484 NR5A1
NM_004959 20070192 D-003429-01 GATTTGAAGTTCCTGAATA 1485 NR5A1
NM_004959 20070192 D-003429-02 GGAGCGAGCTGCTGGTGTT 1486 NR5A1
NM_004959 20070192 D-003429-03 GGAGGTGGCCGACCAGATG 1487 NR5A1
NM_004959 20070192 D-003429-04 CAACGTGCCTGAGCTCATC 1488 NR5A2
NM_003822 20070161 D-003430-01 CCAAACATATGGCCACTTT 1489 NR5A2
NM_003822 20070161 D-003430-02 TCAGAGAACTTAAGGTTGA 1490 NR5A2
NM_003822 20070161 D-003430-03 GGATCCATCTTCCTGGTTA 1491 NR5A2
NM_003822 20070161 D-003430-04 AAGAATACCTCTACTACAA 1492 NR6A1
NM_033334 15451847 D-003431-01 CAACGAACCTGTCTCATTT 1493 NR6A1
NM_033334 15451847 D-003431-02 GAAGAACTACACAGATTTA 1494 NR6A1
NM_033334 15451847 D-003431-03 GAAGATGGATACGCTGTGA 1495 NR6A1
NM_033334 15451847 D-003431-04 AAACGATACTGGTACATTT 1496 null D16815
2116671 D-003432-01 GAAGAATGATCGAATAGAT 1497 null D16815 2116671
D-003432-02 GAACATGGAGCAATATAAT 1498 null D16815 2116671
D-003432-03 GAGGAGCTCTTGGCCTTTA 1499 null D16815 2116671
D-003432-04 TAAACAACATGCACTCTGA 1500 PGR NM_000926 4505766
D-003433-01 GAGATGAGGTCAAGCTACA 1501 PGR NM_000926 4505766
D-003433-02 CAGCGTTTCTATCAACTTA 1502 PGR NM_000926 4505766
D-003433-03 AGATAACTCTCATTCAGTA 1503 PGR NM_000926 4505766
D-003433-04 GTAGTCAAGTGGTCTAAAT 1504 PPARA NM_005036 7549810
D-003434-01 TCACGGAGCTCACGGAATT 1505 PPARA NM_005036 7549810
D-003434-02 GAACATGACATAGAAGATT 1506 PPARA NM_005036 7549810
D-003434-03 GGATAGTTCTGGAAGCTTT 1507 PPARA NM_005036 7549810
D-003434-04 GACTCAAGCTGGTGTATGA 1508 PPARD NM_006238 5453939
D-003435-01 GAGCGCAGCTGCAAGATTC 1509 PPARD NM_006238 5453939
D-003435-02 GCATGAAGCTGGAGTACGA 1510 PPARD NM_006238 5453939
D-003435-03 GGAAGCAGTTGGTGAATGG 1511 PPARD NM_006238 5453939
D-003435-04 GCTGCAAGATTCAGAAGAA 1512 PPARG NM_138712 20336234
D-003436-01 AGACTCAGCTCTACAATAA 1513 PPARG NM_138712 20336234
D-003436-02 GATTGAAGCTTATCTATGA 1514 PPARG NM_138712 20336234
D-003436-03 AAGTAACTCTCCTCAAATA 1515 PPARG NM_138712 20336234
D-003436-04 GCATTTCTACTCCACATTA 1516 RARA NM_000964 4506418
D-003437-01 GACAAGAACTGCATCATCA 1517 RARA NM_000964 4506418
D-003437-02 GCAAATACACTACGAACAA 1518 RARA NM_000964 4506418
D-003437-03 GAACAACAGCTCAGAACAA 1519 RARA NM_000964 4506418
D-003437-04 GAGCAGCAGTTCTGAAGAG 1520 RARB NM_000965 14916493
D-003438-01 GCACACTGCTCAATCAATT 1521 RARB NM_000965 14916493
D-003438-02 GCAGAAGTATTCAGAAGAA 1522 RARB NM_000965 14916493
D-003438-03 GGAATGACAGGAACAAGAA 1523 RARB NM_000965 14916493
D-003438-04 GCACAGTCCTAGCATCTCA 1524 RARG NM_000966 21359851
D-003439-01 GAAATGACCGGAACAAGAA 1525 RARG NM_000966 21359851
D-003439-02 TAGAAGAGCTCATCACCAA 1526 RARG NM_000966 21359851
D-003439-03 CAAGGAAGCTGTGCGAAAT 1527 RARG NM_000966 21359851
D-003439-04 TCAGTGAGCTGGCTACCAA 1528 RORA NM_134261 19743902
D-003440-01 GGAAAGAGTTTATGTTCTA 1529 RORA NM_134261 19743902
D-003440-02 CAAGATCTGTGGAGACAAA 1530 RORA NM_134261 19743902
D-003440-03 GCACCTGACTGAAGATGAA 1531 RORA NM_134261 19743902
D-003440-04 CCGAGAAGATGGAATACTA 1532 RORB NM_006914 19743906
D-003441-01 GCACAGAACATCATTAAGT 1533 RORB NM_006914 19743906
D-003441-02 CCACACCTATGAAGAAATT 1534 RORB NM_006914 19743906
D-003441-03 GATCAAATTCTACTTCTGA 1535 RORB NM_006914 19743906
D-003441-04 TCAAACAGATAAAGCAAGA 1536 RORC NM_005060 19743908
D-003442-01 TAGAACAGCTGCAGTACAA 1537 RORC NM_005060 19743908
D-003442-02 TCACCGAGGCCATTCAGTA 1538 RORC NM_005060 19743908
D-003442-03 GAACAGCTGCAGTACAATC 1539 RORC NM_005060 19743908
D-003442-04 CCTCATGCCACCTTGAATA 1540 RXRA NM_002957 21536318
D-003443-01 TGACGGAGCTTGTGTCCAA 1541 RXRA NM_002957 21536318
D-003443-02 CAACAAGGACTGCCTGATT 1542 RXRA NM_002957 21536318
D-003443-03 GCAAGGACCTGACCTACAC 1543 RXRA NM_002957 21536318
D-003443-04 GCAAGGACCGGAACGAGAA 1544 RXRB NM_021976 21687229
D-003444-01 GCAAAGACCTTACATACTC 1545 RXRB NM_021976 21687229
D-003444-02 GCAATCATTCTGTTTAATC 1546 RXRB NM_021976 21687229
D-003444-03 TCACACCGATCCATTGATG 1547 RXRB NM_021976 21687229
D-003444-04 GCAAACGGCTATGTGCAAT 1548 RXRG NM_006917 21361386
D-003445-01 GGAAGGACCTCATCTACAC 1549 RXRG NM_006917 21361386
D-003445-02 GCGGATCTCTGGTTAAACA 1550 RXRG NM_006917 21361386
D-003445-03 GCGAGCCATTGTACTCTTT 1551 RXRG NM_006917 21361386
D-003445-04 GAGCCATTGTACTCTTTAA 1552 THRA NM_003250 20127451
D-003446-01 GGACAAAGACGAGCAGTGT 1553 THRA NM_003250 20127451
D-003446-02 GGAAACAGAGGCGGAAATT 1554 THRA NM_003250 20127451
D-003446-03 GTAAGCTGATTGAGCAGAA 1555 THRA NM_003250 20127451
D-003446-04 GAACCTCCATCCCACCTAT 1556 THRB NM_000461 10835122
D-003447-01 GAATGTCGCTTTAAGAAAT 1557 THRB NM_000461 10835122
D-003447-02 GAACAGTCGTCGCCACATC 1558 THRB NM_000461 10835122
D-003447-03 GGACAAGCACCAATAGTCA 1559 THRB NM_000461 10835122
D-003447-04 GTGGAAAGGTTGACTTGGA 1560 VDR NM_000376 4507882
D-003448-01 TGAAGAAGCTGAACTTGCA 1561 VDR NM_000376 4507882
D-003448-02 GCAACCAAGACTACAAGTA 1562 VDR NM_000376 4507882
D-003448-03 TCAATGCTATGACCTGTGA 1563 VDR NM_000376 4507882
D-003448-04 CCATTGAGGTCATCATGTT 1564
[0616]
15 TABLE X Gene Symbol Sense SEQ. ID NO. ABCB1 GACCAUAAAUGUAAGGUUU
1565 UAGAAGAUCUGAUGUCAAA 1566 GAAAUGUUCACUUCAGUUA 1567
GAAGAUCGCUACUGAAGCA 1568 ABCC1 GGAAGCAACUGCAGAGACA 1569
GAUGACACCUCUCAACAAA 1570 UAAAGUUGCUCAUCAAGUU 1571
CAACGAGUCUGCCGAAGGA 1572 ABCG2 GCAGAUGCCUUCUUCGUUA 1573
AGGCAAAUCUUCGUUAUUA 1574 GGGAAGAAAUCUGGUCUAA 1575
UGACUCAUCCCAACAUUUA 1576 KCNH2 CCGACGUGCUGCCUGAGUA 1577
GAGAAGAGCAGCGACACUU 1578 GAUCAUAGCACCUAAGAUA 1579
GCUAUUUACUGCUCUUAUU 1580 UCACUGGGCUCCUUUAAUU 1581
GUGCGAGCCUUCUGAAUAU 1582 GCUAAGCUAUACUACUGUA 1583
UGACGGCGCUCUACUUCAC 1584 KCNH1 GAGAUGAAUUCCUUUGAAA 1585
GAAGAACGCAUGAAACGAA 1586 GAUAAAGACACGAUUGAAA 1587
GCUGAGAGGUCUAUUUAAA 1588 CLCA1 GAACAACAAUGGCUAUGAA 1589
GUACAUACCUGGCUGGAUU 1590 GAACAGCUCACAAGUAUAU 1591
GGAAACGUGUGUCUAUAUU 1592 SLC6A1 GGAGGUGGGAGGACAGUUA 1593
UCACAGCCCUGGUGGAUGA 1594 GAAGCUGGCUCCUAUGUUC 1595
GGUCAACACUACCAACAUG 1596 SLC6A2 GAACACAAGGUCAACAUUG 1597
AGAAGGAGCUGGCCUAGUG 1598 CGGAAACUCUUCACAUUUG 1599
CAACAAAUUUGACAACAAC 1600 SLC21A2 GUACAUCUCCAUCUUAUUU 1601
GGAAGUGGCUGAGUUAUUA 1602 GAAGGGAGGCUCAAUGUAA 1603
GAAGGAAGUGGCUGAGUUA 1604 SLC21A3 GUAGAAACAGGAGCUAUUA 1605
CAAGAUUACUGUCAAACAA 1606 GCACAAGAGUAUUUGGUAA 1607
GCAAAUGUCCCUUCUGUAU 1608 GCAUGACUCCUAUAUAAUA 1609
AAACAGCAAUUUCCCUUAA 1610 GAAAAUGCCUCUUCAGGAA 1611 SLC28A1
GUUCAUCGCUCUCCUCUUU 1612 GGAUCAAGCUGUUUCUGAA 1613
GGACUGCAGUUUGUACUUG 1614 GAGUGAAACUGACCUAUGG 1615 SLC29A1
GAACGCUGCUCCCGUGGAA 1616 GAAAGCCACUCUAUCAAAG 1617
GAAACCAGGUGCCUUCAGA 1618 CCUCACAGCUGUAUUCAUG 1619 SLC26A1
CCACGGAGCUGCUGGUCAU 1620 GGGUUGACAUCUUAUUUGA 1621
GCACGAGGGUCUCUGUGUU 1622 GGCCAUCGCCUACUCAUUG 1623
CAACACCCAUGGCAAUUAA 1624 GAGGAAAGAUCUUGCUGAU 1625
GAGCAAGCGUCCUCCAAAU 1626 GCAACACCCAUGGCAAUUA 1627 SLC26A2
CCAAAGAACUCAAUGAACA 1628 ACAAGAACCUUCAGACUAA 1629
GAAGGUAGAUAGAAGAAUG 1630 GUAUUGAACUGUACUGUAA 1631 SLC4A4
GCAAUUCUCUUCAUUUAUC 1632 GGAAAGAUGUCCACUGAAA 1633
GGACAAAGCCUUCUUCAAU 1634 GGAAUGGGAUCCAGCAAUU 1635 GLRA1
UGAAAGCCAUUGACAUUUG 1636 CAGACACGCUGGAGUUUAA 1637
CAAUAGCGCUUUCUGGUUU 1638 GCAGGUAGCAGAUGGACUA 1639 KLK1
UCAGAGUGCUGUCUUAUGU 1640 CAACUUGUUUGACGACGAA 1641
UGACAGAGCCUGCUGAUAC 1642 AGGCGGCUCUGUACCAUUU 1643 ADAM2
GAAACAUGCUGUGAUAUUG 1644 GCAGAUGUUUCCUUAUAUA 1645
CAACAGAGAUGCCAUGAUA 1646 GAAAGGCGCUACAUUGAGA 1647 XPNPEP1
GACCUGAGCUUCCCAACAA 1648 GCGACUGGCUCAACAAUUA 1649
GAGAUUGCGUGGCUAUUUA 1650 GACAGCAACUGGACACUUA 1651 GZMA
GGAAGAGACUCGUGCAAUG 1652 GGAACCAUGUGCCAAGUUG 1653
GAAGUAACUCCUCAUUCAA 1654 GAACUCCUAUAGAUUUCUG 1655 CMKLR1
CAUAGAAGCUUUACCAAGA 1656 GAAUGGAGGAUGAAGAUUA 1657
GGUCAAUGCUCUAAGUGAA 1658 GAGAGGACUUCUAUGAAUG 1659 CLN3
CAUCAUGCCUUCUGAAUAA 1660 CAACAGCUCAUCACGAUUU 1661
GCAACAACUUCUCUUAUGU 1662 GGUCUUCGCUAGCAUCUCA 1663 CALCR
GGACCUAGCUGUUGUAAAG 1664 GAAAGACCAUGCAUUUAAA 1665
GCAGGAAGAUGUAUGCUUU 1666 GAAUAAACCAGUAUCGUUA 1667 OXTR
GGACCCAGAUAUCCAAAUA 1668 GCAAUACUAUCCUAACUGA 1669
GAAUAUAGAUUAGCGUUUG 1670 GAUGAGGCAUGACUACUAA 1671 EDG4
GCGAGUCUGUCCACUAUAC 1672 GAGAACGGCCACCCACUGA 1673
GAACGGCCACCCACUGAUG 1674 GGUCAAUGCUGCUGUGUAC 1675 EDG5
UCCAGGAACACUAUAAUUA 1676 GUGACCAUCUUCUCCAUCA 1677
CAUCCUCUGUUGCGCCAUU 1678 CCAACAAGGUCCAGGAACA 1679 EDG7
ACACUGAUACUGUCGAUGA 1680 AAUAGGAGCAACACUGAUA 1681
CAGCAGGAGUUACCUUGUU 1682 GGACACCCAUGAAGCUAAU 1683 PTCH
GCACAGAACUCCACUCAAA 1684 GGACAGCAGUUCAUUGUUA 1685
GAGAAGAGGCUAUGUUUAA 1686 GGACAAACUUCGACCCUUU 1687 SMO
UCGCUACCCUGCUGUUAUU 1688 GCUACAAGAACUACCGAUA 1689
CAAGAAAGCUUCCUUCAAC 1690 GAGAAGAAAUACAGUCAAU 1691 CASP3
CAAUAUAUCUGAAGAGCUA 1692 GAACUGGACUGUGGCAUUG 1693
GUGAGAAGAUGGUAUAUUU 1694 GAGGGUACUUUAAGACAUA 1695 CASP6
CAUGAGGUGUCAACUGUUA 1696 GAAGUGAAAUGCUUUAAUG 1697
AAAUAUGGCUCCUCCUUAG 1698 GCAAUCACAUUUAUGCAUA 1699
CAACAUAACUGAGGUGGAU 1700 CAUGGUACAUUCAAGAUUU 1701 CASP7
GAACUCUACUUCAGUCAAU 1704 GGGCAAAUGCAUCAUAAUA 1703
CAACAGAGGGAGUUUAAUA 1704 GAACAAAGCCACUGACUGA 1705 CASP8
GAAGUGAACUAUGAAGUAA 1706 CAACAAGGAUGACAAGAAA 1707
GGACAAAGUUUACCAAAUG 1708 GAGGGUCGAUCAUCUAUUA 1709
GAAUAUAGAGGGCUUAUGA 1710 CAACGACUAUGAAGAAUUC 1711
GAAGUGAGCAGAUCAGAAU 1712 GAGGAAAUCUCCAAAUGCA 1713 CASP9
CCAGGCAGCUGAUCAUAGA 1714 UCUCAGGUGUUGCCAAAUA 1715
GAACAGCUGUAAUCUAUGA 1716 CCACUGGUCUGUAGGGAUU 1717 DVL1
UCGUAAAGCUGUUGAUAUC 1718 GAGGAGAUCUUUGAUGACA 1719
GUAAAGCUGUUGAUAUCGA 1720 GAUCGUAAAGCUGUUGAUA 1721 DVL2
AGACGAAGGUGAUUUACCA 1722 UGUGAGAGGUACCUAGUCA 1723
GAAGAAAUUUCAGAUGACA 1724 UAAUAGGCAUUUCCUCUUU 1725 PTEN
GUGAAGAUCUUGACCAAUG 1726 GAUCAGCAUACACAAAUUA 1727
GAAUGAACCUUCUGCAACA 1728 GGCGCUAUGUGUAUUAUUA 1729 PDK1
GUACAAAGCUGGUAUAUCC 1730 GAAAGACUCCCAGUGUAUA 1731
GGAAGUCCAUCUCAUCGAA 1732 CCAAAGACAUGACGACGUU 1733 PDK2
GUAAAGAGGAGACUGAAUG 1734 GGUCUGUGAUGGUCCCUAA 1735
CAAAGAUGCCUACGACAUG 1736 GGGCGAUGCCUGAGGGUUA 1737 PPP2CA
UCACACAAGUUUAUGGUUU 1738 CAACAGCCGUGACCACUUU 1739
UAACCAAGCUGCAAUCAUG 1740 GAACUUGACGAUACUCUAA 1741 CTNNA1
GAAGAGAGGUCGUUCUAAG 1742 AAGCAGAUGUGCAUGAUUA 1743
UCUAAUAACUGCAGUGUUU 1744 GUAAAGGGCCCUCUAAUAA 1745 CTNNA2
GAAAGAAUAUGCCCAAGUU 1746 GAAGAAGAAUGCCACAAUG 1747
GCAGGAAGAUUAUGAUGUG 1748 AAAGAAAGCCCAUGUACUA 1749 HSPCA
GGGAAAGAGCUGCAUAUUA 1750 GCUUAGAACUCUUUACUGA 1751
UAUAAGAGCUUGACCAAUG 1752 GCAGAUAUCUCUAUGAUUG 1753 DCTN2
CAACUCAUGUCCAAUACUG 1754 GGAAUGAGCCAGAUGUUUA 1755
GGAGACAGCUGUACGUUGU 1756 UCCAAGAGCUGACAACUGA 1757 CD2
GUAAGGAGAAGCAAUAUAA 1758 AAGAUGAGCUUUCCAUGUA 1759
GGACAUCUAUCUCAUCAUU 1760 GACAAGAGCCCACAGAGUA 1761 BAD
GUACUUCCCUCAGGCCUAU 1762 GCUGUGCCUUGACUACGUA 1763
GUACUUCCCUCAGGCCUAU 1764 GGUCAGGUGCCUCGAGAUC 1765 SMAC
CAGCGUAACUUCAUUCUUC 1766 UAACUUCAUUCUUCAGGUA 1767
CAGCUGCUCUUACCCAUUU 1768 GAUUGAAGCUAUUACUGAA 1769
UAGAAGAGCUCCGUCAGAA 1770 CCACAUAUGCGUUGAUUGA 1771
GCGCAGGGCUCUCUACCUA 1772 MAP3K5 GAACAGCCUUCAAAUCAAA 1773
GAUGUUCUCUACUAUGUUA 1774 GCAAAUACUGGAAGGAUUA 1775
CAGGAAAGCUCGUAAUUUA 1776 PVR CCACACGGCUGACCUCAUA 1777
CAGCAGAAUUCCUCUUAUA 1778 GCAGAAUUCCUCUUAUAAA 1779
GAUCGGGAUUUAUUUCUAU 1780 ERBB2 UGUGGGAGCUGAUGACUUU 1781
UCACAGAGAUCUUGAAAGG 1782 UGGAAGAGAUCACAGGUUA 1783
GCUCAUCGCUCACAACCAA 1784 SOS1 GAGCACCACUUCUAUGAUU 1785
CAAAGAAGCUGUUCAAUAU 1786 UGAAAGCCCUCCCUUAUUA 1787
GAAAUAGCAUGGAGAAGGA 1788 BRCA1 CCAUACAGCUUCAUAAAUA 1789
GAAGAGAACUUAUCUAGUG 1790 GAAGUGGGCUCCAGUAUUA 1791
GCAAGAUGCUGAUUCAUUA 1792 GAAGUGGGCUCCAGUAUUA 1793
GAACGGACACUGAAAUAUU 1794 GCAGAUAGUUCUACCAGUA 1795 CDKN1A
GAACAAGGAGUCAGACAUU 1796 AAACUAGGCGGUUGAAUGA 1797
GAUGGAACUUCGACUUUGU 1798 GUAAACAGAUGGCACUUUG 1799 CDKN1B
GGAAUGGACAUCCUGUAUA 1800 GGAGAAAGAUGUCAAACGU 1801
GAAUGGACAUCCUGUAUAA 1802 GUAAACAGCUCGAAUUAAG 1803 SLC2A4
CAGAUAGGCUCCGAAGAUG 1804 AGACUCAGCUCCAGAAUAC 1805
GAUCGGUUCUUUCAUCUUC 1806 CAGGAUCGGUUCUUUCAUC 1807 NOS2A
CCAGAUAAGUGACAUAAGU 1808 UAAGUGACCUGCUUUGUAA 1809
GAAGAGAGAUUCCAUUGAA 1810 UGAAAGAGCUCAACAACAA 1811 FRAP1
GAGCAUGCCGUCAAUAAUA 1812 CAAGAGAACUCAUCAUAAG 1813
CCAAAGUGCUGCAGUACUA 1814 UAAGAAAGCUAUCCAGAUU 1815 FKBP1A
GAAACAAGCCCUUUAAGUU 1816 GAAUUACUCUCCAAGUUGA 1817
CAGCACAAGUGGUAGGUUA 1818 GUUGAGGACUGAAUUACUC 1819
GAUGGCAGCUGUUUAAAUG 1820 GAGUAUCCUUUCAGUGUUA 1821 TNFRSF1A
CAAAGGAACCUACUUGUAC 1822 GGAACCUACUUGUACAAUG 1823
GAACCUACUUGUACAAUGA 1824 GAGUGUGUCUCCUGUAGUA 1825 IL1R1
GGACAAGAAUCAAUGGAUA 1826 GAACAAGCCUCCAGGAUUC 1827
GGACUUGUGUGCCCUUAUA 1828 GAACACAAAGGCACUAUAA 1829 IRAK1
CGAAGAAAGUGAUGAAUUU 1830 GCUCUUUGCCCAUCUCUUU 1831
UGAAAGACCUGGUGGAAGA 1832 GCAAUUCAGUUUCUACAUC 1833 TRAF2
GAAGACAGAGUUAUUAAAC 1834 UCACGAAGACAGAGUUAUU 1835
AGACAGAGUUAUUAAACCA 1836 CACGAAGACAGAGUUAUUA 1837
GCUGAAGCCUGUCUGAUGU 1838 TRAF6 CAAAUGAUCUGAGGCAGUU 1839
GUUCAUAGUUUGAGCGUUA 1840 GGAGAAACCUGUUGUGAUU 1841
GGACAAAGUUGCUGAAAUC 1842 CAAAUGAUCUGAGGCAGUU 1843
GGAGAAACCUGUUGUGAUU 1844 GGACAAAGUUGCUGAAAUC 1845
GUUCAUAGUUUGAGCGUUA 1846 TRADD UGAAGCACCUUGAUCUUUG 1847
GGGCAGCGCAUACCUGUUU 1848 GAGGAGCGCUGUUUGAGUU 1849
GGACGAGGAGCGCUGUUUG 1850 GAGGAGCGCUGUUUGAGUU 1851
GGAUGUCUCUCUCCUCUUU 1852 GCUCACUCCUUUCUACUAA 1853
UGAAGCACCUUGAUCUUUG 1854 FADD GCACAGAUAUUUCCAUUUC 1855
GCAGUCCUCUUAUUCCUAA 1856 GAACUCAAGCUGCGUUUAU 1857
GGACGAAUUGAGAUAAUAU 1858 IKBKE UAAGAACACUGCUCAUGAA 1859
GAGGCAUCCUGAAGCAUUA 1860 GAAGGCGGCUGCAGAACUG 1861
GGAACAAGGAGAUCAUGUA 1862 IKBKG CUAUCGAGGUCGUUAAAUU 1863
GAAUGCAGCUGGAAGAUCU 1864 GCGGCGAGCUGGACUGUUU 1865
CCAGACCGAUGUGUAUUUA 1866 TNFRSF5 GGUCUCACCUCGCUAUGGU 1867
GAAAGCGAAUUCCUAGACA 1868 GCACAAACAAGACUGAUGU 1869
GAAGGGCACCUCAGAAACA 1870 UCUCCCAACUUGUAUUAAA 1871 RELA
UCAAGUGUCUUCCAUCAUG 1872 UCAAGUGCCUUAAUAGUAG 1873
GGAGUACCCUGAGGCUAUA 1874 GAUGAGAUCUUCCUACUGU 1875 ARHA
GAGCUGGGCUAAGUAAAUA 1876 GACCAAAGAUGGAGUGAGA 1877
GGAAGAAACUGGUGAUUGU 1878 GGCUGUAACUACUUUAUAA 1879 CDC42
GGACAUUUGUUUGCCAUUU 1880 GGAGAACCAUAUACUCUUG 1881
GAACCAAUGCUUUCUCAUG 1882 GAAGACCUGUUAUGUAGAG 1883
GAUCAAGAAUUGCAAUAUC 1884 GAAAAGGGGUGACCUAGUA 1885
UGACAAACCUUAUGGAAAA 1886 ROCK1 GGAAUGAGCUUCAGAUGCA 1887
GGACACAGCUGUAAGAUUG 1888 GACAAGAGAUUACAGAUAA 1889
GAAGAAACAUUCCCUAUUC 1890 PAK1 GAGGGUGGUUUAUGAUUAA 1891
CAACAAAGAACAAUCACUA 1892 GAAGAAAUAUACACGGUUU 1893
UACAUGAGCUUUACAGAUA 1894 PAK2 GGUAGGAGAUGAAUUGUUU 1895
AGAAGGAACUGAUCAUUAA 1896 CUACAGACCUCCAAUAUCA 1897
GAAACUGGCCAAACCGUUA 1898 PAK3 GAUUAUCGCUGCAAAGGAA 1899
GAGAGUGCCUGCAAGCUUU 1900 GACAAGAGGUGGCCAUAAA 1901
UUAAAUCGCUGUCUUGAGA 1902 PAK4 ACUAAGAGGUGAACAUGUA 1903
GAUCAUGAAUGUCCGAAGA 1904 GAUGAGACCCUACUACUGA 1905
CAGCAAAGGUGCCAAAGAU 1906 PAK6 UAAAGGCAGUUGUCCACUA 1907
GAAGGGACCUGCUUUCUUG 1908 GCAAAGACGUCCCUAAGAG 1909
CCAAUGGGCUGGCUGCAAA 1910 PAK7 GAGCACGGCUUUAAUAAGU 1911
CAAACUCCGUUAUGAUAUA 1912 GGAUAAAGUUGUCUGAUUU 1913
GGAAAUGCCUCCAUAAAUA 1914 HDAC1 GGACAUCGCUGUGAAUUGG 1915
AGAAAGAAGUCACCGAAGA 1916 GGACAAGGCCACCCAAUGA 1917
CCACAGCGAUGACUACAUU 1918 HDAC2 GCUGUUAAAUUAUGGCUUA 1919
GCAAAGAAAGCUAGAAUUG 1920 CAUCAGAGAGUCUUAUAUA 1921
CCAAUGAGUUGCCAUAUAA 1922 CREBBP GGCCAUAGCUUAAUUAAUC 1923
GCACAGCCGUUUACCAUGA 1924 GGACAGCCCUUUAGUCAAG 1925
GAACUGAUUCCUGAAAUAA 1926 BTRC CACAUAAACUCGUAUCUUA 1927
GAGAAGGCACUCAAGUUUA 1928 AGACAUAGUUUACAGAGAA 1929
GCAGAGAGAUUUCAUAACU 1930 RIPK2 GAACAUACCUGUAAAUCAU 1931
GGACAUCGACCUGUUAUUA 1932 UAAAUGAACUCCUACAUAG 1933
GGAAUUAUCUCUGAACAUA 1934 VAV1 GCAGAAAUACAUCUACUAA 1935
GCUAUGAGCUGUUCUUCAA 1936 CGACAAAGCUCUACUCAUC 1937
GCUCAACCCUGGAGACAUU 1938 VAV2 GGACAAGACUCGCAGAUUU 1939
GCUGAGCGCUUUGCAAUAA 1940 CAAGAAGUCUCACGGGAAA 1941
UCACAGAGGCCAAGAAAUU 1942 GRB2 UGGAAGCCAUCGCCAAAUA 432
CAUCAGUGCAUGACGUUUA 1943 UGAAUGAGCUGGUGGAUUA 1944
UGCCAAAACUUACCUAUAA 1945 PLCG1 GAGCUGCACUCCMUGAGA 1946
GAAACCAAGCCAUUAAUGA 1947 CCAAGGAGCUACUGACAUU 1948
AGAGAAACAUGGCCCAAUA 1949 ITGB1 CCACAGACAUUUACAUUAA 1950
GAAGGGAGUUUGCUAAAUU 1951 GAACAGAUCUGAUGAAUGA 1952
CAAGAGAGCUGAAGACUAU 1953 ITGA4 GCAUAUAUAUUCAGCAUUG 1954
CAACUUGACUGCAGUAUUG 1955 GAACUUAACUUUCCAUGUU 1956
GACAAGACCUGUAGUAAUU 1957 STAT1 AGAAAGAGCUUGACAGUAA 1958
GGAAGUAGUUCACAAAAUA 1959 UGAAGUAUCUGUAUCCAAA 1960
GAGCUUCACUCCCUUAGUU 1961 KRAS2 UAAGGACUCUGAAGAUGUA 1962
GACAAAGUGUGUAAUUAUG 1963 GCUCAGGACUUAGCAAGAA 1964
GAAACUGAAUACCUAAGAU 1965 GAAACUGAAUACCUAAGAU 1966
UAAGGACUCUGAAGAUGUA 1967 GACAAAGUGUGUAAUUAUG 1968
GCUCAGGACUUAGCAAGAA 1969 HRAS CCAUCCAGCUGAUCCAGAA 1970
GAACCCUCCUGAUGAGAGU 1971 GAGGACAUCCACCAGUACA 1972 BRAF
GAUUAGAGACCAAGGAUUU 2410 CCACUGAUGUGUGUUAAUU 1973
CAAUAGAACCUGUCAAUAU 1974 GAAGACAGGAAUCGAAUGA 1975 ELK1
GAUGUGAGUAGAAGAGUUA 2411 GGAAGAAUUUGUACCAUUU 1976
GAACGACCUUUCUUUCUUU 1977 GGAGUCAUCUCUUCCUAUA 1978 RALGDS
GGAGAAGCCUCACCUCUUG 1979 GCAGAAAGGACUCAAGAUU 1980
GAGAACAACUACUCAUUGA 1981 GAACUUCUCGUCACUGUAU 1982 PRKCA
GGAUUGUUCUUUCUUCAUA 1983 GAAGGGUUCUCGUAUGUCA 1984
GAAGAAGGAUGUGGUGAUU 1985 GGACUGGGAUCGAACAACA 1986 MAP2K4
GGACAGAAGUGGAAAUAUU 1987 UCAAAGAGGUGAACAUUAA 1988
GACCAAAUCUCAGUUGUUU 1989 GGAGAAUGGUGCUGUUUAA 1990 MAP2K7
GAAGAGACCAAAGUAUAAU 1991 GAAGACCGGCCACGUCAUU 1992
GGAAGAGACCAAAGUAUAA 1993 GCAUUGAGAUUGACCAGAA 1994
UGAGAGAACGAGAAAGUUG 1995 GUGAAACCCUGUCUGCAUU 1996
GGAUCUCUCUCAACAACUA 1997 ACAACUAGGUGAACACAUA 1998 MAPK8
UCACAGUCCUGAAACGAUA 1999 GAUUGGAGAUUCUACAUUC 2000
GCUCAUGGAUGCAAAUCUU 2001 GAAGCUAAGCCGACCAUUU 2002 MAPK9
AAAGAGAGCUUAUCGUGAA 2003 GAUGAUAGGUUAGAAAUAG 2004
ACAAAGAAGUCAUGGAUUG 2005 GGAGCUGGAUCAUGAAAGA 2006 AIF1
GAAAAGGGAUGAUGGGAUU 2007 CCUAGACGAUCCCAAAUAU 2008
GAGCCAAACCAGGGAUUUA 2009 UGAAACGAAUGCUGGAGAA 2010
UCACUCACCCAGAGAAAUA 2011 CCAAGAAAGCUAUCUCUGA 2012
AGACUCACCUAGAGCUAAA 2013 BBC3 CCUGGAGGGUCCUGUACAA 2014
GAGCAAAUGAGCCAAACGU 2015 GGAGGGUCCUGUACAAUCU 2016
GACUUUCUCUGCACCAUGU 2017 BCL2L1 CCAGGGAGCUUGAAAGUUU 2018
AAAGUGCAGUUCAGUAAUA 2019 GAGAAUCACUAACCAGAGA 2020
GAGCCCAUCCCUAUUAUAA 2021 BCL2L11 GAGACGAGUUUAACGCUUA 2022
AAAGCAACCUUCUGAUGUA 2023 CCGAGAAGGUAGACAAUUG 2024
GCAAAGCAACCUUCUGAUG 2025 AGACAGAGCCACAAGGUAA 2026
GCAAGGAGGUUAGAGAAAU 2027 CAAGGAGGUUAGAGAAAUA 2028
UCUUACGACUGUUACGUUA 2029 BID GAAGACAUCAUCCGGAAUA 2030
CAACAGCGUUCCUAGAGAA 2031 GAAAUGGGAUGGACUGAAC 2032
ACGAUGAGCUGCAGACUGA 2033 BIRC2 GAAAGAAGCCUGCAUAUAA 2034
GAAAUUGACUCUACAUUGU 2035
ACAAAUAGCACUUAGGUUA 2036 GAAUACACCUGUGGUUAAA 2037 BIRC3
GGAGAUGCCUGCCAUUAAA 2038 UCAAUGAUCUUGUGUUAGA 2039
GAAAGAACAUGUAAAGUGU 2040 GAAGAAAGAACAUGUAAAG 2041 BIRC4
GUAGAUAGAUGGCAAUAUG 2042 GAGGAGGGCUAACUGAUUG 2043
GAGGAACCCUGCCAUGUAU 2044 GCACGGAUCUUUACUUUUG 2045 BIRC5
GGCGUAAGAUGAUGGAUUU 2046 GCAAAGGAAACCAACAAUA 2047
GCACAAAGCCAUUCUAAGU 2048 CAAAGGAAACCAACAAUAA 2049 BRCA1
CCAUACAGCUUCAUAAAUA 2050 GAAGAGAACUUAUCUAGUG 2051
GAAGUGGGCUCCAGUAUUA 2052 GCAAGAUGCUGAUUCAUUA 2053
CCAUACAGCUUCAUAAAUA 2054 CARD4 GAAAGUUAAUGUCAAGGAA 2055
GAGCAACACUGGCAUAACA 2056 UAACAGAGAUUUGCCUAAA 2057
GCGAAGAGCUGACCAAAUA 2058 CASP10 CAAAGGGUUUCUCUGUUUA 2059
GAAAUGACCUCCCUAAGUU 2060 GAAGGCAGCUGGUAUAUUC 2061
GACAUGAUCUUCCUUCUGA 2062 GCACUCUUCUGUUCCCUUA 2063 CASP2
GUAUUAAACUCUCCUUUGA 2064 GCAAGGAGAUGUCUGAAUA 2065
CAACUUCCCUGAUCUUUAA 2066 GCUCAAAGAUGUAAUGUAG 2067 CDKN1A
GAACAAGGAGUCAGACAUU 2068 AAACUAGGCGGUUGAAUGA 2069
GAUGGAACUUCGACUUUGU 2070 GUAAACAGAUGGCACUUUG 2071 CFLAR
GAUGUGUCCUCAUUAAUUU 2072 GAAGAGAGAUACAAGAUGA 2073
GAGCAUACCUGAAGAGAGA 2074 GCUAUGAAGUCCAGAAAUU 2075 CLK2
GUGAAUAUGUGAAAUAGUG 2076 AAAGCAUGCUAGAGUAUGA 2077
UUAAGAAUGUGGAGAAGUA 2078 GAUAACAAGCUGACACAUA 2079 CLSPN
GGACGUAAUUGAUGAAGUA 2080 GCAGAUGGGUUCUUAAAUG 2081
CAAAUGAGGUUGAGGAAAU 2082 GGAAAUACCUGGAGGAUGA 2083 CSNK2A1
GAUCCACGUUUCAAUGAUA 2084 GCAUUUAGGUGGAGACUUC 2085
GAUGUACGAUUAUAGUUUG 2086 UGAAUUAGAUCCACGUUUC 2087 CTNNB1
GCACAAGAAUGGAUCACAA 2088 GCUGAAACAUGCAGUUGUA 2089
GUACGUACCAUGCAGAAUA 2090 GAACUUGCAUUGUGAUUGG 2091 CXCR4
GAAGCAUGACGGACAAGUA 2092 GAACAUUCCAGAGCGUGUA 2093
GUUCUUAGUUGCUGUAUGU 2094 CAUCAUGGUUGGCCUUAUC 2095 CXCR6
GGAACAAACUGGCAAAGCA 2096 GAUCAGAGCAGCAGUGAAA 2097
GGGCAAAACUGAAUUAUAA 2098 GAUCUCAGGUUCUCCUUGA 2099 DAXX
CUACAGAUCUCCAAUGAAA 2100 GCUACAAGCUGGAGAAUGA 2101
GGAAACAGCUAUGUGGAAA 2102 GGAGUUGGAUCUCUCAGAA 2103 GAS41
GUAGUAAGCUAAACUGAAA 2104 GACAAUAUGUUCAAGAGAA 2105
GACAACAUCUCGUCAGCUA 2106 UAUAUGAUGUGUCCAGUAA 2107 GTSE1
CAAAGAAGCUCACUUACUG 2108 GAACAGCCCUAAAGUGGUU 2109
GAACAUGGAUGACCCUAAG 2110 GGGCAAAGCUAAAUCAAGU 2111 HDAC3
GGAAAGCGAUGUGGAGAUU 2112 CCAAGACCGUGGCCUAUUU 2113
AAAGCGAUGUGGAGAUUUA 2114 GUGAGGAGCUUCCCUAUAG 2115 HDAC5
GAAUUCCUCUUGUCGAAGU 2116 GUUAUUAGCACCUUUAAGA 2117
GGAGGGAGGCCAUGACUUG 2118 CAGGAGAGCUCAAGAAUGG 2119
GGAUAUGGAUUUCAGUUAA 2120 GGAAGUCGGUGCCUUGGUU 2121
GGAAGGAGAGGACUGGUUU 2122 HEC GCAGAUACUUGCACGGUUU 2123
GAGUAGAACUAGAAUGUGA 2124 GCGAAUAAAUCAUGAAAGA 2125
GAAGAUGGAAUUAUGCAUA 2126 HIST1H2 GGCAAUGCGUCUCGCGAUA 2127 AA
GAUCCGCAAUGAUGAGGAA 2128 GCAAUGCGUCUCGCGAUAA 2129
GAGGAACUCAAUAAGCUUU 2130 LMNB1 AAUAGAAGCUGUGCAAUUA 2131
CAACUGACCUCAUCUGGAA 2132 GAAGGAAUCUGAUCUUAAU 2133
GGGAAGGGUUUCUCUAUUA 2134 LMNB2 GGAGGUUCAUUGAGAAUUG 2134
GGCAAUAGCUCACCGUUUA 2135 CAAAUACGCUUAGCUGUGU 2136
GGAGAUCGCCUACAAGUUC 2137 MYB GCAGAAACACUCCAAUUUA 2138
GUAAAUACGUGAAUGCAUU 2139 GCACUGAACUUUUGAGAUA 2140
GAAGAACAGUCAUUUGAUG 2141 MYT1 GAGGUGAGCUGUUAAAUCA 2142
GCAGGGUGAUUUCCUAAUA 2143 GGGAGAAGAUAUUUAAUUG 2144
CAACUUCUCUCCUGAACUU 2145 NFKBIB GGACACGGCACUGCACUUG 2146
GCACUUGGCUGUGAUUCAU 2148 GAGACGAGGGCGAUGAAUA 2149
CAUGAACCCUUCCUGGAUU 2150 NFKBIA GAACAUGGACUUGUAUAUU 2151
GAUGUGGGGUGAAAAGUUA 2152 GGACGAGAAAGAUCAUUGA 2153
AGGACGAGCUGCCCUAUGA 2154 NFKBIE GAAGGGAAGUUUCAGUAAC 2155
GGAAGGGAAGUUUCAGUAA 2156 GAAACUGCUGCUGUGUAC 2157
GAACCAACCACUCAUGGAA 2158 NUMA1 GGGAACAGUUUGAAUAUAA 2159
GCAGUAGCCUGAAGCAGAA 2160 CGAGAAGGAUGCACAGAUA 2161
GCAAGAGGCUGAGAGGAAA 2162 NUP153 GAAGACAAAUGAAAGCUAA 2163
GAUAAAGACUGCUGUUAGA 2164 GAGGAGAGCUCUAAUAUUA 2165
GAGGAAGCCUGAUUAAAGA 2166 OPA1 GAAAGAGCAUGAUGACAUA 2167
GAGGAGAGCUCUAUUAUGU 2168 GAAACUGAAUGGAAGAAUA 2169
AAAGAAGGCUGUACCGUUA 2170 PARVA CUACAUGUCUUUGCUCUUA 2171
GCUAAGUCCUGUAAGAAUA 2172 CAAAGGCAAUGUACUGUUU 2173
GAACAAUGGUGGAUCCAAA 2174 PIK3CG AAGUUCAGCUUCUCUAUUA 2175
GAAGAAAUCUCUGAUGGAU 2176 GAACACCUUUACUCUAUAA 2177
GCAUGGAGCUGGAGAACUA 2178 PRKDC AUGAAAGCUCUAAAGAUG 2179
AAAGGAGGUUCUAAACUA 2180 GAAGAAGCUCAUUUGAUU 2181 GCAAAGAGGUGGCAGUUAA
2182 RASA1 GGAAGAAGAUCCACAUGAA 2183 GAACAUACUUUCAGAGCUU 2184
GAACAAUCUUUGCUGUAUA 2185 UAACAGAACUGCUUCAACA 2186 SLC9A1
GAAGAGAUCCACACACAGU 2187 UCAAUGAGCUGCUGCACAU 2188
GAAGAUAGGUUUCCAUGUG 2189 GAAUUACCCUUCCUCAUCU 2190 TEGT
CUACAGAGCUUCAGUGUGA 2191 GAACAUAUUUGAUCGAAAG 2192
GAGCAAACCUAGAUAAGGA 2193 GCAUUGAUCUCUUCUUAGA 2194 TERT
GGAAGACAGUGGUGAACUU 2195 GCAAAGCAUUGGAAUCAGA 2196
GAGCUGACGUGGAAGAUGA 2197 GAACGGGCCUGGAACCAUA 2198 TNFRSF6
GAUACUAACUGCUCUCAGA 2199 GAAAGAAUGGUGUCAAUGA 2200
UCAAUAAUGUCCCAUGUAA 2201 UCAUGAAUCUCCAACCUUA 2202
GAUGUUGACUUGAGUAAAU 2203 TOP1 AAAGGAAAUGACUAAUGA 2204
AAGAAGGCUGUUCAGAGA 2205 GAAGUAGCUACGUUCUUU 2206 GACAUAAGUGGAAAGAAG
2207 TOP2A GAAAGAGUCCAUCAGAUUU 2208 CAAACUACAUUGGCAUUUA 2209
AAACAGACAUGGAUGGAUA 2210 CGAAAGGAAUGGUUAACUA 2211 TOP3A
CCAGAAAUCUUCCACAGAA 2212 GAAACUAUCUGGAUGUGUA 2213
CCACAAAGAUGGUAUCGUA 2214 GGAAAUGGCUGUGGUAACA 2215 TOP3B
GAGACAAGAUGAAGACUGU 2216 GCACAUGGGCUGCGUCUUU 2217
CCAGUGCGCUUCAAGAUGA 2218 GAACAUCUGCUUUGAGGUU 2219 WEE1
GGUAUUGCCUUGUGAAUUU 2220 GCAGAACAAUUACGAAUAG 2221
GUACAUAGCUGUUUGAAAU 2222 GCUGUAAACUUGUAGCAUU 2223
[0617] In addition, to identifying functional siRNA against gene
families or pathways, it is possible to design duplexes against
genes known to be involved in specific diseases. For example when
dealing with human disorders associated with allergies, it will be
beneficial to develop siRNA against a number of genes including but
not limited to:
16 the interleukin 4 receptor gene (SEQ. ID NO. 2224:
UAGAGGUGCUCAUUCAUUU, SEQ. ID NO. 2225: GGUAUAAGCCUUUCCAAGA, SEQ. ID
NO. 2412: ACACACAGCUGGAAGAAAU, SEQ. ID NO. 2226:
UAACAGAGCUUCCUUAGGU), the Beta-arrestin-2 (SEQ. ID NO. 2227:
GGAUGAAGGAUGACGACUA, SEQ. ID NO. 2228: ACACCAACCUCAUUGAAUU, SEQ. ID
NO. 2229: CGAACAAGAUGACCAGGUA, SEQ. ID NO. 2230:
GAUGAAGGAUGACGACUAU,), the interferon-gamma receptor 1 gene (SEQ.
ID NO. 2231: CAGCAUGGCUCUCCUCUUU, SEQ. ID NO. 2232:
GUAAAGAACUAUGGUGUUA, SEQ. ID NO. 2233: GAAACUACCUGUUACAUUA, SEQ. ID
NO. 2234: GAAGUGAGAUCCAGUAUAA), the matrix metalloproteinase MMP-9
(SEQ. ID NO. 2235: GGAACCAGCUGUAUUUGUU, SEQ. ID NO. 2236:
GUUGGAGUGUUUCUAAUAA, SEQ. ID NO. 2237: GCGCUGGGCUUAGAUCAUU, SEQ. ID
NO. 2238: GGAGCCAGUUUGCCGGAUA), the Slclla1 (Nrampl) gene (SEQ. ID
NO. 2239: CCAAUGGCCUGCUGAACAA, SEQ. ID NO. 2240:
GGGCCUGGCUUCCUCAUGA, SEQ. ID NO. 2241: GGGCAGAGCUCCACCAUGA, SEQ. ID
NO. 2242: GCACGGCCAUUGCAUUCAA), SPINK5 (SEQ. ID NO. 2243:
CCAACUGCCUGUUCAAUAA, SEQ. ID NO. 2244: GGAUACAUGUGAUGAGUUU, SEQ. ID
NO. 2245: GGACGAAUGUGCUGAGUAU, SEQ. ID NO. 2246:
GAGC1JUGUCUUAUUUGCUA,), the CYP1A2 gene (SEQ. ID NO. 2247:
GAAAUGCUGUGUCUUCGUA, SEQ. ID NO. 2248: GGACAGCACUUCCCUGAGA, SEQ. ID
NO. 2249: GAAGACACCACCAUUCUGA, SEQ. ID NO. 2250:
GGCCAGAGCUUGACCUUCA), thymosin-beta4Y (SEQ. ID NO. 2251:
GGACAGGCCUGCGUUGUUU, SEQ. ID NO. 2252: GGAAAGAGGAAGCUCAUGA, SEQ. ID
NO. 2253: GCAAACACGUUGGAUGAGU, SEQ. ID NO. 2254:
GGACUAUGCUGCCCUUUUG, activin A receptor IB (SEQ. ID NO. 2255:
ACAAGACGCUCCAGGAUCU, SEQ. ID NO. 2413: GCAACAGGAUCGACUUGAG, SEQ. ID
NO. 2414: GAAGCUGCGUCCCAACAUC, SEQ. ID NO. 2256:
GCAUAGGCCUGUAAUCGUA, SEQ. ID NO. 2257: UCAGAGAGUUCGAGACAAA, SEQ. ID
NO. 2258: UGCGAAAGGUUGUAUGUGA, SEQ. ID NO. 2259:
GCAACAGGAUCGACUUGAG, SEQ. ID NO. 2260: GAAUAGCGUUGUGUGUUAU, SEQ. ID
NO. 2261: UGAAUAGCGUUGUGUGUUA, SEQ. ID NO. 2262:
GGGAUCAGUUUGUUGAAUA, SEQ. ID NO. 2263: GAGCCUGAAUCAUCGUUUA,),
ADAM33 (SEQ. ID NO. 2264: GGAAGUACCUGGAACUGUA, SEQ. ID NO. 2265:
GGACAGAGGGAACCAUUUA, SEQ. ID NO. 2266: GGUGAGAGGUAGCUCCUAA, SEQ. ID
NO. 2267: AAAGACAGGUGGCCACUGA), the TAP 1 gene (SEQ. ID NO. 2268:
GAAAGAUGAUCAGCUAUUU, SEQ. ID NO. 2269: CAACAGAACCAGACAGGUA, SEQ. ID
NO. 2270: UGAGAAAUGUUCAGAAUGU, SEQ. ID NO. 2271:
UACCUUCACUCGAAACUUA, COX-2 (SEQ. ID NO. 2272: GAACGAAAGUAAAGAUGUU,
SEQ. ID NO. 2273: GGACUUAUGGGUAAUGUUA, SEQ. ID NO. 2274:
UGAAAGGACUUAUGGGUAA, SEQ. ID NO. 2275: GAUCAGAGUUCACUUUCUU), ADPRT
(SEQ. ID NO. 2276: GGAAAGAUGUUAAGCAUUU, SEQ. ID NO. 2277:
CAUGGGAGCUCUUGAAAUA, SEQ. ID NO. 2278: GAACAAGGAUGAAGUGAAG, SEQ. ID
NO. 2279: UGAAGAAGCUCACAGUAAA,), HDC (SEQ. ID NO. 2280:
CAGCAGACCUUCAGUGUGA, SEQ. ID NO. 2281: GGAGAGAGAUGGUGGAUUA, SEQ. ID
NO. 2282: GUACAGAGCUGGAGAUGAA, SEQ. ID NO. 2283:
GAACGUCCCUUCAGUCUGU), HnmT (SEQ. ID NO. 2284: CAAAUUCUCUCCAAAGUUC,
SEQ. ID NO. 2285: GGAUAUAUCUGACUGCUUU, SEQ. ID NO. 2286:
GAGCAGAGCUUGGGAAAGA, SEQ. ID NO. 2287: GAUAUGAGAUGUAGCAAAU), GATA-3
(SEQ. ID NO. 2288: GAACUGCUUUCUUUCGUUU, SEQ. ID NO. 2289:
GCAGUAUCAUGAAGCCUAA, SEQ. ID NO. 2290: GAAACUAGGUCUGAUAUUC, SEQ. ID
NO. 2291: GUACAGCUCCGGACUCUUC), Gab2 (SEQ. ID NO. 2292:
GCACAACCAUUCUGAAGUU, SEQ. ID NO. 2293: GGACUUAGAUGCCCAGAUG, SEQ. ID
NO. 2294: GAAGGUGGAUUCUAGGAAA, SEQ. ID NO. 2295:
GGACUAGCCCUGCUGUUUA), and STAT6 (SEQ. ID NO. 2296:
GAUAGAAACUCCUGCUAAU, SEQ. ID NO. 2297: GGACAUUUAUUCCCAGCUA, SEQ. ID
NO. 2298: GGACAGAGCUACAGACCUA, SEQ. ID NO. 2299:
GGAUGGCUCUCCACAGAUA).
[0618] In addition, rationally designed siRNA or siRNA pools can be
directed against genes involved in anemia, hemophila or
hypercholesterolemia. Such genes would include, but are not be
limited to:
17 APOA5 (SEQ. ID NO. 2300: GAAAGACAGCCUUGAGCAA, SEQ. ID NO. 2301:
GGACAGGGAGGCCACCAAA, SEQ. ID NO. 2302: GGACGAGGCUUGGGCUUUG, SEQ. ID
NO. 2303: AGCAAGACCUCAACAAUAU), HMG-CoA reductase (SEQ. ID NO.
2304: GAAUGAAGCUUUGCCCUUU, SEQ. ID NO. 2305: GAACACAGUUUAGUGCUUU,
SEQ. ID NO. 2306: UAUCAGAGCUCUUAAUGUU, SEQ. ID NO. 2307:
UGAAGAAUGUCUACAGAUA), NOS3 (SEQ. ID NO. 2308: UGAAGCACCUGGAGAAUGA,
SEQ. ID NO. 2309: CGGAACAGCACAAGAGUUA, SEQ. ID NO. 2310:
GGAAGAAGACCUUUAAAGA, SEQ. ID NO. 2415: GCACAAGAGUUAUAAGAUC), ARH
(SEQ. ID NO. 2416: CGAUACAGCUUGGCACUUU, SEQ. ID NO. 2311:
GAGAAGCGCUGCCCUGUGA, SEQ. ID NO. 2312: GAAUCAUGCUGUUCUCUUU, SEQ. ID
NO. 2313: GGAGUAACCGGACACCUUA), CYP7A1 (SEQ. ID NO. 2314:
UAAGGUGACUCGAGUGUUU, SEQ. ID NO. 2315: AAACGACACUUUCAUCAAA, SEQ. ID
NO. 2316: GGACUCAAGUUAAAGUAUU, SEQ. ID NO. 2317:
GUAAUGGACUCAAGUUAAA), FANCA (SEQ. ID NO. 2318: GGACAUCACUGCCCACUUC,
SEQ. ID NO. 2319: AGAGGAAGAUGUUCACUUA, SEQ. ID NO. 2320:
GAUCGUGGCUCUUCAGGAA, SEQ. ID NO. 2321: GGACAGAGGCAGAUAAGAA), FANCG
(SEQ. ID NO. 2322: GCACUAAGCAGCCUUCAUG, SEQ. ID NO. 2323:
GCAAGCAGGUGCCUACAGA, SEQ. ID NO. 2324: GGAAUUAGAUGCUCCAUUG, SEQ. ID
NO. 2325: GGACAUCUCUGCCAAAGUC), ALAS (SEQ. ID NO. 2326:
CAAUAUGCCUGGAAACUAU, SEQ. ID NO. 2327: GGUUAAGACUCACCAGUUC, SEQ. ID
NO. 2328: CAACAGGACUUUAGGUUCA, SEQ. ID NO. 2329:
GCAUAAGAUUGACAUCAUC), PIGA (SEQ. ID NO. 2330: GAAAGAGGGCAUAAGGUUA,
SEQ. ID NO. 2331: GGACUGAUCUUUAAACUAU, SEQ. ID NO. 2332:
UCAAAUGGCUUACUUCAUC, SEQ. ID NO. 2333: UCUAAGAACUGAUGUCUAA), and
factor VIII (SEQ. ID NO. 2334: GCAAAUAGAUCUCCAUUAC, SEQ. ID NO.
2335: CCAGAUAUGUCGUUCUUUA, SEQ. ID NO. 2336: GAAAGGCUGUGCUCUCAAA,
SEQ. ID NO. 2337: GGAGAAACCUGCAUGAAAG, SEQ. ID NO. 2338:
CUUGAAGCCUCCUGAAUUA, SEQ. ID NO. 2339: GAGGAAGCAUCCAAAGAUU, SEQ. ID
NO. 2340: GAUAGGAGAUACAAACUUU).
[0619] Furthermore, rationally designed siRNA or siRNA pools can be
directed against genes involved in disorders of the brain and
nervous system. Such genes would include, but are not be limited
to:
18 APBB1 (SEQ. ID NO. 2341: CUACGUAGCUCGUGAUAAG, SEQ. ID NO. 2342:
GCAGAGAUGUCCACAGGUU, SEQ. ID NO. 2343: CAUGAGAUCUGCUCUAAGA, SEQ. ID
NO. 2344: GGGCACCUCUGCUGUAUUG), BACE1 (SEQ. ID NO. 2345:
CCACAGAGCAAGUGAUUUA, SEQ. ID NO. 2346: GCAGAAAGGAGAUCAUUUA, SEQ. ID
NO. 2347: GUAGCAAGAUCUUUACAUA, SEQ. ID NO. 2348:
UGUCAGAGCUUGAUUAGAA), PSEN1 (SEQ. ID NO. 2349: GAGCUGACAUUGAAAUAUG,
SEQ. ID NO. 2350: GUACAGCUAUUUCUCAUCA, SEQ. ID NO. 2351:
GAGGUUAGGUGAAGUGGUU, SEQ. ID NO. 2352: GAAAGGGAGUCACAAGACA, SEQ. ID
NO. 2353: GAACUGGAGUGGAGUAGGA, SEQ. ID NO. 2354:
CAGCAGGCAUAUCUCAUUA, SEQ. ID NO. 2355: UCAAGUACCUCCCUGAAUG), PSEN2
(SEQ. ID NO. 2356: GCUGGGAAGUGGCUUAAUA, SEQ. ID NO. 2357:
CAUAUUCCCUGCCCUGAUA, SEQ. ID NO. 2358: GGGAAGUGCUCAAGACCUA, SEQ. ID
NO. 2359: CAUAGAAAGUGACGUGUUA), MASS 1 (SEQ. ID NO. 2360:
GGAAGGAGCUGUUAUGAGA, SEQ. ID NO. 2361: GAAAGGAGAAGCUAAAUUA, SEQ. ID
NO. 2362: GGAGGAAGGUCAAGAUUUA, SEQ. ID NO. 2363:
GGAAAUAGCUGAGAUAAUG,), ARX (SEQ. ID NO. 2364: CCAGACGCCUGAUAUUGAA,
SEQ. ID NO. 2365: CAGCACCACUCAAGACCAA, SEQ. ID NO. 2366:
CGCCUGAUAUUGAAGUAAA, SEQ. ID NO. 2367: CAACAUCCACUCUCUCUUG) and
NNMT (SEQ. ID NO. 2368: GGGCAGUGCUCCAGUGGUA, SEQ. ID NO. 2369:
GAAAGAGGCUGGCUACACA, SEQ. ID NO. 2370: GUACAGAAGUGAGACAUAA, SEQ. ID
NO. 2371: GAGGUGAUCUCGCAAAGUU).
[0620] In addition, rationally designed siRNA or siRNA pools can be
directed against genes involved in hypertension and related
disorders. Such genes would include, but are not be limited to:
19 angiotensin II type 1 receptor (SEQ. ID NO. 2372:
CAAGAAGCCUGCACCAUGU, SEQ. ID NO. 2373: GCACUUCACUACCAAAUGA, SEQ. ID
NO. 2374: GCACUGGUCCCAAGUAGUA, SEQ. ID NO. 2375:
CCAAAGGGCAGUAAAGUUU, SEQ. ID NO. 2376: GCUCAGAGGAGGUGUAUUU, SEQ. ID
NO. 2377: GCACUUCACUACCAAAUGA, SEQ. ID NO. 2378:
AAAGGGCAGUAAAGUUU), AGTR2 (SEQ. ID NO. 2379: GAACAUCUCUGGCAACAAU,
SEQ. ID NO. 2380: GGUGAUAUAUCUCAAAUUG, SEQ. ID NO. 2381:
GCAAGCAUCUUAUAUAGUU, SEQ. ID NO. 2382: GAACCAGUCUUUCAACUCA),
[0621] and other related targets.
Example XIII
Validation of Multigene Knockout Using Rab5 and Eps
[0622] Two or more genes having similar, overlapping functions
often leads to genetic redundancy. Mutations that knockout only one
of, e.g., a pair of such genes (also referred to as homologs)
results in little or no phenotype due to the fact that the
remaining intact gene is capable of fulfilling the role of the
disrupted counterpart. To fully understand the function of such
genes in cellular physiology, it is often necessary to knockout or
knockdown both homologs simultaneously. Unfortunately, concomitant
knockdown of two or more genes is frequently difficult to achieve
in higher organisms (e.g., mice) thus it is necessary to introduce
new technologies dissect gene function. One such approach to
knocking down multiple genes simultaneously is by using siRNA. For
example, FIG. 11 showed that rationally designed siRNA directed
against a number of genes involved in the clathrin-mediated
endocytosis pathway resulted in significant levels of protein
reduction (e.g., >80%). To determine the effects of gene
knockdown on clathrin-related endocytosis, internalization assays
were performed using epidermal growth factor and transferrin.
Specifically, mouse receptor-grade EGF (Collaborative Research
Inc.) and iron-saturated human transferrin (Sigma) were iodinated
as described previously (Jiang, X., Huang, F., Marusyk, A. &
Sorkin, A. (2003) Mol Biol Cell 14, 858-70). HeLa cells grown in
12-well dishes were incubated with .sup.125I-EGF (1 .mu.g/ml) or
.sup.125I-transferrin (1 .mu.g/ml) in binding medium (DMEM, 0.1%
bovine serum albumin) at 37.degree. C., and the ratio of
internalized and surface radioactivity was determined during 5-min
time course to calculate specific internalization rate constant
k.sub.e as described previously (Jiang, X et al.). The measurements
of the uptakes of radiolabeled transferrin and EGF were performed
using short time-course assays to avoid influence of the recycling
on the uptake kinetics, and using low ligand concentration to avoid
saturation of the clathrin-dependent pathway (for EGF Lund, K. A.,
Opresko, L. K., Strarbuck, C., Walsh, B. J. & Wiley, H. S.
(1990) J. Biol. Chem. 265, 15713-13723).
[0623] The effects of knocking down Rab5a, 5b, 5c, Eps, or Eps 15R
(individually) are shown in FIG. 22 and demonstrate that disruption
of single genes has little or no effect on EGF or Tfn
internalization. In contrast, simultaneous knock down of Rab5a, 5b,
and 5c, or Eps and Eps 15R, leads to a distinct phenotype (note:
total concentration of siRNA in these experiments remained constant
with that in experiments in which a single siRNA was introduced,
see FIG. 23). These experiments demonstrate the effectiveness of
using rationally designed siRNA to knockdown multiple genes and
validates the utility of these reagents to override genetic
redundancy.
Example XIV
Validation of Multigene Targeting Using G6PD, GAPDH, PLK, and
UQC
[0624] Further demonstration of the ability to knock down
expression of multiple genes using rationally designed siRNA was
performed using pools of siRNA directed against four separate
genes. To achieve this, siRNA were transfected into cells (total
siRNA concentration of 100 nM) and assayed twenty-four hours later
by B-DNA. Results shown in FIG. 24 show that pools of rationally
designed molecules are capable of simultaneously silencing four
different genes.
Example XV
Validation of Multigene Knockouts as Demonstrated by Gene
Expression Profiling, a Prophetic Example
[0625] To further demonstrate the ability to concomitantly
knockdown the expression of multiple gene targets, single siRNA or
siRNA pools directed against a collection of genes (e.g., 4, 8, 16,
or 23 different targets) are simultaneously transfected into cells
and cultured for twenty-four hours. Subsequently, mRNA is harvested
from treated (and untreated) cells and labeled with one of two
fluorescent probes dyes (e.g., a red fluorescent probe for the
treated cells, a green fluorescent probe for the control cells.).
Equivalent amounts of labeled RNA from each sample is then mixed
together and hybridized to sequences that have been linked to a
solid support (e.g., a slide, "DNA CHIP"). Following hybridization,
the slides are washed and analyzed to assess changes in the levels
of target genes induced by siRNA.
Example XVI
Identifying Hyperfunctional siRNA
[0626] Identification of Hyperfunctional Bcl-2 siRNA
[0627] The ten rationally designed Bcl2 siRNA (identified in FIG.
13, 14) were tested to identify hyperpotent reagents. To accomplish
this, each of the ten Bcl-2 siRNA were individually transfected
into cells at a 300 pM (0.3 nM) concentrations. Twenty-four hours
later, transcript levels were assessed by B-DNA assays and compared
with relevant controls. As shown in FIG. 25, while the majority of
Bcl-2 siRNA failed to induce functional levels of silencing at this
concentration, siRNA 1 and 8 induced >80% silencing, and siRNA 6
exhibited greater than 90% silencing at this subnanomolar
concentration.
[0628] By way of prophetic examples, similar assays could be
performed with any of the groups of rationally designed genes
described in Example VII or Example VIII. Thus for instance,
rationally designed siRNA sequences directed against
[0629] PDGFA
[0630] (SEQ. ID NO. 2383: GGUAAGAUAUUGUGCUUUA,
[0631] SEQ. ID NO. 2384: CCGCAAAUAUGCAGAAUUA,
[0632] SEQ. ID NO. 2385: GGAUGUACAUGGCGUGUUA,
[0633] SEQ. ID NO. 2386: GGUGAAGUUUGUAUGUUUA), or
[0634] PDGFB
[0635] (SEQ. ID NO. 2387: GCUCCGCGCUUUCCGAUUU,
[0636] SEQ. ID NO. 2388: GAGCAGGAAUGGUGAGAUG,
[0637] SEQ. ID NO. 2389: GAACUUGGGAUAAGAGUGU,
[0638] SEQ. ID NO. 2390: CCGAGGAGCUUUAUGAGAU,
[0639] SEQ. ID NO. 2391: UUUAUGAGAUGCUGAGUGA)
[0640] could be introduced into cells at increasingly limiting
concentrations to determine whether any of the duplexes are
hyperfunctional. Similarly, rationally designed sequences directed
against
[0641] HIF1 Alpha
[0642] (SEQ. ID NO. 2392: GAAGGAACCUGAUGCUUUA,
[0643] SEQ. ID NO. 2393: GCAUAUAUCUAGAAGGUAU,
[0644] SEQ. ID NO. 2394: GAACAAAUACAUGGGAUUA,
[0645] SEQ. ID NO. 2395: GGACACAGAUUUAGACUUG), or
[0646] VEGF
[0647] (SEQ. ID NO. 2396: GAACGUACUUGCAGAUGUG,
[0648] SEQ. ID NO. 2397: GAGAAAGCAUUUGUUUGUA,
[0649] SEQ. ID NO. 2398: GGAGAAAGCAUUUGUUUGU,
[0650] SEQ. ID NO. 2399: CGAGGCAGCUUGAGUUAAA) could be introduced
into cells at increasingly limiting concentrations and screened for
hyperfunctional duplexes.
Example XVII
Gene Silencing: Prophetic Example
[0651] Below is an example of how one might transfect a cell.
[0652] a. Select a cell line. The selection of a cell line is
usually determined by the desired application. The most important
feature to RNAi is the level of expression of the gene of interest.
It is highly recommended to use cell lines for which siRNA
transfection conditions have been specified and validated.
[0653] b. Plate the cells. Approximately 24 hours prior to
transfection, plate the cells at the appropriate density so that
they will be approximately 70-90% confluent, or approximately
1.times.10.sup.5 cells/ml at the time of transfection. Cell
densities that are too low may lead to toxicity due to excess
exposure and uptake of transfection reagent-siRNA complexes. Cell
densities that are too high may lead to low transfection
efficiencies and little or no silencing. Incubate the cells
overnight. Standard incubation conditions for mammalian cells are
37.degree. C. in 5% CO.sub.2. Other cell types, such as insect
cells, require different temperatures and CO.sub.2 concentrations
that are readily ascertainable by persons skilled in the art. Use
conditions appropriate for the cell type of interest.
[0654] c. siRNA re-suspension. Add 20 .mu.l siRNA universal buffer
to each siRNA to generate a final concentration of 50 .mu.M.
[0655] d. SiRNA-lipid complex formation. Use RNase-free solutions
and tubes. Using the following table, Table XI:
20 TABLE XI 96-well 24-well Mixture 1 (TransIT-TKO-Plasmid dilution
mixture) Opti-MEM 9.3 .mu.l 46.5 .mu.l TransIT-TKO (1 .mu.g/.mu.l)
0.5 .mu.l 2.5 .mu.l Mixture 1 Final Volume 10.0 .mu.l 50.0 .mu.l
Mixture 2 (siRNA dilution mixture) Opti-MEM 9.0 .mu.l 45.0 .mu.l
siRNA (1 .mu.M) 1.0 .mu.l 5.0 .mu.l Mixture 2 Final Volume 10.0
.mu.l 50.0 .mu.l Mixture 3 (siRNA-Transfection reagent mixture)
Mixture 1 10 .mu.l 50 .mu.l Mixture 2 10 .mu.l 50 .mu.l Mixture 3
Final Volume 20 .mu.l 100 .mu.l Incubate 20 minutes at room
temperature. Mixture 4 (Media-siRNA/Transfection reagent mixture)
Mixture 3 20 .mu.l 100 .mu.l Complete media 80 .mu.l 400 .mu.l
Mixture 4 Final Volume 100 .mu.l 500 .mu.l Incubate 48 hours at
37.degree. C.
[0656] Transfection. Create a Mixture 1 by combining the specified
amounts of OPTI-MEM serum free media and transfection reagent in a
sterile polystyrene tube. Create a Mixture 2 by combining specified
amounts of each siRNA with OPTI-MEM media in sterile 1 ml tubes.
Create a Mixture 3 by combining specified amounts of Mixture 1 and
Mixture 2. Mix gently (do not vortex) and incubate at room
temperature for 20 minutes. Create a Mixture 4 by combining
specified amounts of Mixture 3 to complete media. Add appropriate
volume to each cell culture well. Incubate cells with transfection
reagent mixture for 24-72 hours at 37.degree. C. This incubation
time is flexible. The ratio of silencing will remain consistent at
any point in the time period. Assay for gene silencing using an
appropriate detection method such as RT-PCR, Western blot analysis,
immunohistochemistry, phenotypic analysis, mass spectrometry,
fluorescence, radioactive decay, or any other method that is now
known or that comes to be known to persons skilled in the art and
that from reading this disclosure would useful with the present
invention. The optimal window for observing a knockdown phenotype
is related to the mRNA turnover of the gene of interest, although
24-72 hours is standard. Final Volume reflects amount needed in
each well for the desired cell culture format. When adjusting
volumes for a Stock Mix, an additional 10% should be used to
accommodate variability in pipetting, etc. Duplicate or triplicate
assays should be carried out when possible.
[0657] While the invention has been described in connection with
specific embodiments thereof, it will be understood that it is
capable of further modifications and this application is intended
to cover any variations, uses, or adaptations of the invention
following, in general, the principles of the invention and
including such departure from the present disclosure as come within
known or customary practice within the art to which the invention
pertains and as may be applied to the essential features
hereinbefore set forth and as follows in the scope of the appended
claims.
Sequence CWU 0
0
* * * * *