U.S. patent application number 10/738642 was filed with the patent office on 2004-12-02 for sirna-mediated gene silencing.
Invention is credited to Davidson, Beverly L., Gouvion, Cynthia, Miller, Victor, Paulson, Henry.
Application Number | 20040241854 10/738642 |
Document ID | / |
Family ID | 46205048 |
Filed Date | 2004-12-02 |
United States Patent
Application |
20040241854 |
Kind Code |
A1 |
Davidson, Beverly L. ; et
al. |
December 2, 2004 |
siRNA-mediated gene silencing
Abstract
The present invention is directed to small interfering RNA
molecules (siRNA) targeted against an allele of interest, and
methods of using these siRNA molecules.
Inventors: |
Davidson, Beverly L.; (North
Liberty, IA) ; Paulson, Henry; (Iowa City, IA)
; Miller, Victor; (Iowa City, IA) ; Gouvion,
Cynthia; (Iowa City, IA) |
Correspondence
Address: |
FISH & RICHARDSON P.C.
3300 DAIN RAUSCHER PLAZA
60 SOUTH SIXTH STREET
MINNEAPOLIS
MN
55402
US
|
Family ID: |
46205048 |
Appl. No.: |
10/738642 |
Filed: |
December 16, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10738642 |
Dec 16, 2003 |
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PCT/US03/16887 |
May 26, 2003 |
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PCT/US03/16887 |
May 26, 2003 |
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10430351 |
May 5, 2003 |
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10430351 |
May 5, 2003 |
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10322086 |
Dec 17, 2002 |
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10322086 |
Dec 17, 2002 |
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10212322 |
Aug 5, 2002 |
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Current U.S.
Class: |
435/455 ;
435/325 |
Current CPC
Class: |
Y02A 50/30 20180101;
C12N 2330/30 20130101; A01K 2217/05 20130101; C12N 2799/022
20130101; A61K 48/00 20130101; C12N 2799/021 20130101; C12N
2310/111 20130101; C12Y 302/01031 20130101; C12N 15/1137 20130101;
A61K 38/00 20130101; C12N 2310/53 20130101; Y02A 50/465 20180101;
C12N 15/111 20130101; C12N 2310/14 20130101; C12N 15/113
20130101 |
Class at
Publication: |
435/455 ;
435/325 |
International
Class: |
C12N 015/85 |
Goverment Interests
[0002] Work relating to this application was supported by grants
from the National Institutes of Health (NS044494 and NS38712). The
government may have certain rights in the invention.
Claims
What is claimed is:
1. A mammalian cell comprising an isolated first strand of RNA of
15 to 30 nucleotides in length having a 5' end and a 3' end,
wherein the first strand is complementary to at least 15
nucleotides of a targeted gene of interest, and wherein the 5' end
of the first strand of RNA is operably linked to a G nucleotide to
form a first segment of RNA, and an isolated second strand of RNA
of 15 to 30 nucleotides in length having a 5' end and a 3' end,
wherein at least 12 nucleotides of the first and second strands are
complementary to each other and form a small interfering RNA
(siRNA) duplex under physiological conditions, and wherein the
siRNA silences only one allele of the targeted gene in the
cell.
2. The mammalian cell of claim 1, wherein the duplex is between 15
and 25 base pairs in length.
3. The mammalian cell of claim 1, wherein the duplex is 20 base
pairs in length.
4. The mammalian cell of claim 1, wherein the first strand is 20
nucleotides in length, and the second strand is 20 nucleotides in
length.
5. The mammalian cell of claim 4, wherein the first strand is
complementary to 19 out of 20 contiguous nucleotides of the
targeted gene and is non-complementary to one nucleotide of the
targeted gene.
6. The mammalian cell of claim 5, wherein the one non-complementary
nucleotide is at position 9, 10, or 11, as measured from the 5' end
of the first strand of RNA.
7. The mammalian cell of claim 5, wherein the one non-complementary
nucleotide is at position 10, as measured from the 5' end of the
first strand of RNA.
8. The mammalian cell of claim 4, wherein the first strand is
complementary to 18 out of 20 contiguous nucleotides of the
targeted gene and is non-complementary to two nucleotides of the
targeted gene.
9. The mammalian cell of claim 8, wherein two non-complementary
nucleotides are at nucleotide position 9, 10, 11, or 12 as measured
from the 5' end of the first strand of RNA.
10. The mammalian cell of claim 5, wherein the two
non-complementary nucleotides are at nucleotide position 10 and 11,
as measured from the 5' end of the first strand of RNA.
11. The mammalian cell of claim 1, wherein the 5' end of the second
strand of RNA is operably linked to a G nucleotide.
12. The mammalian cell of claim 1, wherein the first strand and the
second strand are operably linked by means of an RNA loop strand to
form a hairpin structure comprising a duplex structure and a loop
structure.
13. The mammalian cell of claim 12, wherein the loop structure
contains from 4 to 10 nucleotides.
14. The mammalian cell of claim 13, wherein the loop structure
contains 4, 5 or 6 nucleotides.
15. The mammalian cell of claim 1, wherein the targeted gene is a
gene associated with a condition amenable to siRNA therapy.
16. The mammalian cell of claim 15, wherein the gene encodes a
transcript for Swedish double amyloid precursor protein (APPsw)
mutation or a transcript for Tau.
17. A mammalian cell comprising an expression cassette encoding an
isolated first strand of RNA of 15 to 30 nucleotides in length
having a 5' end and a 3' end, wherein the first strand is
complementary to at least 15 nucleotides of a targeted gene of
interest, and wherein the 5' end of the first strand of RNA is
operably linked to a G nucleotide to form a first strand of RNA,
and an isolated second strand of RNA of 15 to 30 nucleotides in
length having a 5' end and a 3' end, and wherein at least 12
nucleotides of the first and second strands are complementary to
each other and form a small interfering RNA (siRNA) duplex under
physiological conditions, and wherein the siRNA silences only one
allele of the targeted gene in the cell.
18. The mammalian cell of claim 17, wherein the expression cassette
is contained in a vector.
19. The mammalian cell of claim 18, wherein the vector is an
adenoviral, lentiviral, adeno-associated viral (AAV), poliovirus,
HSV, or murine Maloney-based viral vector.
20. The mammalian cell of claim 18, wherein the vector is an
adenoviral vector.
21. An isolated RNA duplex comprising a first strand of RNA having
a 5' end and a 3' end, and a second strand of RNA, wherein the
first strand comprises 20 nucleotides complementary to mutant Tau
transcript encoded by siA10 GGTGGCCAGATGGAAGTAAA (SEQ ID NO:63),
wherein the 5' end of the first strand of RNA is operably linked to
a G nucleotide to form a first segment of RNA, and wherein the
second strand is complementary to all the nucleotides of the first
strand.
22. The RNA duplex of claim 21, wherein the first strand and the
second strand are operably linked by means of an RNA loop strand to
form a hairpin structure comprising a duplex structure and a loop
structure.
23. The RNA duplex of claim 21, wherein the loop structure contains
from 4 to 10 nucleotides.
24. The RNA duplex of claim 21, wherein the loop structure contains
4, 5 or 6 nucleotides.
25. An expression cassette comprising a nucleic acid encoding at
least one strand of the RNA duplex of claims 21.
26. A vector comprising the expression cassette of claim 25.
27. A vector comprising two expression cassettes, a first
expression cassette comprising a nucleic acid encoding the first
strand of the RNA duplex of claim 21 and a second expression
cassette comprising a nucleic acid encoding the second strand of
the RNA duplex of claim 21.
28. A cell comprising the expression cassette of claim 25.
29. The cell of claim 28, wherein the cell is a mammalian cell.
30. A non-human mammal comprising the expression cassette of claim
25.
31. An isolated RNA duplex comprising a first strand of RNA having
a 5' end and a 3' end, and a second strand of RNA, wherein the
first strand comprises 20 nucleotides complementary to Swedish
double amyloid precursor protein (APPsw) mutation transcript
encoded by siT10/C11 TGAAGTGAATCTGGATGCAG (SEQ ID NO:64), wherein
the 5' end of the first strand of RNA is operably linked to a G
nucleotide to form a first segment of RNA, and wherein the second
strand is complementary to all the nucleotides of the first
strand.
32. The RNA duplex of claim 31, wherein the first strand and the
second strand are operably linked by means of an RNA loop strand to
form a hairpin structure comprising a duplex structure and a loop
structure.
33. The RNA duplex of claim 31, wherein the loop structure contains
from 4 to 10 nucleotides.
34. The RNA duplex of claim 31, wherein the loop structure contains
4, 5 or 6 nucleotides.
35. An expression cassette comprising a nucleic acid encoding at
least one strand of the RNA duplex of claims 22.
36. A vector comprising the expression cassette of claim 35.
37. A vector comprising two expression cassettes, a first
expression cassette comprising a nucleic acid encoding the first
strand of the RNA duplex of claim 31 and a second expression
cassette comprising a nucleic acid encoding the second strand of
the RNA duplex of claim 31.
38. A cell comprising the expression cassette of claim 26.
39. The cell of claim 38, wherein the cell is a mammalian cell.
40. A method of performing allele-specific gene silencing in a
mammal comprising administering to the mammal an isolated first
strand of RNA of 15 to 30 nucleotides in length having a 5' end and
a 3' end, wherein the first strand is complementary to at least 15
nucleotides of a targeted gene of interest, and wherein the 5' end
of the first strand of RNA is operably linked to a G nucleotide to
form a first segment of RNA, and an isolated second strand of RNA
of 15 to 30 nucleotides in length having a 5' end and a 3' end,
wherein at least 12 nucleotides of the first and second strands are
complementary to each other and form a small interfering RNA
(siRNA) duplex under physiological conditions, and wherein the
siRNA silences only one allele of the targeted gene in the
mammal.
41. The method of claim 40, wherein the duplex is between 15 and 25
base pairs in length.
42. The method of claim 40, wherein the duplex is 20 base pairs in
length.
43. The method of claim 40, wherein the first strand is 20
nucleotides in length, and the second strand is 20 nucleotides in
length.
44. The method of claim 43, wherein the first strand is
complementary to 19 out of 20 contiguous nucleotides of the
targeted gene and is non-complementary to one nucleotide of the
targeted gene.
45. The method of claim 44, wherein the one non-complementary
nucleotide is at position 9, 10, or 11, as measured from the 5' end
of the first strand of RNA.
46. The method of claim 44, wherein the one non-complementary
nucleotide is at position 10, as measured from the 5' end of the
first strand of RNA.
47. The method of claim 43, wherein the first strand is
complementary to 18 out of 20 contiguous nucleotides of the
targeted gene and is non-complementary to two nucleotides of the
targeted gene.
48. The method of claim 47, wherein two non-complementary
nucleotides are at nucleotide position 9, 10, 11, or 12 as measured
from the 5' end of the first strand of RNA.
49. The method of claim 44, wherein the two non-complementary
nucleotides are at nucleotide position 10 and 11, as measured from
the 5' end of the first strand of RNA.
50. The method of claim 40, wherein the 5' end of the second strand
of RNA is operably linked to a G nucleotide.
51. The method of claim 40, wherein the first strand and the second
strand are operably linked by means of an RNA loop strand to form a
hairpin structure comprising a duplex structure and a loop
structure.
52. The method of claim 51, wherein the loop structure contains
from 4 to 10 nucleotides.
53. The method of claim 52, wherein the loop structure contains 4,
5 or 6 nucleotides.
54. The method of claim 40, wherein the targeted gene is a gene
associated with a condition amenable to siRNA therapy.
55. The method of claim 54, wherein the gene encodes a transcript
for Swedish double amyloid precursor protein (APPsw) mutation or a
transcript for Tau.
56. A method of producing an RNA comprising (a) producing an
isolated first strand of RNA of 15 to 30 nucleotides in length
having a 5' end and a 3' end, wherein the first strand is
complementary to at least 15 nucleotides of a targeted gene of
interest, and wherein the 5' end of the first strand of RNA is
operably linked to a G nucleotide to form a first segment of RNA,
(b) producing an isolated second strand of RNA of 15 to 30
nucleotides in length having a 5' end and a 3' end, and (c)
contacting the first strand and the second strand under hybridizing
conditions to form a siRNA duplex, wherein the siRNA silences only
one allele of the targeted gene in the cell.
57. The method of claim 56, wherein the duplex is between 15 and 25
base pairs in length.
58. The method of claim 56, wherein the duplex is 20 base pairs in
length.
59. The method of claim 56, wherein the first strand is 20
nucleotides in length, and the second strand is 20 nucleotides in
length.
60. The method of claim 59, wherein the first strand is
complementary to 19 out of 20 contiguous nucleotides of the
targeted gene and is non-complementary to one nucleotide of the
targeted gene.
61. The method of claim 60, wherein the one non-complementary
nucleotide is at position 9, 10, or 11, as measured from the 5' end
of the first strand of RNA.
62. The method of claim 60, wherein the one non-complementary
nucleotide is at position 10, as measured from the 5' end of the
first strand of RNA.
63. The method of claim 59, wherein the first strand is
complementary to 18 out of 20 contiguous nucleotides of the
targeted gene and is non-complementary to one nucleotide of the
targeted gene.
64. The method of claim 63, wherein the two non-complementary
nucleotides are at nucleotide position 9, 10, 11, or 12 as measured
from the 5' end of the first strand of RNA.
65. The method of claim 63, wherein the two non-complementary
nucleotides are at nucleotide position 10 and 11, as measured from
the 5' end of the first strand of RNA.
66. The method of claim 63, wherein the 5' end of the second strand
of RNA is operably linked to a G nucleotide.
Description
CLAIM OF PRIORITY
[0001] This is a continuation-in-part of International Application
No. PCT/US03/16887 filed on May 26, 2003, which is a
continuation-in-part of application U.S. application Ser. No.
10/430,351 filed on May 5, 2003, which is a continuation of U.S.
application Ser. No. 10/322,086 filed on Dec. 17, 2002, which is a
continuation-in-part application of U.S. application Ser. No.
10/212,322, filed Aug. 5, 2002, all of which applications are
incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0003] Double-stranded RNA (dsRNA) can induce sequence-specific
posttranscriptional gene silencing in many organisms by a process
known as RNA interference (RNAi). However, in mammalian cells,
dsRNA that is 30 base pairs or longer can induce
sequence-nonspecific responses that trigger a shut-down of protein
synthesis. Recent work suggests that RNA fragments are the
sequence-specific mediators of RNAi (Elbashir et al., 2001).
Interference of gene expression by these small interfering RNA
(siRNA) is now recognized as a naturally occurring strategy for
silencing genes in C. elegans, Drosophila, plants, and in mouse
embryonic stem cells, oocytes and early embryos (Cogoni et al.,
1994; Baulcombe, 1996; Kennerdell, 1998; Timmons, 1998; Waterhouse
et al., 1998; Wianny and Zernicka-Goetz, 2000; Yang et al., 2001;
Svoboda et al., 2000). In mammalian cell culture, a siRNA-mediated
reduction in gene expression has been accomplished only by
transfecting cells with synthetic RNA oligonucleotides (Caplan et
al., 2001; Elbashir et al., 2001).
SUMMARY OF THE INVENTION
[0004] The present invention provides a mammalian cell containing
an isolated first strand of RNA of 15 to 30 nucleotides in length
having a 5' end and a 3' end, wherein the first strand is
complementary to at least 15 nucleotides of a targeted gene of
interest, and wherein the 5' end of the first strand of RNA is
operably linked to a G nucleotide to form a first segment of RNA,
and an isolated second strand of RNA of 15 to 30 nucleotides in
length having a 5' end and a 3' end, wherein at least 12
nucleotides of the first and second strands are complementary to
each other and form a small interfering RNA (siRNA) duplex under
physiological conditions, and wherein the siRNA silences only one
allele of the targeted gene in the cell. The duplex formed by the
two strands of RNA may be between 15 and 25 base pairs in length,
such as 20 base pairs in length. The first strand may be 20
nucleotides in length, and the second strand may be 20 nucleotides
in length. In one embodiment, the 5' end of the second strand of
RNA is operably linked to a G nucleotide. This G nucleotide may be
directly linked to the second strand of RNA (i.e., no intervening
nucleotides are present).
[0005] In one embodiment, the first strand is complementary to 19
out of 20 contiguous nucleotides of the targeted gene and is
non-complementary to one nucleotide of the targeted gene. For
example, the one non-complementary nucleotide is at position 9, 10,
or 11, as measured from the 5' end of the first strand of RNA. In
one embodiment, the one non-complementary nucleotide is at position
10, as measured from the 5' end of the first strand of RNA. In an
alternative embodiment, the first strand is complementary to 18 out
of 20 contiguous nucleotides of the targeted gene and is
non-complementary to two nucleotides of the targeted gene. For
example, the two non-complementary nucleotides are at nucleotide
position 9, 10, 11, or 12 as measured from the 5' end of the first
strand of RNA. In one embodiment, the two non-complementary
nucleotides are at nucleotide position 10 and 11, as measured from
the 5' end of the first strand of RNA.
[0006] In the present invention, the first and second strand of RNA
may be operably linked together by means of an RNA loop strand to
form a hairpin structure to form a "duplex structure" and a "loop
structure." These loop structures may be from 4 to 10 nucleotides
in length. For example, the loop structure may be 4, 5 or 6
nucleotides long.
[0007] In the mammalian cell of the present invention, the targeted
gene may be a gene associated with a condition amenable to siRNA
therapy. In one embodiment, the gene encodes a transcript for
Swedish double amyloid precursor protein (APPsw) mutation or a
transcript for Tau.
[0008] The present invention also provides a mammalian cell
containing an expression cassette encoding an isolated first strand
of RNA of 15 to 30 nucleotides in length having a 5' end and a 3'
end, wherein the first strand is complementary to at least 15
nucleotides of a targeted gene of interest, and wherein the 5' end
of the first strand of RNA is operably linked to a G nucleotide to
form a first strand of RNA, and an isolated second strand of RNA of
15 to 30 nucleotides in length having a 5' end and a 3' end, and
wherein at least 12 nucleotides of the first and second strands are
complementary to each other and form a small interfering RNA
(siRNA) duplex under physiological conditions, and wherein the
siRNA silences only one allele of the targeted gene in the cell.
These expression cassettes may further contain a promoter. Such
promoters can be regulatable promoters or constitutive promoters.
Examples of suitable promoters include a CMV, RSV, pol II or pol
III promoter. The expression cassette may further contain a
polyadenylation signal, such as a synthetic minimal polyadenylation
signal. The expression cassette may further contain a marker gene.
The expression cassette may be contained in a vector. Examples of
appropriate vectors include adenoviral, lentiviral,
adeno-associated viral (AAV), poliovirus, HSV, or murine
Maloney-based viral vectors. In one embodiment, the vector is an
adenoviral vector.
[0009] The present invention further provides an isolated RNA
duplex containing a first strand of RNA having a 5' end and a
3'end, and a second strand of RNA, -transcript encoded by siA10
GGTGGCCAGATGGAAGTAAA (SEQ ID NO:63), wherein the 5' end of the
first strand of RNA is operably linked to a G nucleotide to form a
first segment of RNA, and wherein the second strand is
complementary to all the nucleotides of the first strand. In one
embodiment, the first strand and the second strand are operably
linked by means of an RNA loop strand to form a hairpin structure
comprising a duplex structure and a loop structure.
[0010] The present invention also provides an expression cassette
comprising a nucleic acid encoding at least one strand of the RNA
duplex described above. As used herein the term "encoded by" means
that the DNA sequence in the SEQ ID NO is transcribed into the RNA
of interest.
[0011] The present invention provides a vector containing the
expression cassette described above. Further, the vector may
contain two expression cassettes, a first expression cassette
containing a nucleic acid encoding a first strand of the RNA duplex
and a second expression cassette containing a nucleic acid encoding
a second strand of the RNA duplex. The present invention also
provides cells containing these expression cassettes (such as a
mammalian cell), and a non-human mammal that has a cell containing
one of these expression cassettes.
[0012] The present invention provides an isolated RNA duplex
containing a first strand of RNA having a 5' end and a 3' end, and
a second strand of RNA, wherein the first strand is made of 20
nucleotides complementary to Swedish double amyloid precursor
protein (APPsw) mutation transcript encoded by siT10/C11
TGAAGTGAATCTGGATGCAG (SEQ ID NO:64), wherein the 5' end of the
first strand of RNA is operably linked to a G nucleotide to form a
first segment of RNA, and wherein the second strand is
complementary to all the nucleotides of the first strand. In this
RNA duplex, the first strand and the second strand may be operably
linked by means of an RNA loop strand to form a hairpin structure
comprising a duplex structure and a loop structure. The loop
structure may contain from 4 to 10 nucleotides, such as 4, 5 or 6
nucleotides.
[0013] The present invention provides an expression cassette
containing a nucleic acid encoding at least one strand of the RNA
duplex described above. It also provides a vector that contains
this expression cassette. Further, the vector may contain two
expression cassettes, a first expression cassette containing a
nucleic acid encoding the first strand of the RNA duplex as
described above and a second expression cassette containing a
nucleic acid encoding the second strand of the RNA duplex. The
present invention also provides a cell (such as a mammalian cell)
containing this expression cassette.
[0014] In the present invention, an expression cassette may contain
a nucleic acid encoding at least one strand of the RNA duplex
described above. Such an expression cassette may further contain a
promoter. The expression cassette may be contained in a vector.
These cassettes and vectors may be contained in a cell, such as a
mammalian cell. A cell in a non-human mammal may contain the
cassette or vector. The vector may contain two expression
cassettes, the first expression cassette containing a nucleic acid
encoding the first strand of the RNA duplex, and a second
expression cassette containing a nucleic acid encoding the second
strand of the RNA duplex.
[0015] The present invention further provides a method of
performing allele-specific gene silencing in a mammal by
administering to the mammal an isolated first strand of RNA of 15
to 30 nucleotides in length having a 5' end and a 3' end, wherein
the first strand is complementary to at least 15 nucleotides of a
targeted gene of interest, and wherein the 5' end of the first
strand of RNA is operably linked to a G nucleotide to form a first
segment of RNA, and an isolated second strand of RNA of 15 to 30
nucleotides in length having a 5' end and a 3' end, wherein at
least 12 nucleotides of the first and second strands are
complementary to each other and form a small interfering RNA
(siRNA) duplex under physiological conditions, and wherein the
siRNA preferentially silences one allele of the targeted gene in
the mammal. In one embodiment of the present invention, the duplex
is between 15 and 25 base pairs in length.
[0016] In one embodiment, the duplex may be 20 base pairs in
length. In one embodiment of the present invention, the first
strand is 20 nucleotides in length, and the second strand is 20
nucleotides in length. For example, the first strand is
complementary to 19 out of 20 contiguous nucleotides of the
targeted gene and is non-complementary to one nucleotide of the
targeted gene. The one non-complementary nucleotide may be at
position 9, 10, or 11, as measured from the 5' end of the first
strand of RNA. For instance, the one non-complementary nucleotide
is at position 10, as measured from the 5' end of the first strand
of RNA.
[0017] In another embodiment, the first strand is complementary to
18 out of 20 contiguous nucleotides of the targeted gene and is
non-complementary to two nucleotides of the targeted gene. The two
non-complementary nucleotides may be at nucleotide position 9, 10,
11, or 12 as measured from the 5' end of the first strand of RNA.
For instance, the two non-complementary nucleotides may be at
nucleotide position 10 and 11, as measured from the 5' end of the
first strand of RNA. In this method, the 5' end of the second
strand of RNA may be operably linked to a G nucleotide. In one
embodiment, the first strand and the second strand are operably
linked by means of an RNA loop strand to form a hairpin structure
comprising a duplex structure and a loop structure. In one
embodiment, the targeted gene is a gene associated with a condition
amenable to siRNA therapy. For example, gene may encode a
transcript for Swedish double amyloid precursor protein (APPsw)
mutation or a transcript for Tau.
[0018] The targeted gene may be a gene associated with a condition
amenable to siRNA therapy. For example, the condition amenable to
siRNA therapy could be a disabling neurological disorder.
"Neurological disease" and "neurological disorder" refer to both
hereditary and sporadic conditions that are characterized by
nervous system dysfunction, and which may be associated with
atrophy of the affected central or peripheral nervous system
structures, or loss of function without atrophy. A neurological
disease or disorder that results in atrophy is commonly called a
"neurodegenerative disease" or "neurodegenerative disorder."
Neurodegenerative diseases and disorders include, but are not
limited to, amyotrophic lateral sclerosis (ALS), hereditary spastic
hemiplegia, primary lateral sclerosis, spinal muscular atrophy,
Kennedy's disease, Alzheimer's disease, Parkinson's disease,
multiple sclerosis, and repeat expansion neurodegenerative
diseases, e.g., diseases associated with expansions of DNA repeats
such as the polyglutamine (polyQ) repeat diseases, e.g.,
Huntington's disease (HD), specific spinocerebellar ataxias (SCA1,
SCA2, SCA3, SCA6, SCA7, and SCA17), spinal and bulbar muscular
atrophy (SBMA), dentatorubropallidoluysian atrophy (DRPLA).
[0019] The present invention also provides a method of producing an
RNA by (a) producing an isolated first strand of RNA of 15 to 30
nucleotides in length having a 5' end and a 3' end, wherein the
first strand is complementary to at least 15 nucleotides of a
targeted gene of interest, and wherein the 5' end of the first
strand of RNA is operably linked to a G nucleotide to form a first
segment of RNA, (b) producing an isolated second strand of RNA of
15 to 30 nucleotides in length having a 5' end and a 3' end, and
(c) contacting the first strand and the second strand under
hybridizing conditions to form a siRNA duplex, wherein the siRNA
silences only one allele of the targeted gene in the cell.
[0020] In the present method, the duplex may be between 15 and 25
base pairs in length, such as 20 base pairs in length. In one
embodiment, the first strand is 20 nucleotides in length, and the
second strand is 20 nucleotides in length. The first strand may be
complementary to 19 out of 20 contiguous nucleotides of the
targeted gene and is non-complementary to one nucleotide of the
targeted gene. In one embodiment, the one non-complementary
nucleotide is at position 9, 10, or 11, as measured from the 5' end
of the first strand of RNA (such as at position 10). Alternatively,
the first strand may be complementary to 18 out of 20 contiguous
nucleotides of the targeted gene and is non-complementary to one
nucleotide of the targeted gene. In one embodiment, the two
non-complementary nucleotides are at nucleotide position 9, 10, 11,
or 12 as measured from the 5' end of the first strand of RNA (such
as at nucleotide position 10 and 11). In one embodiment, the 5' end
of the second strand of RNA is operably linked (directly or
indirectly) to a G nucleotide.
BRIEF DESCRIPTION OF THE FIGURES
[0021] This patent or application file contains at least one
drawing executed in color. Copies of this patent or patent
application publication with color drawing(s) will be provided by
the Office upon request and payment of the necessary fee.
[0022] FIG. 1. siRNA expressed from CMV promoter constructs and in
vitro effects. (A) A cartoon of the expression plasmid used for
expression of functional siRNA in cells. The CMV promoter was
modified to allow close juxtaposition of the hairpin to the
transcription initiation site, and a minimal polyadenylation signal
containing cassette was constructed immediately 3' of the MCS
(mCMV, modified CMV; mpA, minipA). (B, C) Fluorescence
photomicrographs of HEK293 cells 72 h after transfection of pEGFPN1
and pCMV.beta.gal (control), or pEGFPN1 and pmCMVsiGFPmpA,
respectively. (D) Northern blot evaluation of transcripts harvested
from pmCMVsiGFPmpA (lanes 3, 4) and pmCMVsi.beta.galmpA (lane 2)
transfected HEK293 cells. Blots were probed with .sup.32P-labeled
sense oligonucleotides. Antisense probes yielded similar results
(not shown). Lane 1, .sup.32P-labeled RNA markers. AdsiGFP infected
cells also possessed appropriately sized transcripts (not shown).
(E) Northern blot for evaluation of target mRNA reduction by siRNA
(upper panel). The internal control GAPDH is shown in the lower
panel. HEK293 cells were transfected with pEGFPN1 and
pmCMVsiGFPmpA, expressing siGFP, or plasmids expressing the control
siRNA as indicated. pCMVeGFPx, which expresses siGFPx, contains a
large poly(A) cassette from SV40 large T and an unmodified CMV
promoter, in contrast to pmCMVsiGFPmpA shown in (A). (F) Western
blot with anti-GFP antibodies of cell lysates harvested 72 h after
transfection with pEGFPN1 and pCMVsiGFPmpA, or pEGFPN1 and
pmCMVsi.beta.glucmpA. (G, H) Fluorescence photomicrographs of
HEK293 cells 72 h after transfection of pEGFPN1 and pCMVsiGFPx, or
pEGFPN1 and pmCMVsi.beta.glucmpA, respectively. (I, J) siRNA
reduces expression from endogenous alleles. Recombinant
adenoviruses were generated from pmCMVsi.beta.glucmpA and
pmCMVsiGFPmpA and purified. HeLa cells were infected with 25
infectious viruses/cell (MOI=25) or mock-infected (control) and
cell lysates harvested 72 h later. (I) Northern blot for
.beta.-glucuronidase mRNA levels in Adsi.beta.gluc and AdsiGFP
transduced cells. GAPDH was used as an internal control for
loading. (J) The concentration of .beta.-glucuronidase activity in
lysates quantified by a fluorometric assay. Stein, C. S. et al., J.
Virol. 73:3424-3429 (1999).
[0023] FIG. 2. Viral vectors expressing siRNA reduce expression
from transgenic and endogenous alleles in vivo. Recombinant
adenovirus vectors were prepared from the siGFP and si.beta.gluc
shuttle plasmids described in FIG. 1. (A) Fluorescence microscopy
reveals diminution of eGFP expression in vivo. In addition to the
siRNA sequences in the E1 region of adenovirus, RFP expression
cassettes in E3 facilitate localization of gene transfer.
Representative photomicrographs of eGFP (left), RFP (middle), and
merged images (right) of coronal sections from mice injected with
adenoviruses expressing siGFP (top panels) or si.beta.gluc (bottom
panels) demonstrate siRNA specificity in eGFP transgenic mice
striata after direct brain injection. (B) Full coronal brain
sections (1 mm) harvested from AdsiGFP or Adsi.beta.gluc injected
mice were split into hemisections and both ipsilateral (il) and
contralateral (cl) portions evaluated by western blot using
antibodies to GFP. Actin was used as an internal control for each
sample. (C) Tail vein injection of recombinant adenoviruses
expressing si.beta.gluc directed against mouse
.alpha.-glucuronidase (AdsiMu.beta.gluc) reduces endogenous
.beta.-glucuronidase RNA as determined by Northern blot in contrast
to control-treated (Adsi.beta.gal) mice.
[0024] FIG. 3. siGFP gene transfer reduces Q19-eGFP expression in
cell lines. PC12 cells expressing the polyglutamine repeat Q19
fused to eGFP (eGFP-Q19) under tetracycline repression (A, bottom
left) were washed and dox-free media added to allow eGFP-Q19
expression (A, top left). Adenoviruses were applied at the
indicated multiplicity of infection (MOI) 3 days after dox removal.
(A) eGFP fluorescence 3 days after adenovirus-mediated gene
transfer of Adsi.beta.gluc (top panels) or AdsiGFP (bottom panels).
(B, C) Western blot analysis of cell lysates harvested 3 days after
infection at the indicated MOIs demonstrate a dose-dependent
decrease in GFP-Q19 protein levels. NV, no virus. Top lanes,
eGFP-Q19. Bottom lanes, actin loading controls. (D) Quantitation of
eGFP fluorescence. Data represent mean total area fluorescence
+standard deviation in 4 low power fields/well (3 wells/plate).
[0025] FIG. 4. siRNA mediated reduction of expanded polyglutamine
protein levels and intracellular aggregates. PC12 cells expressing
tet-repressible eGFP-Q80 fusion proteins were washed to remove
doxycycline and adenovirus vectors expressing siRNA were applied 3
days later. (A-D) Representative punctate eGFP fluorescence of
aggregates in mock-infected cells (A), or those infected with 100
MOI of Adsi.beta.gluc (B), AdsiGFPx (C) or Adsi.beta.gal (D). (E)
Three days after infection of dox-free eGFP-Q80 PC12 cells with
AdsiGFP, aggregate size and number are notably reduced. (F) Western
blot analysis of eGFP-Q80 aggregates (arrowhead) and monomer
(arrow) following Adsi.beta.gluc or AdsiGFP infection at the
indicated MOIs demonstrates dose dependent siGFP-mediated reduction
of GFP-Q80 protein levels. (G) Quantification of the total area of
fluorescent inclusions measured in 4 independent fields/well 3 days
after virus was applied at the indicated MOIs. The data are
mean.+-.standard deviation. FIG. 5. RNAi-mediated suppression of
expanded CAG repeat containing genes. Expanded CAG repeats are not
direct targets for preferential inactivation (A), but a linked SNP
can be exploited to generate siRNA that selectively silences mutant
ataxin-3 expression (B-F). (A) Schematic of cDNA encoding
generalized polyQ-fluorescent protein fusions. Bars indicate
regions targeted by siRNAs. HeLa cells co-transfected with Q80-GFP,
Q19-RFP and the indicated siRNA. Nuclei are visualized by DAPI
staining (blue) in merged images. (B)Schematic of human ataxin-3
cDNA with bars indicating regions targeted by siRNAs. The targeted
SNP (G987C) is shown in color. In the displayed siRNAs, red or blue
bars denote C or G respectively. In this Figure,
AGCAGCAGCAGGGGGACCTATCAGGAC is SEQ ID NO:7, and
CAGCAGCAGCAGCGGGACCTATCAG- GAC is SEQ ID NO:8. (C) Quantitation of
fluorescence in Cos-7 cells transfected with wild type or mutant
ataxin-3-GFP expression plasmids and the indicated siRNA.
Fluorescence from cells co-transfected with siMiss was set at one.
Bars depict mean total fluorescence from three independent
experiments +/-standard error of the mean (SEM). (D) Western blot
analysis of cells co-transfected with the indicated ataxin-3
expression plasmids (top) and siRNAs (bottom). Appearance of
aggregated, mutant ataxin-3 in the stacking gel (seen with siMiss
and siG10) is prevented by siRNA inhibition of the mutant allele.
(E) Allele specificity is retained in the simulated heterozygous
state. Western blot analysis of Cos-7 cells cotransfected with
wild-type (atx-3-Q28-GFP) and mutant (atx-Q166) expression plasmids
along with the indicated siRNAs. (Mutant ataxin-3 detected with
1C2, an antibody specific for expanded polyQ, and wild-type
ataxin-3 detected with anti-ataxin-3 antibody.) (F) Western blot of
Cos-7 cells transfected with Atx-3-GFP expression plasmids and
plasmids encoding the indicated shRNA. The negative control
plasmid, phU6-LacZi, encodes siRNA specific for LacZ. Both normal
and mutant protein were detected with anti-ataxin-3 antibody.
Tubulin immunostaining shown as a loading control in panels
(D)-(F).
[0026] FIG. 6. Primer sequences (SEQ ID NOs:11-40) for in vitro
synthesis of siRNAs using T7 polymerase. All primers contain the
following T7 promoter sequence at their 3' ends:
5'-TATAGTGAGTCGTATTA-3' (SEQ ID NO:9). The following primer was
annealed to all oligos to synthesize siRNAs:
1 5'-TAATACGACTCACTATAG-3'. (SEQ ID NO:10)
[0027] FIG. 7. Inclusion of either two (siC7/8) or three (siC10)
CAG triplets at the 5' end of ataxin-3 siRNA does not inhibit
expression of unrelated CAG repeat containing genes. (A) Western
blot analysis of Cos-7 cells transfected with CAG repeat-GFP fusion
proteins and the indicated siRNA. Immunostaining with monoclonal
anti-GFP antibody (MBL) at 1:1000 dilution. (B) Western blot
analysis of Cos-7 cells transfected with Flag-tagged ataxin-1-Q30,
which is unrelated to ataxin-3, and the indicated siRNA.
Immunostaining with anti-Flag monoclonal antibody (Sigma St. Louis,
Mo.) at 1:1000 dilution. In panels (A) and (B), lysates were
collected 24 hours after transfection. Tubulin immunostaining shown
as a loading control.
[0028] FIG. 8. shRNA-expressing adenovirus mediates allele-specific
silencing in transiently transfected Cos-7 cells simulating the
heterozygous state. (A) Representative images of cells
cotransfected to express wild type and mutant ataxin-3 and infected
with the indicated adenovirus at 50 multiplicities of infection
(MOI). Atx-3-Q28-GFP (green) is directly visualized and Atx-3-Q166
(red) is detected by immunofluorescence with 1C2 antibody. Nuclei
visualized with DAPI stain in merged images. An average of 73.1% of
cells co-expressed both ataxin-3 proteins with siMiss. (B)
Quantitation of mean fluorescence from 2 independent experiments
performed as in (A). (C) Western blot analysis of viral-mediated
silencing in Cos-7 cells expressing wild type and mutant ataxin-3
as in (A). Mutant ataxin-3 detected with 1 C2 antibody and
wild-type human and endogenous primate ataxin-3 detected with
anti-ataxin-3 antibody. (D) shRNA-expressing adenovirus mediates
allele-specific silencing in stably transfected neural cell lines.
Differentiated PC12 neural cells expressing wild type (left) or
mutant (right) ataxin-3 were infected with adenovirus (100 MOI)
engineered to express the indicated hairpin siRNA. Shown are
Western blots immunostained for ataxin-3 and GAPDH as loading
control.
[0029] FIG. 9. Allele-specific siRNA suppression of a missense Tau
mutation. (A) Schematic of human tau cDNA with bars indicating
regions and mutations tested for siRNA suppression. Of these, the
V337M region showed effective suppression and was further studied.
Vertical bars represent microtubule binding repeat elements in Tau.
In the displayed siRNAs, blue and red bars denote A and C
respectively. In this Figure, GTGGCCAGATGGAAGTAAAATC is SEQ ID
NO:35, and GTGGCCAGGTGGAAGTAAAATC is SEQ ID NO:41. (B) Western blot
analysis of cells co-transfected with WT or V337M Tau-EGFP fusion
proteins and the indicated siRNAs. Cells were lysed 24 hr after
transfection and probed with anti-tau antibody. Tubulin
immunostaining is shown as loading control. (C) Quantitation of
fluorescence in Cos-7 cells transfected with wild type tau-EGFP or
mutant V337M tau-EGFP expression plasmids and the indicated siRNAs.
Bars depict mean fluorescence and SEM from three independent
experiments. Fluorescence from cells co-transfected with siMiss was
set at one.
[0030] FIG. 10. Allele-specific silencing of Tau in cells
simulating the heterozygous state. (A) Representative fluorescent
images of fixed Hela cells co-transfected with flag-tagged WT-Tau
(red), V337M-Tau-GFP (green), and the indicated siRNAs. An average
of 73.7% of cells co-expressed both Tau proteins with siMiss. While
siA9 suppresses both alleles, siA9/C12 selectively decreased
expression of mutant Tau only. Nuclei visualized with DAPI stain in
merged images. (B) Quantitation of mean fluorescence from 2
independent experiments performed as in (A). (C) Western blot
analysis of cells co-transfected with Flag-WT-Tau and
V337M-Tau-EGFP fusion proteins and the indicated siRNAs. Cells were
lysed 24 hr after transfection and probed with anti-tau antibody.
V337M-GFP Tau was differentiated based on reduced electrophoretic
mobility due to the addition of GFP. Tubulin immunostaining is
shown as a loading control.
[0031] FIG. 11. Schematic diagram of allele-specific silencing of
mutant TorsinA by small interfering RNA (siRNA). In the disease
state, wild type and mutant alleles of TOR1A are both transcribed
into mRNA. siRNA with sequence identical to the mutant allele
(deleted of GAG) should bind mutant mRNA selectively and mediate
its degradation by the RNA-induced silencing complex (RISC)
(circle). Wild type mRNA, not recognized by the mutant-specific
siRNA, will remain and continue to be translated into normal
TorsinA (FIG. 11A). The two adjacent GAG's in wild type TOR1A
alleles are shown as two parallelograms, one of which is deleted in
mutant TOR1A alleles (FIG. 11B).
[0032] FIG. 12. Design and targeted sequences of siRNAs. Shown are
the relative positions and targeted mRNA sequences for each primer
used in this study. Mis-siRNA (negative control; SEQ ID NOs:42-43)
does not target TA; com-siRNA (SEQ ID NOs:44-45) targets a sequence
present in wild type and mutant TA; wt-siRNA (SEQ ID NOs:47-48)
targets only wild type TA; and three mutant-specific siRNAs (Mut A
(SEQ ID NOs:49-50), B (SEQ ID NOs:51-52), C (SEQ ID NOs:53-54))
preferentially target mutant TA. The pair of GAG codons near the
c-terminus of wild type mRNA (SEQ ID NO:46) are shown in underlined
gray and black, with one codon deleted in mutant mRNA.
[0033] FIG. 13. siRNA silencing of TAwt and TAmut in Cos-7 cells.
(A) Western blot results showing the effect of different siRNAs on
GFP-TAwt expression levels. Robust suppression is achieved with
wt-siRNA and com-siRNA, while the mutant-specific siRNAs MutA, (B)
and (C) have modest or no effect on GFP-TAwt expression. Tubulin
loading controls are also shown. (B) Similar experiments with cells
expressing HA-TAmut, showing significant suppression by
mutant-specific siRNAs and com-siRNA but no suppression by the wild
type-specific siRNA, wt-siRNA. (C) Quantification of results from
at least three separate experiments as in A and B. (D) Cos-7 cells
transfected with GFP-TAwt or GFP-TAmut and different siRNAs
visualized under fluorescence microscopy (200.times.).
Representative fields are shown indicating allele-specific
suppression. (E) Quantification of fluorescence signal from two
different experiments as in D.
[0034] FIG. 14. Allele-specific silencing by siRNA in the simulated
heterozygous state. Cos-7 cells were cotransfected with plasmids
encoding differentially tagged TAwt and TAmut, together with the
indicated siRNA. (A) Western blot results analysis showing
selective suppression of the targeted allele by wt-siRNA or
mutC-siRNA. (B) Quantification of results from three experiments as
in (A).
[0035] FIG. 15. Allele-specific silencing of mutant huntingtin by
siRNA. PC6-3 cells were co-transfected with plasmids expressing
siRNA specific for the polymorphism encoding the transcript for
mutant huntingtin.
[0036] FIG. 16. Primer sequences for in vitro generation of siRNA
duplexes using T7 polymerase (SEQ ID NOs:11-12, 13-14, 63-90). All
primers used for T7 synthesis contain the following promoter
sequence at their 3' ends: 5'-CTATAGTGAGTCGTATTA-3' (SEQ ID NO:62).
The following primer was annealed to all templates to synthesize
siRNA duplexes:
2 5'-TAATACGACTCACTATAG-3'. (SEQ ID NO:10)
[0037] FIG. 17. siRNA+G duplexes silence endogenous and reporter
genes. (A) Schematic of siRNA synthesis depicting DNA template and
structure of synthesized duplexes (SEQ ID NOs:10 and 62). Blue
indicates the RNA product synthesized from the DNA template
(upper). For the siRNA duplex, gray indicates the region with
perfect complementarity to the intended target while black depicts
the antisense sequence and additional non-complementary nucleotides
added by the synthesis method. N represents any ribonucleotide. (B)
Comparison of GFP silencing by perfectly complementary siRNA versus
siRNA of the "+G" design. Images depict Cos-7 transfected with a
GFP expression construct and the indicated siRNA. Images of GFP
fluorescence are merged with images of the same field showing
DAPI-stained nuclei. Shown on the left are results with negative
control, mistargeted siRNAs (siMiss and siMiss+G respectively),
which fail to silence GFP expression. On the right, GFP expression
is efficiently suppressed by siRNA of both configurations. (C)
Western blot analysis of lysates from the same experiment as in B.
Tubulin staining is shown as a loading control. (D) Efficient
silencing of endogenous lamin gene expression with siRNA+G
duplexes. HeLa cells were transfected with the indicated siRNA and
expression of lamin A/C was evaluated by western blot 72 hr later.
The siRNA+G against human lamin markedly decreased protein levels
relative to the mistargeted control siRNA.
[0038] FIG. 18. Optimization of allele-specific silencing of mutant
tau. Cos-7 cells were cotransfected with expression constructs
encoding mutant (V337M-GFP) and WT (Flag-WT) tau and the indicated
siRNAs or shRNA plasmids. (A) Western blot results showing the
efficacy of allele-specific silencing when varying the placement of
the point mutation (G to A) in the siRNA from positions 9-12. (B)
Silencing tau with shRNA plasmid expressed from the tRNA-valine
promoter. Shown is a western blot analysis of cells cotransfected
with mutant and wild type tau and the indicated shRNA plasmids.
Placing the mutation at position 10 (tvA10) of the hairpin results
in strong preferential silencing of mutant tau. shRNA directed
against wild type (mismatched at position 9 relative to mutant tau)
tau inhibits expression from both alleles but shows a preference
for the wild type sequence.
[0039] FIG. 19. Optimization of allele-specific silencing of mutant
APP. Cos-7 cells were transfected with expression constructs
encoding wild type APP (APP) or mutant (APPsw) and the indicated
siRNAs or shRNA plasmids. (A) Immunofluorescence of Cos-7 cells
cotransfected with plasmids encoding APP or APPsw and the indicated
siRNA+G. Representative images of fields (630.times.) reveals that
allele specificity is optimal when the double mismatch is placed at
the central position (siT O/CI 1) of the targeted sequence. APP
proteins are visualized with APP antibody followed by secondary
antibody labeled with FITC (green). Nuclei are stained with DAPI
(blue). (B) Lanes 5-10 show a Western blot of cells transfected as
in A, confirming preferential silencing of APPsw with siRNA
containing central mismatches. Lane 4 is APP or APPsw transfected
without siRNA. Lane 11 represents untransfected cells showing
endogenous APP. Also shown in lanes 1-3 is comparable silencing of
APP with siRNA or siRNA+G duplexes targeted to APP. Tubulin is
shown as a loading control. (C) Western blot analysis of Cos-7
cells transfected with APP or APPsw and the indicated shRNA
plasmids. tvAPP silences APP whereas tvT10/C11 selectively
suppresses APPsw expression. Endogenous APP in untransfected cells
is shown in the last lane. Tubulin loading control is also
shown.
DETAILED DESCRIPTION OF THE INVENTION
[0040] Modulation of gene expression by endogenous, noncoding RNAs
is increasingly appreciated as a mechanism playing a role in
eukaryotic development, maintenance of chromatin structure and
genomic integrity (McManus, 2002). Recently, techniques have been
developed to trigger RNA interference (RNAi) against specific
targets in mammalian cells by introducing exogenously produced or
intracellularly expressed siRNAs (Elbashir, 2001; Brummelkamp,
2002). These methods have proven to be quick, inexpensive and
effective for knockdown experiments in vitro and in vivo (2
Elbashir, 2001; Brummelkamp, 2002; McCaffrey, 2002; Xia, 2002). The
ability to accomplish selective gene silencing has led to the
hypothesis that siRNAs might be employed to suppress gene
expression for therapeutic benefit (Xia, 2002; Jacque, 2002;
Gitlin, 2002).
[0041] RNA interference is now established as an important
biological strategy for gene silencing, but its application to
mammalian cells has been limited by nonspecific inhibitory effects
of long double-stranded RNA on translation. Moreover, delivery of
interfering RNA has largely been limited to administration of RNA
molecules. Hence, such administration must be performed repeatedly
to have any sustained effect. The present inventors have developed
a delivery mechanism that results in specific silencing of targeted
genes through expression of small interfering RNA (siRNA). The
inventors have markedly diminished expression of exogenous and
endogenous genes in vitro and in vivo in brain and liver, and
further apply this novel strategy to a model system of a major
class of neurodegenerative disorders, the polyglutamine diseases,
to show reduced polyglutamine aggregation in cells. This strategy
is generally useful in reducing expression of target genes in order
to model biological processes or to provide therapy for dominant
human diseases.
[0042] Disclosed herein is a strategy that results in substantial
silencing of targeted alleles via siRNA. Use of this strategy
results in markedly diminished in vitro and in vivo expression of
targeted alleles. This strategy is useful in reducing expression of
targeted alleles in order to model biological processes or to
provide therapy for human diseases. For example, this strategy can
be applied to a major class of neurodegenerative disorders, the
polyglutamine diseases, as is demonstrated by the reduction of
polyglutamine aggregation in cells following application of the
strategy. As used herein the term "substantial silencing" means
that the mRNA of the targeted allele is inhibited and/or degraded
by the presence of the introduced siRNA, such that expression of
the targeted allele is reduced by about 10% to 100% as compared to
the level of expression seen when the siRNA is not present.
Generally, when an allele is substantially silenced, it will have
at least 40%, 50%, 60%, to 70%, e.g., 71%, 72%, 73%, 74%, 75%, 76%,
77%, 78%, to 79%, generally at least 80%, e.g., 81%-84%, at least
85%, e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99% or even 100% reduction expression as compared to when
the siRNA is not present. As used herein the term "substantially
normal activity" means the level of expression of an allele when an
siRNA has not been introduced to a cell.
[0043] Dominantly inherited diseases are ideal candidates for
siRNA-based therapy. To explore the utility of siRNA in inherited
human disorders, the present inventors employed cellular models to
test whether mutant alleles responsible for these
dominantly-inherited human disorders could be specifically
targeted. First, different classes of dominantly inherited,
untreatable neurodegenerative diseases were examined: polyglutamine
(polyQ) neurodegeneration in MJD/SCA3, Huntington's disease and
frontotemporal dementia with parkinsonism linked to chromosome 17
(FTDP-17). Machado-Joseph disease is also known as Spinocerebellar
Ataxia Type 3 (The HUGO official name is MJD). The gene involved is
MJD1, which encodes for the protein ataxin-3 (also called Mjd1p).
Huntington's disease is due to expansion of the CAG repeat motif in
exon 1 of huntingtin. In 38% of patients a polymorphism exists in
exon 58 of the huntingtin gene, allowing for allele specific
targeting. Frontotemporal dementia (sometimes with parkinonism, and
linked to chromosome 17, so sometimes called FTDP-17) is due to
mutations in the MAPT1 gene that encodes the protein tau. The
inventors also examined amyloid precursor protein (APP) as a target
of RNAi.
[0044] APP and tau were chosen as candidate RNAi targets because of
their central role in inherited and acquired forms of age-related
dementia, including Alzheimer's disease (AD) (Hardy et al., 2002;
Lee et al., 2001; Mullan et al., 1992; Poorkaj et al., 1998; Hutton
et al., 1998). AD is characterized by two major pathological
hallmarks: senile plaques, which contain beta-amyloid (AP) derived
from cleavage of APP; and neurofibrillary tangles, which contain
filamentous tau protein. Rare inherited forms of AD have revealed
an essential role for A.beta. production in the pathogenesis of all
forms of AD, both sporadic and inherited (Hardy et al., 2002).
Mutations in the three genes known to cause familial AD--the genes
encoding APP, presenilin 1 and presenilin 2--act dominantly to
enhance the production of neurotoxic A.beta. (Hardy et al.,
2002).
[0045] The best studied AD mutation is the Swedish double mutation
in APP (APPsw), two consecutive missense changes that alter
adjacent amino acids near the .beta. cleavage site (Mullan et al.,
1992). APPsw has been used to create several widely used transgenic
mouse models of AD (Lewis et al., 2001; Oddo et al., 2003), thus
the inventors chose it as an ideal mutation against which to
generate allele-specific siRNAs for AD research. Such siRNA might
also have therapeutic value because RNAi-mediated silencing of APP
should inhibit A.beta. deposition.
[0046] Tau, the major component of neurofibrillary tangles,
likewise plays a significant role in AD pathogenesis (Lee et al.,
2001). Mutations in tau cause a similar dominantly inherited
neurodegenerative disease, frontotemporal dementia with
parkinsonism linked to chromosome 17 (FTDP-17). In FTDP-17, tau
mutations either alter the tau protein sequence or lead to aberrant
splicing (Lee et al., 2001; Lewis et al., 2001; Oddo et al., 2003).
Abnormalities of tau expression also contribute to several other
important neurodegenerative disorders, including progressive
supranuclear palsy and cortical-basal ganglionic degeneration
(Houlden et al., 2001). Thus, efforts to reduce tau expression,
either generally or in an allele-specific manner, may prove to be
therapeutically useful in FTDP-17, AD or other tau-related
diseases.
[0047] The polyQ neurodegenerative disorders include at least nine
diseases caused by CAG repeat expansions that encode polyQ in the
disease protein. PolyQ expansion confers a dominant toxic property
on the mutant protein that is associated with aberrant accumulation
of the disease protein in neurons (Zoghbi, 2000). In FTDP-17, Tau
mutations lead to the formation of neurofibrillary tangles
accompanied by neuronal dysfunction and degeneration (Poorkaj,
1998; Hutton, 1998). The precise mechanisms by which these mutant
proteins cause neuronal injury are unknown, but considerable
evidence suggests that the abnormal proteins themselves initiate
the pathogenic process (Zoghbi, 2000). Accordingly, eliminating
expression of the mutant protein by siRNA or other means slows or
prevents disease (Yamamoto, 2000). However, because many dominant
disease genes also encode essential proteins (e.g. Nasir, 1995)
siRNA-mediated approaches were developed that selectively
inactivate mutant alleles, while allowing continued expression of
the wild type proteins ataxin-3 and huntingtin.
[0048] Second, the dominantly-inherited disorder DYT1 dystonia was
studied. DYT1 dystonia is also known as Torsion dystonia type 1,
and is caused by a GAG deletion in the TOR1A gene encoding torsinA.
DYT1 dystonia is the most common cause of primary generalized
dystonia. DYT1 usually presents in childhood as focal dystonia that
progresses to severe generalized disease (Fahn, 1998; Klein,
2002a). With one possible exception (Leung, 2001; Doheny, 2002;
Klein, 2002), all cases of DYT1 result from a common GAG deletion
in TOR1A, eliminating one of two adjacent glutamic acids near the
C-terminus of the protein TorsinA (TA) (Ozelius, 1997). Although
the precise cellular function of TA is unknown, it seems clear that
mutant TA (TAmut) acts through a dominant-negative or
dominant-toxic mechanism (Breakefield, 2001).
[0049] Several characteristics of DYT1 make it an ideal disease in
which to use siRNA-mediated gene silencing as therapy. Of greatest
importance, the dominant nature of the disease suggests that a
reduction in mutant TA, whatever the precise pathogenic mechanism
proves to be, is helpful. Moreover, the existence of a single
common mutation that deletes a full three nucleotides suggested it
might be feasible to design siRNA that specifically targets the
mutant allele and is applicable to all affected persons. Finally,
there is no effective therapy for DYT1, a relentless and disabling
disease.
[0050] As outlined in the strategy in FIG. 11, the inventors
developed siRNA that would specifically eliminate production of
protein from the mutant allele. By exploiting the three base pair
difference between wild type and mutant alleles, the inventors
successfully silenced expression of the mutant protein (TAmut)
without interfering with expression of the wild type protein
(TAwt). Because TAwt may be an essential protein it is critically
important that efforts be made to silence only the mutant allele.
This allele-specific strategy has obvious therapeutic potential for
DYT1 and represents a novel and powerful research tool with which
to investigate the function of TA and its dysfunction in the
disease state.
[0051] Expansions of poly-glutamine tracts in proteins that are
expressed in the central nervous system can cause neurodegenerative
diseases. Some neurodegenerative diseases are caused by a
(CAG).sub.n repeat that encodes poly-glutamine in a protein include
Huntington disease (HD), spinocerebellar ataxia (SCA1, SCA2, SCA3,
SCA6, SCA7), spinal and bulbar muscular atrophy (SBMA), and
dentatorubropallidoluysian atrophy (DRPLA). In these diseases, the
poly-glutamine expansion in a protein confers a novel toxic
property upon the protein. Studies indicate that the toxic property
is a tendency for the disease protein to misfold and form
aggregates within neurons.
[0052] The gene involved in Huntington's disease (IT-15) is located
at the end of the short arm of chromosome 4. This gene is
designated HD and encodes the protein huntingtin (also known as
Htt). A mutation occurs in the coding region of this gene and
produces an unstable expanded trinucleotide repeat
(cytosine-adenosine-guanosine), resulting in a protein with an
expanded glutamate sequence. The normal and abnormal functions of
this protein (termed huntingtin) are unknown. The abnormal
huntingtin protein appears to accumulate in neuronal nuclei of
transgenic mice, but the causal relationship of this accumulation
to neuronal death is uncertain.
[0053] One of skill in the art can select additional target sites
for generating siRNA specific for other alleles beyond those
specifically described in the experimental examples. Such
allele-specific siRNAs made be designed using the guidelines
provided by Ambion (Austin, Tex.). Briefly, the target cDNA
sequence is scanned for target sequences that had AA
di-nucleotides. Sense and anti-sense oligonucleotides are generated
to these targets (AA+3' adjacent 19 nucleotides) that contained a
G/C content of 35 to 55%. These sequences are then compared to
others in the human genome database to minimize homology to other
known coding sequences (BLAST search). (is this paragraph
required?)
[0054] To accomplish intracellular expression of the therapeutic
siRNA, an RNA molecule is constructed containing two complementary
strands or a hairpin sequence (such as a 21-bp hairpin)
representing sequences directed against the gene of interest. The
siRNA, or a nucleic acid encoding the siRNA, is introduced to the
target cell, such as a diseased brain cell. The siRNA reduces
target mRNA and protein expression.
[0055] The construct encoding the therapeutic siRNA is configured
such that the one or more strands of the siRNA are encoded by a
nucleic acid that is immediately contiguous to a promoter. In one
example, the promoter is a pol II promoter. If a pol II promoter is
used in a particular construct, it is selected from readily
available pol II promoters known in the art, depending on whether
regulatable, inducible, tissue or cell-specific expression of the
siRNA is desired. The construct is introduced into the target cell,
such as by injection, allowing for diminished target-gene
expression in the cell.
[0056] It was surprising that a pol II promoter would be effective.
While small RNAs with extensive secondary structure are routinely
made from Pol III promoters, there is no a priori reason to assume
that small interfering RNAs could be expressed from pol II
promoters. Pol III promoters terminate in a short stretch of Ts (5
or 6), leaving a very small 3' end and allowing stabilization of
secondary structure. Polymerase II transcription extends well past
the coding and polyadenylation regions, after which the transcript
is cleaved. Two adenylation steps occur, leaving a transcript with
a tail of up to 200 As. This string of As would of course
completely destabilize any small, 21 base pair hairpin. Therefore,
in addition to modifying the promoter to minimize sequences between
the transcription start site and the siRNA sequence (thereby
stabilizing the hairpin), the inventors also extensively modified
the polyadenylation sequence to test if a very short
polyadenylation could occur. The results, which were not predicted
from prior literature, showed that it could.
[0057] The present invention provides an expression cassette
containing an isolated nucleic acid sequence encoding a small
interfering RNA molecule (siRNA) targeted against a gene of
interest. The siRNA may form a hairpin structure that contains a
duplex structure and a loop structure. The loop structure may
contain from 4 to 10 nucleotides, such as 4, 5 or 6 nucleotides.
The duplex is less than 30 nucleotides in length, such as from 19
to 25 nucleotides. The siRNA may further contain an overhang
region. Such an overhang may be a 3' overhang region or a 5'
overhang region. The overhang region may be, for example, from 1 to
6 nucleotides in length. The expression cassette may further
contain a pol II promoter, as described herein. Examples of pol II
promoters include regulatable promoters and constitutive promoters.
For example, the promoter may be a CMV or RSV promoter. The
expression cassette may further contain a polyadenylation signal,
such as a synthetic minimal polyadenylation signal. The nucleic
acid sequence may further contain a marker gene. The expression
cassette may be contained in a viral vector. An appropriate viral
vector for use in the present invention may be an adenoviral,
lentiviral, adeno-associated viral (AAV), poliovirus, herpes
simplex virus (HSV) or murine Maloney-based viral vector. The gene
of interest may be a gene associated with a condition amenable to
siRNA therapy. Examples of such conditions include
neurodegenerative diseases, such as a trinucleotide-repeat disease
(e.g., polyglutamine repeat disease). Examples of these diseases
include Huntington's disease, several spinocerebellar ataxias, and
Alzheimer's disease. Alternatively, the gene of interest may encode
a ligand for a chemokine involved in the migration of a cancer
cell, or a chemokine receptor.
[0058] The present invention also provides an expression cassette
containing an isolated nucleic acid sequence encoding a first
segment, a second segment located immediately 3' of the first
segment, and a third segment located immediately 3' of the second
segment, wherein the first and third segments are each less than 30
base pairs in length and each more than 10 base pairs in length,
and wherein the sequence of the third segment is the complement of
the sequence of the first segment, and wherein the isolated nucleic
acid sequence functions as a small interfering RNA molecule (siRNA)
targeted against a gene of interest. The expression cassette may be
contained in a vector, such as a viral vector.
[0059] The present invention provides a method of reducing the
expression of a gene product in a cell by contacting a cell with an
expression cassette described above. It also provides a method of
treating a patient by administering to the patient a composition of
the expression cassette described above.
[0060] The present invention further provides a method of reducing
the expression of a gene product in a cell by contacting a cell
with an expression cassette containing an isolated nucleic acid
sequence encoding a first segment, a second segment located
immediately 3' of the first segment, and a third segment located
immediately 3' of the second segment, wherein the first and third
segments are each less than 30 base pairs in length and each more
than 10 base pairs in length, and wherein the sequence of the third
segment is the complement of the sequence of the first segment, and
wherein the isolated nucleic acid sequence functions as a small
interfering RNA molecule (siRNA) targeted against a gene of
interest.
[0061] The present invention also provides a method of treating a
patient, by administering to the patient a composition containing
an expression cassette, wherein the expression cassette contains an
isolated nucleic acid sequence encoding a first segment, a second
segment located immediately 3' of the first segment, and a third
segment located immediately 3' of the second segment, wherein the
first and third segments are each less than 30 bases in length and
each more than 10 bases in length, and wherein the sequence of the
third segment is the complement of the sequence of the first
segment, and wherein the isolated nucleic acid sequence functions
as a small interfering RNA molecule (siRNA) targeted against a gene
of interest.
[0062] RNAi holds promise as a potential therapy for human
diseases. Yet a limitation to successfully developing gene-specific
or allele-specific siRNAs is the selection and design of siRNAs
with the desired silencing characteristics. Individual siRNAs
targeted to different regions of a transcript often display
striking differences in efficacy and specificity (Miller et al.,
2003; Ding et al., 2003). Typically, several target sites and
designs need to be tested before optimal silencing is achieved
(Miller et al., 2003). Here the inventors have described a simple
method that not only circumvents the time and cost disadvantages of
chemically synthesizing siRNA duplexes but also removes the
sequence restrictions imposed by in vitro transcription with T7
polymerase.
[0063] The insertion of a single G mismatch at the 5' of the siRNA
duplex permitted efficient priming by T7 polymerase without
compromising the silencing efficacy of the resultant siRNA. Such
"+G" siRNAs can rapidly be generated to essentially any point in a
targeted gene and tested for efficacy. This approach to siRNA
design facilitates the in vitro generation of effective siRNAs. As
demonstrated here for two important disease targets, tau and APP,
these in vitro transcribed duplexes can then serve as guides for
producing shRNA plasmids that retain silencing capability and
allele specificity. This approach represents an improved, stepwise
method for optimized silencing of essentially any gene of
interest.
[0064] Indeed, based on new insights into RISC assembly,
manipulating the 5' terminal nucleotide of the guide strand in this
way may be highly advantageous. Schwarz et al. (Schwarz et al.,
2003) recently discovered marked asymmetry in the rate at which
each strand of an RNA duplex enters the RISC complex. Preferential
entry of the guide, or antisense, strand into RISC can be achieved
by introducing 5' mismatches in the antisense strand while
maintaining perfect base pairing at the 5' terminus of the sense
strand. This maximizes entry of the antisense strand into the RISC
complex, while also reducing potential off-target inhibition by the
sense strand. The "+G" approach to siRNA design is perfectly suited
to engineering dsRNAs based on this principle that should display
preferred RISC entry of the guide strand.
[0065] The inventors have also discovered that central placement of
mismatches is required for allelic discrimination. Using the
present approach to in vitro siRNA production, the inventors
systematically tested the effect of placing mismatches at each
point along the guide strand of the siRNA. The inventors have found
that central placement of mismatches resulted in optimal
allele-specific silencing of mutant alleles.
[0066] I. Definitions
[0067] The term "nucleic acid" refers to deoxyribonucleotides or
ribonucleotides and polymers thereof in either single- or
double-stranded form, composed of monomers (nucleotides) containing
a sugar, phosphate and a base that is either a purine or
pyrimidine. Unless specifically limited, the term encompasses
nucleic acids containing known analogs of natural nucleotides that
have similar binding properties as the reference nucleic acid and
are metabolized in a manner similar to naturally occurring
nucleotides. Unless otherwise indicated, a particular nucleic acid
sequence also encompasses conservatively modified variants thereof
(e.g., degenerate codon substitutions) and complementary sequences,
as well as the sequence explicitly indicated. Specifically,
degenerate codon substitutions may be achieved by generating
sequences in which the third position of one or more selected (or
all) codons is substituted with mixed-base and/or deoxyinosine
residues (Batzer et al., (1991); Ohtsuka et al., (1985); Rossolini
et al., (1994)).
[0068] A "nucleic acid fragment" is a portion of a given nucleic
acid molecule. Deoxyribonucleic acid (DNA) in the majority of
organisms is the genetic material while ribonucleic acid (RNA) is
involved in the transfer of information contained within DNA into
proteins.
[0069] The term "nucleotide sequence" refers to a polymer of DNA or
RNA which can be single- or double-stranded, optionally containing
synthetic, non-natural or altered nucleotide bases capable of
incorporation into DNA or RNA polymers.
[0070] The terms "nucleic acid", "nucleic acid molecule", "nucleic
acid fragment", "nucleic acid sequence or segment", or
"polynucleotide" are used interchangeably and may also be used
interchangeably with gene, cDNA, DNA and RNA encoded by a gene.
[0071] The invention encompasses isolated or substantially purified
nucleic acid or protein compositions. In the context of the present
invention, an "isolated" or "purified" DNA molecule or RNA molecule
or an "isolated" or "purified" polypeptide is a DNA molecule, RNA
molecule, or polypeptide that exists apart from its native
environment and is therefore not a product of nature. An isolated
DNA molecule, RNA molecule or polypeptide may exist in a purified
form or may exist in a non-native environment such as, for example,
a transgenic host cell. For example, an "isolated" or "purified"
nucleic acid molecule or protein, or biologically active portion
thereof, is substantially free of other cellular material, or
culture medium when produced by recombinant techniques, or
substantially free of chemical precursors or other chemicals when
chemically synthesized. In one embodiment, an "isolated" nucleic
acid is free of sequences that naturally flank the nucleic acid
(i.e., sequences located at the 5' and 3' ends of the nucleic acid)
in the genomic DNA of the organism from which the nucleic acid is
derived. For example, in various embodiments, the isolated nucleic
acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1
kb, 0.5 kb, or 0.1 kb of nucleotide sequences that naturally flank
the nucleic acid molecule in genomic DNA of the cell from which the
nucleic acid is derived. A protein that is substantially free of
cellular material includes preparations of protein or polypeptide
having less than about 30%, 20%, 10%, or 5% (by dry weight) of
contaminating protein. When the protein of the invention, or
biologically active portion thereof, is recombinantly produced,
preferably culture medium represents less than about 30%, 20%, 10%,
or 5% (by dry weight) of chemical precursors or
non-protein-of-interest chemicals. Fragments and variants of the
disclosed nucleotide sequences and proteins or partial-length
proteins encoded thereby are also encompassed by the present
invention. By "fragment" or "portion" is meant a full length or
less than full length of the nucleotide sequence encoding, or the
amino acid sequence of, a polypeptide or protein.
[0072] The term "gene" is used broadly to refer to any segment of
nucleic acid associated with a biological function. Thus, genes
include coding sequences and/or the regulatory sequences required
for their expression. For example, "gene" refers to a nucleic acid
fragment that expresses mRNA, functional RNA, or specific protein,
including regulatory sequences. "Genes" also include nonexpressed
DNA segments that, for example, form recognition sequences for
other proteins. "Genes" can be obtained from a variety of sources,
including cloning from a source of interest or synthesizing from
known or predicted sequence information, and may include sequences
designed to have desired parameters. An "allele" is one of several
alternative forms of a gene occupying a given locus on a
chromosome.
[0073] "Naturally occurring" is used to describe an object that can
be found in nature as distinct from being artificially produced.
For example, a protein or nucleotide sequence present in an
organism (including a virus), which can be isolated from a source
in nature and which has not been intentionally modified by a person
in the laboratory, is naturally occurring.
[0074] The term "chimeric" refers to a gene or DNA that contains 1)
DNA sequences, including regulatory and coding sequences, that are
not found together in nature, or 2) sequences encoding parts of
proteins not naturally adjoined, or 3) parts of promoters that are
not naturally adjoined. Accordingly, a chimeric gene may include
regulatory sequences and coding sequences that are derived from
different sources, or include regulatory sequences and coding
sequences derived from the same source, but arranged in a manner
different from that found in nature.
[0075] A "transgene" refers to a gene that has been introduced into
the genome by transformation. Transgenes include, for example, DNA
that is either heterologous or homologous to the DNA of a
particular cell to be transformed. Additionally, transgenes may
include native genes inserted into a non-native organism, or
chimeric genes.
[0076] The term "endogenous gene" refers to a native gene in its
natural location in the genome of an organism.
[0077] A "foreign" gene refers to a gene not normally found in the
host organism that has been introduced by gene transfer.
[0078] The terms "protein," "peptide" and "polypeptide" are used
interchangeably herein.
[0079] A "variant" of a molecule is a sequence that is
substantially similar to the sequence of the native molecule. For
nucleotide sequences, variants include those sequences that,
because of the degeneracy of the genetic code, encode the identical
amino acid sequence of the native protein. Naturally occurring
allelic variants such as these can be identified with the use of
molecular biology techniques, as, for example, with polymerase
chain reaction (PCR) and hybridization techniques. Variant
nucleotide sequences also include synthetically derived nucleotide
sequences, such as those generated, for example, by using
site-directed mutagenesis, which encode the native protein, as well
as those that encode a polypeptide having amino acid substitutions.
Generally, nucleotide sequence variants of the invention will have
at least 40%, 50%, 60%, to 70%, e.g., 71%, 72%, 73%, 74%, 75%, 76%,
77%, 78%, to 79%, generally at least 80%, e.g., 81%-84%, at least
85%, e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, to 98%, sequence identity to the native (endogenous)
nucleotide sequence.
[0080] "Conservatively modified variations" of a particular nucleic
acid sequence refers to those nucleic acid sequences that encode
identical or essentially identical amino acid sequences. Because of
the degeneracy of the genetic code, a large number of functionally
identical nucleic acids encode any given polypeptide. For instance,
the codons CGT, CGC, CGA, CGG, AGA and AGG all encode the amino
acid arginine. Thus, at every position where an arginine is
specified by a codon, the codon can be altered to any of the
corresponding codons described without altering the encoded
protein. Such nucleic acid variations are "silent variations,"
which are one species of "conservatively modified variations."
Every nucleic acid sequence described herein that encodes a
polypeptide also describes every possible silent variation, except
where otherwise noted. One of skill in the art will recognize that
each codon in a nucleic acid (except ATG, which is ordinarily the
only codon for methionine) can be modified to yield a functionally
identical molecule by standard techniques. Accordingly, each
"silent variation" of a nucleic acid that encodes a polypeptide is
implicit in each described sequence.
[0081] "Recombinant DNA molecule" is a combination of DNA sequences
that are joined together using recombinant DNA technology and
procedures used to join together DNA sequences as described, for
example, in Sambrook and Russell (2001).
[0082] The terms "heterologous gene", "heterologous DNA sequence",
"exogenous DNA sequence", "heterologous RNA sequence", "exogenous
RNA sequence" or "heterologous nucleic acid" each refer to a
sequence that either originates from a source foreign to the
particular host cell, or is from the same source but is modified
from its original or native form. Thus, a heterologous gene in a
host cell includes a gene that is endogenous to the particular host
cell but has been modified through, for example, the use of DNA
shuffling. The terms also include non-naturally occurring multiple
copies of a naturally occurring DNA or RNA sequence. Thus, the
terms refer to a DNA or RNA segment that is foreign or heterologous
to the cell, or homologous to the cell but in a position within the
host cell nucleic acid in which the element is not ordinarily
found. Exogenous DNA segments are expressed to yield exogenous
polypeptides.
[0083] A "homologous" DNA or RNA sequence is a sequence that is
naturally associated with a host cell into which it is
introduced.
[0084] "Wild-type" refers to the normal gene or organism found in
nature.
[0085] "Genome" refers to the complete genetic material of an
organism.
[0086] A "vector" is defined to include, inter alia, any viral
vector, as well as any plasmid, cosmid, phage or binary vector in
double or single stranded linear or circular form that may or may
not be self transmissible or mobilizable, and that can transform
prokaryotic or eukaryotic host either by integration into the
cellular genome or exist extrachromosomally (e.g., autonomous
replicating plasmid with an origin of replication).
[0087] "Expression cassette" as used herein means a nucleic acid
sequence capable of directing expression of a particular nucleotide
sequence in an appropriate host cell, which may include a promoter
operably linked to the nucleotide sequence of interest that may be
operably linked to termination signals. It also may include
sequences required for proper translation of the nucleotide
sequence. The coding region usually codes for a protein of interest
but may also code for a functional RNA of interest, for example an
antisense RNA, a nontranslated RNA in the sense or antisense
direction, or a siRNA. The expression cassette including the
nucleotide sequence of interest may be chimeric. The expression
cassette may also be one that is naturally occurring but has been
obtained in a recombinant form useful for heterologous expression.
The expression of the nucleotide sequence in the expression
cassette may be under the control of a constitutive promoter or of
an regulatable promoter that initiates transcription only when the
host cell is exposed to some particular stimulus. In the case of a
multicellular organism, the promoter can also be specific to a
particular tissue or organ or stage of development.
[0088] Such expression cassettes can include a transcriptional
initiation region linked to a nucleotide sequence of interest. Such
an expression cassette is provided with a plurality of restriction
sites for insertion of the gene of interest to be under the
transcriptional regulation of the regulatory regions. The
expression cassette may additionally contain selectable marker
genes.
[0089] "Coding sequence" refers to a DNA or RNA sequence that codes
for a specific amino acid sequence. It may constitute an
"uninterrupted coding sequence", i.e., lacking an intron, such as
in a cDNA, or it may include one or more introns bounded by
appropriate splice junctions. An "intron" is a sequence of RNA that
is contained in the primary transcript but is removed through
cleavage and re-ligation of the RNA within the cell to create the
mature mRNA that can be translated into a protein.
[0090] The term "open reading frame" (ORF) refers to the sequence
between translation initiation and termination codons of a coding
sequence. The terms "initiation codon" and "termination codon"
refer to a unit of three adjacent nucleotides (a `codon`) in a
coding sequence that specifies initiation and chain termination,
respectively, of protein synthesis (mRNA translation).
[0091] "Functional RNA" refers to sense RNA, antisense RNA,
ribozyme RNA, siRNA, or other RNA that may not be translated but
yet has an effect on at least one cellular process.
[0092] The term "RNA transcript" refers to the product resulting
from RNA polymerase catalyzed transcription of a DNA sequence. When
the RNA transcript is a perfect complementary copy of the DNA
sequence, it is referred to as the primary transcript or it may be
a RNA sequence derived from posttranscriptional processing of the
primary transcript and is referred to as the mature RNA. "Messenger
RNA" (mRNA) refers to the RNA that is without introns and that can
be translated into protein by the cell. "cDNA" refers to a single-
or a double-stranded DNA that is complementary to and derived from
mRNA.
[0093] "Regulatory sequences" and "suitable regulatory sequences"
each refer to nucleotide sequences located upstream (5' non-coding
sequences), within, or downstream (3' non-coding sequences) of a
coding sequence, and which influence the transcription, RNA
processing or stability, or translation of the associated coding
sequence. Regulatory sequences include enhancers, promoters,
translation leader sequences, introns, and polyadenylation signal
sequences. They include natural and synthetic sequences as well as
sequences that may be a combination of synthetic and natural
sequences. As is noted above, the term "suitable regulatory
sequences" is not limited to promoters. However, some suitable
regulatory sequences useful in the present invention will include,
but are not limited to constitutive promoters, tissue-specific
promoters, development-specific promoters, regulatable promoters
and viral promoters. Examples of promoters that may be used in the
present invention include CMV, RSV, polII and polIII promoters.
[0094] "5' non-coding sequence" refers to a nucleotide sequence
located 5' (upstream) to the coding sequence. It is present in the
fully processed mRNA upstream of the initiation codon and may
affect processing of the primary transcript to mRNA, mRNA stability
or translation efficiency (Turner et al., 1995).
[0095] "3' non-coding sequence" refers to nucleotide sequences
located 3' (downstream) to a coding sequence and may include
polyadenylation signal sequences and other sequences encoding
regulatory signals capable of affecting mRNA processing or gene
expression. The polyadenylation signal is usually characterized by
affecting the addition of polyadenylic acid tracts to the 3' end of
the mRNA precursor.
[0096] The term "translation leader sequence" refers to that DNA
sequence portion of a gene between the promoter and coding sequence
that is transcribed into RNA and is present in the fully processed
mRNA upstream (5) of the translation start codon. The translation
leader sequence may affect processing of the primary transcript to
mRNA, mRNA stability or translation efficiency.
[0097] The term "mature" protein refers to a post-translationally
processed polypeptide without its signal peptide. "Precursor"
protein refers to the primary product of translation of an mRNA.
"Signal peptide" refers to the amino terminal extension of a
polypeptide, which is translated in conjunction with the
polypeptide forming a precursor peptide and which is required for
its entrance into the secretory pathway. The term "signal sequence"
refers to a nucleotide sequence that encodes the signal
peptide.
[0098] "Promoter" refers to a nucleotide sequence, usually upstream
(5) to its coding sequence, which directs and/or controls the
expression of the coding sequence by providing the recognition for
RNA polymerase and other factors required for proper transcription.
"Promoter" includes a minimal promoter that is a short DNA sequence
comprised of a TATA-box and other sequences that serve to specify
the site of transcription initiation, to which regulatory elements
are added for control of expression. "Promoter" also refers to a
nucleotide sequence that includes a minimal promoter plus
regulatory elements that is capable of controlling the expression
of a coding sequence or functional RNA. This type of promoter
sequence consists of proximal and more distal upstream elements,
the latter elements often referred to as enhancers. Accordingly, an
"enhancer" is a DNA sequence that can stimulate promoter activity
and may be an innate element of the promoter or a heterologous
element inserted to enhance the level or tissue specificity of a
promoter. It is capable of operating in both orientations (normal
or flipped), and is capable of functioning even when moved either
upstream or downstream from the promoter. Both enhancers and other
upstream promoter elements bind sequence-specific DNA-binding
proteins that mediate their effects. Promoters may be derived in
their entirety from a native gene, or be composed of different
elements derived from different promoters found in nature, or even
be comprised of synthetic DNA segments. A promoter may also contain
DNA sequences that are involved in the binding of protein factors
that control the effectiveness of transcription initiation in
response to physiological or developmental conditions.
[0099] The "initiation site" is the position surrounding the first
nucleotide that is part of the transcribed sequence, which is also
defined as position +1. With respect to this site all other
sequences of the gene and its controlling regions are numbered.
Downstream sequences (i.e., further protein encoding sequences in
the 3' direction) are denominated positive, while upstream
sequences (mostly of the controlling regions in the 5' direction)
are denominated negative.
[0100] Promoter elements, particularly a TATA element, that are
inactive or that have greatly reduced promoter activity in the
absence of upstream activation are referred to as "minimal or core
promoters." In the presence of a suitable transcription factor, the
minimal promoter functions to permit transcription. A "minimal or
core promoter" thus consists only of all basal elements needed for
transcription initiation, e.g., a TATA box and/or an initiator.
[0101] "Constitutive expression" refers to expression using a
constitutive or regulated promoter. "Conditional" and "regulated
expression" refer to expression controlled by a regulated
promoter.
[0102] "Operably-linked" refers to the association of nucleic acid
sequences on single nucleic acid fragment so that the function of
one of the sequences is affected by another. For example, a
regulatory DNA sequence is said to be "operably linked to" or
"associated with" a DNA sequence that codes for an RNA or a
polypeptide if the two sequences are situated such that the
regulatory DNA sequence affects expression of the coding DNA
sequence (i.e., that the coding sequence or functional RNA is under
the transcriptional control of the promoter). Coding sequences can
be operably-linked to regulatory sequences in sense or antisense
orientation.
[0103] "Expression" refers to the transcription and/or translation
of an endogenous gene, heterologous gene or nucleic acid segment,
or a transgene in cells. For example, in the case of siRNA
constructs, expression may refer to the transcription of the siRNA
only. In addition, expression refers to the transcription and
stable accumulation of sense (mRNA) or functional RNA. Expression
may also refer to the production of protein.
[0104] "Altered levels" refers to the level of expression in
transgenic cells or organisms that differs from that of normal or
untransformed cells or organisms.
[0105] "Overexpression" refers to the level of expression in
transgenic cells or organisms that exceeds levels of expression in
normal or untransformed cells or organisms.
[0106] "Antisense inhibition" refers to the production of antisense
RNA transcripts capable of suppressing the expression of protein
from an endogenous gene or a transgene.
[0107] "Transcription stop fragment" refers to nucleotide sequences
that contain one or more regulatory signals, such as
polyadenylation signal sequences, capable of terminating
transcription. Examples include the 3' non-regulatory regions of
genes encoding nopaline synthase and the small subunit of ribulose
bisphosphate carboxylase.
[0108] "Translation stop fragment" refers to nucleotide sequences
that contain one or more regulatory signals, such as one or more
termination codons in all three frames, capable of terminating
translation. Insertion of a translation stop fragment adjacent to
or near the initiation codon at the 5' end of the coding sequence
will result in no translation or improper translation. Excision of
the translation stop fragment by site-specific recombination will
leave a site-specific sequence in the coding sequence that does not
interfere with proper translation using the initiation codon.
[0109] The terms "cis-acting sequence" and "cis-acting element"
refer to DNA or RNA sequences whose functions require them to be on
the same molecule. An example of a cis-acting sequence on the
replicon is the viral replication origin.
[0110] The terms "trans-acting sequence" and "trans-acting element"
refer to DNA or RNA sequences whose function does not require them
to be on the same molecule.
[0111] "Chromosomally-integrated" refers to the integration of a
foreign gene or nucleic acid construct into the host DNA by
covalent bonds. Where genes are not "chromosomally integrated" they
may be "transiently expressed." Transient expression of a gene
refers to the expression of a gene that is not integrated into the
host chromosome but functions independently, either as part of an
autonomously replicating plasmid or expression cassette, for
example, or as part of another biological system such as a
virus.
[0112] The following terms are used to describe the sequence
relationships between two or more nucleic acids or polynucleotides:
(a) "reference sequence", (b) "comparison window", (c) "sequence
identity", (d) "percentage of sequence identity", and (e)
"substantial identity".
[0113] (a) As used herein, "reference sequence" is a defined
sequence used as a basis for sequence comparison. A reference
sequence may be a subset or the entirety of a specified sequence;
for example, as a segment of a full-length cDNA or gene sequence,
or the complete cDNA or gene sequence.
[0114] (b) As used herein, "comparison window" makes reference to a
contiguous and specified segment of a polynucleotide sequence,
wherein the polynucleotide sequence in the comparison window may
comprise additions or deletions (i.e., gaps) compared to the
reference sequence (which does not comprise additions or deletions)
for optimal alignment of the two sequences. Generally, the
comparison window is at least 20 contiguous nucleotides in length,
and optionally can be 30, 40, 50, 100, or longer. Those of skill in
the art understand that to avoid a high similarity to a reference
sequence due to inclusion of gaps in the polynucleotide sequence a
gap penalty is typically introduced and is subtracted from the
number of matches.
[0115] Methods of alignment of sequences for comparison are
well-known in the art. Thus, the determination of percent identity
between any two sequences can be accomplished using a mathematical
algorithm. Preferred, non-limiting examples of such mathematical
algorithms are the algorithm of Myers and Miller (1988); the local
homology algorithm of Smith et al. (1981); the homology alignment
algorithm of Needleman and Wunsch (1970); the
search-for-similarity-method of Pearson and Lipman (1988); the
algorithm of Karlin and Altschul (1990), modified as in Karlin and
Altschul (1993).
[0116] Computer implementations of these mathematical algorithms
can be utilized for comparison of sequences to determine sequence
identity. Such implementations include, but are not limited to:
CLUSTAL in the PC/Gene program (available from Intelligenetics,
Mountain View, Calif.); the ALIGN program (Version 2.0) and GAP,
BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics
Software Package, Version 8 (available from Genetics Computer Group
(GCG), 575 Science Drive, Madison, Wis., USA). Alignments using
these programs can be performed using the default parameters. The
CLUSTAL program is well described by Higgins et al. (1988); Higgins
et al. (1989); Corpet et al. (1988); Huang et al. (1992); and
Pearson et al. (1994). The ALIGN program is based on the algorithm
of Myers and Miller, supra. The BLAST programs of Altschul et al.
(1990), are based on the algorithm of Karlin and Altschul
supra.
[0117] Software for performing BLAST analyses is publicly available
through the National Center for Biotechnology Information
(http://www.ncbi.nlm.nih.gov/). This algorithm involves first
identifying high scoring sequence pairs (HSPs) by identifying short
words of length W in the query sequence, which either match or
satisfy some positive-valued threshold score T when aligned with a
word of the same length in a database sequence. T is referred to as
the neighborhood word score threshold. These initial neighborhood
word hits act as seeds for initiating searches to find longer HSPs
containing them. The word hits are then extended in both directions
along each sequence for as far as the cumulative alignment score
can be increased. Cumulative scores are calculated using, for
nucleotide sequences, the parameters M (reward score for a pair of
matching residues; always >0) and N (penalty score for
mismatching residues; always <0). For amino acid sequences, a
scoring matrix is used to calculate the cumulative score. Extension
of the word hits in each direction are halted when the cumulative
alignment score falls off by the quantity X from its maximum
achieved value, the cumulative score goes to zero or below due to
the accumulation of one or more negative-scoring residue
alignments, or the end of either sequence is reached.
[0118] In addition to calculating percent sequence identity, the
BLAST algorithm also performs a statistical analysis of the
similarity between two sequences. One measure of similarity
provided by the BLAST algorithm is the smallest sum probability
(P(N)), which provides an indication of the probability by which a
match between two nucleotide or amino acid sequences would occur by
chance. For example, a test nucleic acid sequence is considered
similar to a reference sequence if the smallest sum probability in
a comparison of the test nucleic acid sequence to the reference
nucleic acid sequence is less than about 0.1, more preferably less
than about 0.01, and most preferably less than about 0.001.
[0119] To obtain gapped alignments for comparison purposes, Gapped
BLAST (in BLAST 2.0) can be utilized as described in Altschul et
al. (1997). Alternatively, PSI-BLAST (in BLAST 2.0) can be used to
perform an iterated search that detects distant relationships
between molecules. See Altschul et al., supra. When utilizing
BLAST, Gapped BLAST, PSI-BLAST, the default parameters of the
respective programs (e.g. BLASTN for nucleotide sequences, BLASTX
for proteins) can be used. The BLASTN program (for nucleotide
sequences) uses as defaults a wordlength (W) of 11, an expectation
(E) of 10, a cutoff of 100, M=5, N=-4, and a comparison of both
strands. For amino acid sequences, the BLASTP program uses as
defaults a wordlength (W) of 3, an expectation (E) of 10, and the
BLOSUM62 scoring matrix. See http://www.ncbi.nlm.nih.gov. Alignment
may also be performed manually by inspection.
[0120] For purposes of the present invention, comparison of
nucleotide sequences for determination of percent sequence identity
to the promoter sequences disclosed herein is preferably made using
the BlastN program (version 1.4.7 or later) with its default
parameters or any equivalent program. By "equivalent program" is
intended any sequence comparison program that, for any two
sequences in question, generates an alignment having identical
nucleotide or amino acid residue matches and an identical percent
sequence identity when compared to the corresponding alignment
generated by the preferred program.
[0121] (c) As used herein, "sequence identity" or "identity" in the
context of two nucleic acid or polypeptide sequences makes
reference to a specified percentage of residues in the two
sequences that are the same when aligned for maximum correspondence
over a specified comparison window, as measured by sequence
comparison algorithms or by visual inspection. When percentage of
sequence identity is used in reference to proteins it is recognized
that residue positions which are not identical often differ by
conservative amino acid substitutions, where amino acid residues
are substituted for other amino acid residues with similar chemical
properties (e.g., charge or hydrophobicity) and therefore do not
change the functional properties of the molecule. When sequences
differ in conservative substitutions, the percent sequence identity
may be adjusted upwards to correct for the conservative nature of
the substitution. Sequences that differ by such conservative
substitutions are said to have "sequence similarity" or
"similarity." Means for making this adjustment are well known to
those of skill in the art. Typically this involves scoring a
conservative substitution as a partial rather than a full mismatch,
thereby increasing the percentage sequence identity. Thus, for
example, where an identical amino acid is given a score of 1 and a
non-conservative substitution is given a score of zero, a
conservative substitution is given a score between zero and 1. The
scoring of conservative substitutions is calculated, e.g., as
implemented in the program PC/GENE (Intelligenetics, Mountain View,
Calif.).
[0122] (d) As used herein, "percentage of sequence identity" means
the value determined by comparing two optimally aligned sequences
over a comparison window, wherein the portion of the polynucleotide
sequence in the comparison window may comprise additions or
deletions (i.e., gaps) as compared to the reference sequence (which
does not comprise additions or deletions) for optimal alignment of
the two sequences. The percentage is calculated by determining the
number of positions at which the identical nucleic acid base or
amino acid residue occurs in both sequences to yield the number of
matched positions, dividing the number of matched positions by the
total number of positions in the window of comparison, and
multiplying the result by 100 to yield the percentage of sequence
identity.
[0123] (e)(i) The term "substantial identity" of polynucleotide
sequences means that a polynucleotide comprises a sequence that has
at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%,
preferably at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or
89%, more preferably at least 90%, 91%, 92%, 93%, or 94%, and most
preferably at least 95%, 96%, 97%, 98%, or 99% sequence identity,
compared to a reference sequence using one of the alignment
programs described using standard parameters. One of skill in the
art will recognize that these values can be appropriately adjusted
to determine corresponding identity of proteins encoded by two
nucleotide sequences by taking into account codon degeneracy, amino
acid similarity, reading frame positioning, and the like.
Substantial identity of amino acid sequences for these purposes
normally means sequence identity of at least 70%, more preferably
at least 80%, 90%, and most preferably at least 95%.
[0124] Another indication that nucleotide sequences are
substantially identical is if two molecules hybridize to each other
under stringent conditions. Generally, stringent conditions are
selected to be about 5.degree. C. lower than the thermal melting
point (Tm) for the specific sequence at a defined ionic strength
and pH. However, stringent conditions encompass temperatures in the
range of about 1.degree. C. to about 20.degree. C., depending upon
the desired degree of stringency as otherwise qualified herein.
Nucleic acids that do not hybridize to each other under stringent
conditions are still substantially identical if the polypeptides
they encode are substantially identical. This may occur, e.g., when
a copy of a nucleic acid is created using the maximum codon
degeneracy permitted by the genetic code. One indication that two
nucleic acid sequences are substantially identical is when the
polypeptide encoded by the first nucleic acid is immunologically
cross reactive with the polypeptide encoded by the second nucleic
acid.
[0125] (e)(ii) The term "substantial identity" in the context of a
peptide indicates that a peptide comprises a sequence with at least
70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%, preferably
80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more
preferably at least 90%, 91%, 92%, 93%, or 94%, or even more
preferably, 95%, 96%, 97%, 98% or 99%, sequence identity to the
reference sequence over a specified comparison window. Preferably,
optimal alignment is conducted using the homology alignment
algorithm of Needleman and Wunsch (1970). An indication that two
peptide sequences are substantially identical is that one peptide
is immunologically reactive with antibodies raised against the
second peptide. Thus, a peptide is substantially identical to a
second peptide, for example, where the two peptides differ only by
a conservative substitution.
[0126] For sequence comparison, typically one sequence acts as a
reference sequence to which test sequences are compared. When using
a sequence comparison algorithm, test and reference sequences are
input into a computer, subsequence coordinates are designated if
necessary, and sequence algorithm program parameters are
designated. The sequence comparison algorithm then calculates the
percent sequence identity for the test sequence(s) relative to the
reference sequence, based on the designated program parameters.
[0127] As noted above, another indication that two nucleic acid
sequences are substantially identical is that the two molecules
hybridize to each other under stringent conditions. The phrase
"hybridizing specifically to" refers to the binding, duplexing, or
hybridizing of a molecule only to a particular nucleotide sequence
under stringent conditions when that sequence is present in a
complex mixture (e.g., total cellular) DNA or RNA. "Bind(s)
substantially" refers to complementary hybridization between a
probe nucleic acid and a target nucleic acid and embraces minor
mismatches that can be accommodated by reducing the stringency of
the hybridization media to achieve the desired detection of the
target nucleic acid sequence.
[0128] "Stringent hybridization conditions" and "stringent
hybridization wash conditions" in the context of nucleic acid
hybridization experiments such as Southern and Northern
hybridizations are sequence dependent, and are different under
different environmental parameters. Longer sequences hybridize
specifically at higher temperatures. The Tm is the temperature
(under defined ionic strength and pH) at which 50% of the target
sequence hybridizes to a perfectly matched probe. Specificity is
typically the function of post-hybridization washes, the critical
factors being the ionic strength and temperature of the final wash
solution. For DNA-DNA hybrids, the Tm can be approximated from the
equation of Meinkoth and Wahl (1984); T.sub.m 81.5.degree. C.+16.6
(log M)+0.41 (% GC)-0.61 (% form)-500/L; where M is the molarity of
monovalent cations, % GC is the percentage of guanosine and
cytosine nucleotides in the DNA, % form is the percentage of
formamide in the hybridization solution, and L is the length of the
hybrid in base pairs. Tm is reduced by about 1.degree. C. for each
1% of mismatching; thus, T.sub.m, hybridization, and/or wash
conditions can be adjusted to hybridize to sequences of the desired
identity. For example, if sequences with >90% identity are
sought, the T.sub.m can be decreased 10.degree. C. Generally,
stringent conditions are selected to be about 5.degree. C. lower
than the thermal melting point (T.sub.m) for the specific sequence
and its complement at a defined ionic strength and pH. However,
severely stringent conditions can utilize a hybridization and/or
wash at 1, 2, 3, or 4.degree. C. lower than the thermal melting
point (T.sub.m); moderately stringent conditions can utilize a
hybridization and/or wash at 6, 7, 8, 9, or 10.degree. C. lower
than the thermal melting point (T.sub.m); low stringency conditions
can utilize a hybridization and/or wash at 11, 12, 13, 14, 15, or
20.degree. C. lower than the thermal melting point (T.sub.m). Using
the equation, hybridization and wash compositions, and desired T,
those of ordinary skill will understand that variations in the
stringency of hybridization and/or wash solutions are inherently
described. If the desired degree of mismatching results in a T of
less than 45.degree. C. (aqueous solution) or 32.degree. C.
(formamide solution), it is preferred to increase the SSC
concentration so that a higher temperature can be used. An
extensive guide to the hybridization of nucleic acids is found in
Tijssen (1993). Generally, highly stringent hybridization and wash
conditions are selected to be about 5.degree. C. lower than the
thermal melting point (Tm) for the specific sequence at a defined
ionic strength and pH.
[0129] An example of highly stringent wash conditions is 0.15 M
NaCl at 72.degree. C. for about 15 minutes. An example of stringent
wash conditions is a 0.2.times.SSC wash at 65.degree. C. for 15
minutes (see, Sambrook and Russell, infra, for a description of SSC
buffer). Often, a high stringency wash is preceded by a low
stringency wash to remove background probe signal. An example
medium stringency wash for a duplex of, e.g., more than 100
nucleotides, is 1.times.SSC at 45.degree. C. for 15 minutes. An
example low stringency wash for a duplex of, e.g., more than 100
nucleotides, is 4-6.times.SSC at 40.degree. C. for 15 minutes. For
short probes (e.g., about 10 to 50 nucleotides), stringent
conditions typically involve salt concentrations of less than about
1.5 M, more preferably about 0.01 to 1.0 M, Na ion concentration
(or other salts) at pH 7.0 to 8.3, and the temperature is typically
at least about 30.degree. C. and at least about 60.degree. C. for
long probes (e.g., >50 nucleotides). Stringent conditions may
also be achieved with the addition of destabilizing agents such as
formamide. In general, a signal to noise ratio of 2.times.(or
higher) than that observed for an unrelated probe in the particular
hybridization assay indicates detection of a specific
hybridization. Nucleic acids that do not hybridize to each other
under stringent conditions are still substantially identical if the
proteins that they encode are substantially identical. This occurs,
e.g., when a copy of a nucleic acid is created using the maximum
codon degeneracy permitted by the genetic code.
[0130] Very stringent conditions are selected to be equal to the Tm
for a particular probe. An example of stringent conditions for
hybridization of complementary nucleic acids which have more than
100 complementary residues on a filter in a Southern or Northern
blot is 50% formamide, e.g., hybridization in 50% formamide, 1 M
NaCl, 1% SDS at 37.degree. C., and a wash in 0.1.times.SSC at 60 to
65.degree. C. Exemplary low stringency conditions include
hybridization with a buffer solution of 30 to 35% formamide, 1M
NaCl, 1% SDS (sodium dodecyl sulfate) at 37.degree. C., and a wash
in 1.times. to 2.times.SSC (20.times.SSC=3.0 M NaCl/0.3 M trisodium
citrate) at 50 to 55.degree. C. Exemplary moderate stringency
conditions include hybridization in 40 to 45% formamide, 1.0 M
NaCl, 1% SDS at 37.degree. C., and a wash in 0.5.times. to
1.times.SSC at 55 to 60.degree. C.
[0131] By "variant" polypeptide is intended a polypeptide derived
from the native protein by deletion (also called "truncation") or
addition of one or more amino acids to the N-terminal and/or
C-terminal end of the native protein; deletion or addition of one
or more amino acids at one or more sites in the native protein; or
substitution of one or more amino acids at one or more sites in the
native protein. Such variants may results from, for example,
genetic polymorphism or from human manipulation. Methods for such
manipulations are generally known in the art.
[0132] Thus, the polypeptides of the invention may be altered in
various ways including amino acid substitutions, deletions,
truncations, and insertions. Methods for such manipulations are
generally known in the art. For example, amino acid sequence
variants of the polypeptides can be prepared by mutations in the
DNA. Methods for mutagenesis and nucleotide sequence alterations
are well known in the art. See, for example, Kunkel (1985); Kunkel
et al. (1987); U.S. Pat. No. 4,873,192; Walker and Gaastra (1983),
and the references cited therein. Guidance as to appropriate amino
acid substitutions that do not affect biological activity of the
protein of interest may be found in the model of Dayhoff et al.
(1978). Conservative substitutions, such as exchanging one amino
acid with another having similar properties, are preferred.
[0133] Thus, the genes and nucleotide sequences of the invention
include both the naturally occurring sequences as well as variant
forms. Likewise, the polypeptides of the invention encompass both
naturally occurring proteins as well as variations and modified
forms thereof. Such variants will continue to possess the desired
activity. The deletions, insertions, and substitutions of the
polypeptide sequence encompassed herein are not expected to produce
radical changes in the characteristics of the polypeptide. However,
when it is difficult to predict the exact effect of the
substitution, deletion, or insertion in advance of doing so, one
skilled in the art will appreciate that the effect will be
evaluated by routine screening assays.
[0134] Individual substitutions deletions or additions that alter,
add or delete a single amino acid or a small percentage of amino
acids (typically less than 5%, more typically less than 1%) in an
encoded sequence are "conservatively modified variations," where
the alterations result in the substitution of an amino acid with a
chemically similar amino acid. Conservative substitution tables
providing functionally similar amino acids are well known in the
art. The following five groups each contain amino acids that are
conservative substitutions for one another: Aliphatic: Glycine (G),
Alanine (A), Valine (V), Leucine (L), Isoleucine (I); Aromatic:
Phenylalanine (F), Tyrosine (Y), Tryptophan (W); Sulfur-containing:
Methionine (M), Cysteine (C); Basic: Arginine (R), Lysine (K),
Histidine (H); Acidic: Aspartic acid (D), Glutamic acid (E),
Asparagine (N), Glutamine (Q). In addition, individual
substitutions, deletions or additions which alter, add or delete a
single amino acid or a small percentage of amino acids in an
encoded sequence are also "conservatively modified variations."
[0135] The term "transformation" refers to the transfer of a
nucleic acid fragment into the genome of a host cell, resulting in
genetically stable inheritance. A "host cell" is a cell that has
been transformed, or is capable of transformation, by an exogenous
nucleic acid molecule. Host cells containing the transformed
nucleic acid fragments are referred to as "transgenic" cells, and
organisms comprising transgenic cells are referred to as
"transgenic organisms".
[0136] "Transformed", "transduced", "transgenic", and "recombinant"
refer to a host cell or organism into which a heterologous nucleic
acid molecule has been introduced. The nucleic acid molecule can be
stably integrated into the genome generally known in the art and
are disclosed in Sambrook and Russell, infra. See also Innis et al.
(1995); and Gelfand (1995); and Innis and Gelfand (1999). Known
methods of PCR include, but are not limited to, methods using
paired primers, nested primers, single specific primers, degenerate
primers, gene-specific primers, vector-specific primers, partially
mismatched primers, and the like. For example, "transformed,"
"transformant," and "transgenic" cells have been through the
transformation process and contain a foreign gene integrated into
their chromosome. The term "untransformed" refers to normal cells
that have not been through the transformation process.
[0137] A "transgenic" organism is an organism having one or more
cells that contain an expression vector.
[0138] "Genetically altered cells" denotes cells which have been
modified by the introduction of recombinant or heterologous nucleic
acids (e.g., one or more DNA constructs or their RNA counterparts)
and further includes the progeny of such cells which retain part or
all of such genetic modification.
[0139] The term "fusion protein" is intended to describe at least
two polypeptides, typically from different sources, which are
operably linked. With regard to polypeptides, the term operably
linked is intended to mean that the two polypeptides are connected
in a manner such that each polypeptide can serve its intended
function. Typically, the two polypeptides are covalently attached
through peptide bonds. The fusion protein is preferably produced by
standard recombinant DNA techniques. For example, a DNA molecule
encoding the first polypeptide is ligated to another DNA molecule
encoding the second polypeptide, and the resultant hybrid DNA
molecule is expressed in a host cell to produce the fusion protein.
The DNA molecules are ligated to each other in a 5' to 3'
orientation such that, after ligation, the translational frame of
the encoded polypeptides is not altered (i.e., the DNA molecules
are ligated to each other in-frame).
[0140] As used herein, the term "derived" or "directed to" with
respect to a nucleotide molecule means that the molecule has
complementary sequence identity to a particular molecule of
interest.
[0141] "Gene silencing" refers to the suppression of gene
expression, e.g., transgene, heterologous gene and/or endogenous
gene expression. Gene silencing may be mediated through processes
that affect transcription and/or through processes that affect
post-transcriptional mechanisms. In some embodiments, gene
silencing occurs when siRNA initiates the degradation of the mRNA
of a gene of interest in a sequence-specific manner via RNA
interference (for a review, see Brantl, 2002). In some embodiments,
gene silencing may be allele-specific. "Allele-specific" gene
silencing refers to the specific silencing of one allele of a
gene.
[0142] "Knock-down," "knock-down technology" refers to a technique
of gene silencing in which the expression of a target gene is
reduced as compared to the gene expression prior to the
introduction of the siRNA, which can lead to the inhibition of
production of the target gene product. The term "reduced" is used
herein to indicate that the target gene expression is lowered by
1-100%. For example, the expression may be reduced by 10, 20, 30,
40, 50, 60, 70, 80, 90, 95, or even 99%. Knock-down of gene
expression can be directed by the use of dsRNAs or siRNAs. For
example, "RNA interference (RNAi)," which can involve the use of
siRNA, has been successfully applied to knockdown the expression of
specific genes in plants, D. melanogaster, C. elegans,
trypanosomes, planaria, hydra, and several vertebrate species
including the mouse. For a review of the mechanisms proposed to
mediate RNAi, please refer to Bass et al., 2001, Elbashir et al.,
2001 or Brantl 2002.
[0143] "RNA interference (RNAi)" is the process of
sequence-specific, post-transcriptional gene silencing initiated by
siRNA. RNAi is seen in a number of organisms such as Drosophila,
nematodes, fungi and plants, and is believed to be involved in
anti-viral defense, modulation of transposon activity, and
regulation of gene expression. During RNAi, siRNA induces
degradation of target mRNA with consequent sequence-specific
inhibition of gene expression.
[0144] A "small interfering" or "short interfering RNA" or siRNA is
a RNA duplex of nucleotides that is targeted to a gene interest. A
"RNA duplex" refers to the structure formed by the complementary
pairing between two regions of a RNA molecule. siRNA is "targeted"
to a gene in that the nucleotide sequence of the duplex portion of
the siRNA is complementary to a nucleotide sequence of the targeted
gene. In some embodiments, the length of the duplex of siRNAs is
less than 30 nucleotides. In some embodiments, the duplex can be
29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13,
12, 11 or 10 nucleotides in length. In some embodiments, the length
of the duplex is 19-25 nucleotides in length. The RNA duplex
portion of the siRNA can be part of a hairpin structure. In
addition to the duplex portion, the hairpin structure may contain a
loop portion positioned between the two sequences that form the
duplex. The loop can vary in length. In some embodiments the loop
is 5, 6, 7, 8, 9, 10, 11, 12 or 13 nucleotides in length. The
hairpin structure can also contain 3' or 5' overhang portions. In
some embodiments, the overhang is a 3' or a 5' overhang 0, 1, 2, 3,
4 or 5 nucleotides in length.
[0145] The siRNA can be encoded by a nucleic acid sequence, and the
nucleic acid sequence can also include a promoter. The nucleic acid
sequence can also include a polyadenylation signal. In some
embodiments, the polyadenylation signal is a synthetic minimal
polyadenylation signal.
[0146] "Treating" as used herein refers to ameliorating at least
one symptom of, curing and/or preventing the development of a
disease or a condition.
[0147] "Neurological disease" and "neurological disorder" refer to
both hereditary and sporadic conditions that are characterized by
nervous system dysfunction, and which may be associated with
atrophy of the affected central or peripheral nervous system
structures, or loss of function without atrophy. A neurological
disease or disorder that results in atrophy is commonly called a
"neurodegenerative disease" or "neurodegenerative disorder."
Neurodegenerative diseases and disorders include, but are not
limited to, amyotrophic lateral sclerosis (ALS), hereditary spastic
hemiplegia, primary lateral sclerosis, spinal muscular atrophy,
Kennedy's disease, Alzheimer's disease, Parkinson's disease,
multiple sclerosis, and repeat expansion neurodegenerative
diseases, e.g., diseases associated with expansions of
trinucleotide repeats such as polyglutamine (polyQ) repeat
diseases, e.g., Huntington's disease (HD), spinocerebellar ataxia
(SCA1, SCA2, SCA3, SCA6, SCA7, and SCA17), spinal and bulbar
muscular atrophy (SBMA), dentatorubropallidoluysian atrophy
(DRPLA). An example of a neurological disorder that does not appear
to result in atrophy is DYT1 dystonia.
[0148] II. Nucleic Acid Molecules of the Invention
[0149] Sources of nucleotide sequences from which the present
nucleic acid molecules can be obtained include any vertebrate,
preferably mammalian, cellular source.
[0150] As discussed above, the terms "isolated and/or purified"
refer to in vitro isolation of a nucleic acid, e.g., a DNA or RNA
molecule from its natural cellular environment, and from
association with other components of the cell, such as nucleic acid
or polypeptide, so that it can be sequenced, replicated, and/or
expressed. For example, "isolated nucleic acid" may be a DNA
molecule containing less than 31 sequential nucleotides that is
transcribed into an siRNA. Such an isolated siRNA may, for example,
form a hairpin structure with a duplex 21 base pairs in length that
is complementary or hybridizes to a sequence in a gene of interest,
and remains stably bound under stringent conditions (as defined by
methods well known in the art, e.g., in Sambrook and Russell,
2001). Thus, the RNA or DNA is "isolated" in that it is free from
at least one contaminating nucleic acid with which it is normally
associated in the natural source of the RNA or DNA and is
preferably substantially free of any other mammalian RNA or DNA.
The phrase "free from at least one contaminating source nucleic
acid with which it is normally associated" includes the case where
the nucleic acid is reintroduced into the source or natural cell
but is in a different chromosomal location or is otherwise flanked
by nucleic acid sequences not normally found in the source cell,
e.g., in a vector or plasmid.
[0151] In addition to a DNA sequence encoding a siRNA, the nucleic
acid molecules of the invention include double-stranded interfering
RNA molecules, which are also useful to inhibit expression of a
target gene.
[0152] As used herein, the term "recombinant nucleic acid", e.g.,
"recombinant DNA sequence or segment" refers to a nucleic acid,
e.g., to DNA, that has been derived or isolated from any
appropriate cellular source, that may be subsequently chemically
altered in vitro, so that its sequence is not naturally occurring,
or corresponds to naturally occurring sequences that are not
positioned as they would be positioned in a genome which has not
been transformed with exogenous DNA. An example of preselected DNA
"derived" from a source, would be a DNA sequence that is identified
as a useful fragment within a given organism, and which is then
chemically synthesized in essentially pure form. An example of such
DNA "isolated" from a source would be a useful DNA sequence that is
excised or removed from said source by chemical means, e.g., by the
use of restriction endonucleases, so that it can be further
manipulated, e.g., amplified, for use in the invention, by the
methodology of genetic engineering.
[0153] Thus, recovery or isolation of a given fragment of DNA from
a restriction digest can employ separation of the digest on
polyacrylamide or agarose gel by electrophoresis, identification of
the fragment of interest by comparison of its mobility versus that
of marker DNA fragments of known molecular weight, removal of the
gel section containing the desired fragment, and separation of the
gel from DNA. See Lawn et al. (1981), and Goeddel et al. (1980).
Therefore, "recombinant DNA" includes completely synthetic DNA
sequences, semi-synthetic DNA sequences, DNA sequences isolated
from biological sources, and DNA sequences derived from RNA, as
well as mixtures thereof.
[0154] Nucleic acid molecules having base substitutions (i.e.,
variants) are prepared by a variety of methods known in the art.
These methods include, but are not limited to, isolation from a
natural source (in the case of naturally occurring sequence
variants) or preparation by oligonucleotide-mediated (or
site-directed) mutagenesis, PCR mutagenesis, and cassette
mutagenesis of an earlier prepared variant or a non-variant version
of the nucleic acid molecule.
[0155] Oligonucleotide-mediated mutagenesis is a method for
preparing substitution variants. This technique is known in the art
as described by Adelman et al. (1983). Briefly, nucleic acid
encoding a siRNA can be altered by hybridizing an oligonucleotide
encoding the desired mutation to a DNA template, where the template
is the single-stranded form of a plasmid or bacteriophage
containing the unaltered or native gene sequence. After
hybridization, a DNA polymerase is used to synthesize an entire
second complementary strand of the template that will thus
incorporate the oligonucleotide primer, and will code for the
selected alteration in the nucleic acid encoding siRNA. Generally,
oligonucleotides of at least 25 nucleotides in length are used. An
optimal oligonucleotide will have 12 to 15 nucleotides that are
completely complementary to the template on either side of the
nucleotide(s) coding for the mutation. This ensures that the
oligonucleotide will hybridize properly to the single-stranded DNA
template molecule. The oligonucleotides are readily synthesized
using techniques known in the art such as that described by Crea et
al. (1978).
[0156] The DNA template can be generated by those vectors that are
either derived from bacteriophage M13 vectors (the commercially
available M13 mpl8 and M13 mp 19 vectors are suitable), or those
vectors that contain a single-stranded phage origin of replication
as described by Viera et al. (1987). Thus, the DNA that is to be
mutated may be inserted into one of these vectors to generate
single-stranded template. Production of the single-stranded
template is described in Chapter 3 of Sambrook and Russell, 2001.
Alternatively, single-stranded DNA template may be generated by
denaturing double-stranded plasmid (or other) DNA using standard
techniques.
[0157] For alteration of the native DNA sequence (to generate amino
acid sequence variants, for example), the oligonucleotide is
hybridized to the single-stranded template under suitable
hybridization conditions. A DNA polymerizing enzyme, usually the
Klenow fragment of DNA polymerase I, is then added to synthesize
the complementary strand of the template using the oligonucleotide
as a primer for synthesis. A heteroduplex molecule is thus formed
such that one strand of DNA encodes the mutated form of the DNA,
and the other strand (the original template) encodes the native,
unaltered sequence of the DNA. This heteroduplex molecule is then
transformed into a suitable host cell, usually a prokaryote such as
E. coli JM101. After the cells are grown, they are plated onto
agarose plates and screened using the oligonucleotide primer
radiolabeled with 32-phosphate to identify the bacterial colonies
that contain the mutated DNA. The mutated region is then removed
and placed in an appropriate vector, generally an expression vector
of the type typically employed for transformation of an appropriate
host.
[0158] The method described immediately above may be modified such
that a homoduplex molecule is created wherein both strands of the
plasmid contain the mutations(s). The modifications are as follows:
The single-stranded oligonucleotide is annealed to the
single-stranded template as described above. A mixture of three
deoxyribonucleotides, deoxyriboadenosine (dATP), deoxyriboguanosine
(dGTP), and deoxyribothymidine (dTTP), is combined with a modified
thiodeoxyribocytosine called dCTP-(*S) (which can be obtained from
the Amersham Corporation). This mixture is added to the
template-oligonucleotide complex. Upon addition of DNA polymerase
to this mixture, a strand of DNA identical to the template except
for the mutated bases is generated. In addition, this new strand of
DNA will contain dCTP-(*S) instead of dCTP, which serves to protect
it from restriction endonuclease digestion.
[0159] After the template strand of the double-stranded
heteroduplex is nicked with an appropriate restriction enzyme, the
template strand can be digested with ExoIII nuclease or another
appropriate nuclease past the region that contains the site(s) to
be mutagenized. The reaction is then stopped to leave a molecule
that is only partially single-stranded. A complete double-stranded
DNA homoduplex is then formed using DNA polymerase in the presence
of all four deoxyribonucleotide triphosphates, ATP, and DNA ligase.
This homoduplex molecule can then be transformed into a suitable
host cell such as E. coli JM101.
[0160] III. Expression Cassettes of the Invention
[0161] To prepare expression cassettes, the recombinant DNA
sequence or segment may be circular or linear, double-stranded or
single-stranded. Generally, the DNA sequence or segment is in the
form of chimeric DNA, such as plasmid DNA or a vector that can also
contain coding regions flanked by control sequences that promote
the expression of the recombinant DNA present in the resultant
transformed cell.
[0162] A "chimeric" vector or expression cassette, as used herein,
means a vector or cassette including nucleic acid sequences from at
least two different species, or has a nucleic acid sequence from
the same species that is linked or associated in a manner that does
not occur in the "native" or wild type of the species.
[0163] Aside from recombinant DNA sequences that serve as
transcription units for an RNA transcript, or portions thereof, a
portion of the recombinant DNA may be untranscribed, serving a
regulatory or a structural function. For example, the recombinant
DNA may have a promoter that is active in mammalian cells.
[0164] Other elements functional in the host cells, such as
introns, enhancers, polyadenylation sequences and the like, may
also be a part of the recombinant DNA. Such elements may or may not
be necessary for the function of the DNA, but may provide improved
expression of the DNA by affecting transcription, stability of the
siRNA, or the like. Such elements may be included in the DNA as
desired to obtain the optimal performance of the siRNA in the
cell.
[0165] Control sequences are DNA sequences necessary for the
expression of an operably linked coding sequence in a particular
host organism. The control sequences that are suitable for
prokaryotic cells, for example, include a promoter, and optionally
an operator sequence, and a ribosome binding site. Eukaryotic cells
are known to utilize promoters, polyadenylation signals, and
enhancers.
[0166] Operably linked nucleic acids are nucleic acids placed in a
functional relationship with another nucleic acid sequence. For
example, a promoter or enhancer is operably linked to a coding
sequence if it affects the transcription of the sequence; or a
ribosome binding site is operably linked to a coding sequence if it
is positioned so as to facilitate translation. Generally, operably
linked DNA sequences are DNA sequences that are linked are
contiguous. However, enhancers do not have to be contiguous.
Linking is accomplished by ligation at convenient restriction
sites. If such sites do not exist, the synthetic oligonucleotide
adaptors or linkers are used in accord with conventional
practice.
[0167] The recombinant DNA to be introduced into the cells may
contain either a selectable marker gene or a reporter gene or both
to facilitate identification and selection of expressing cells from
the population of cells sought to be transfected or infected
through viral vectors. In other embodiments, the selectable marker
may be carried on a separate piece of DNA and used in a
co-transfection procedure. Both selectable markers and reporter
genes may be flanked with appropriate regulatory sequences to
enable expression in the host cells. Useful selectable markers are
known in the art and include, for example, antibiotic-resistance
genes, such as neo and the like.
[0168] Reporter genes are used for identifying potentially
transfected cells and for evaluating the functionality of
regulatory sequences. Reporter genes that encode for easily
assayable proteins are well known in the art. In general, a
reporter gene is a gene that is not present in or expressed by the
recipient organism or tissue and that encodes a protein whose
expression is manifested by some easily detectable property, e.g.,
enzymatic activity. For example, reporter genes include the
chloramphenicol acetyl transferase gene (cat) from Tn9 of E. coli
and the luciferase gene from firefly Photinus pyralis. Expression
of the reporter gene is assayed at a suitable time after the DNA
has been introduced into the recipient cells.
[0169] The general methods for constructing recombinant DNA that
can transfect target cells are well known to those skilled in the
art, and the same compositions and methods of construction may be
utilized to produce the DNA useful herein. For example, Sambrook
and Russell, infra, provides suitable methods of construction.
[0170] The recombinant DNA can be readily introduced into the host
cells, e.g., mammalian, bacterial, yeast or insect cells by
transfection with an expression vector composed of DNA encoding the
siRNA by any procedure useful for the introduction into a
particular cell, e.g., physical or biological methods, to yield a
cell having the recombinant DNA stably integrated into its genome
or existing as a episomal element, so that the DNA molecules, or
sequences of the present invention are expressed by the host cell.
Preferably, the DNA is introduced into host cells via a vector. The
host cell is preferably of eukaryotic origin, e.g., plant,
mammalian, insect, yeast or fungal sources, but host cells of
non-eukaryotic origin may also be employed.
[0171] Physical methods to introduce a preselected DNA into a host
cell include calcium phosphate precipitation, lipofection, particle
bombardment, microinjection, electroporation, and the like.
Biological methods to introduce the DNA of interest into a host
cell include the use of DNA and RNA viral vectors. For mammalian
gene therapy, as described hereinbelow, it is desirable to use an
efficient means of inserting a copy gene into the host genome.
Viral vectors, and especially retroviral vectors, have become the
most widely used method for inserting genes into mammalian, e.g.,
human cells. Other viral vectors can be derived from poxviruses,
herpes simplex virus I, adenoviruses and adeno-associated viruses,
and the like. See, for example, U.S. Pat. Nos. 5,350,674 and
5,585,362.
[0172] As discussed above, a "transfected", "or "transduced" host
cell or cell line is one in which the genome has been altered or
augmented by the presence of at least one heterologous or
recombinant nucleic acid sequence. The host cells of the present
invention are typically produced by transfection with a DNA
sequence in a plasmid expression vector, a viral expression vector,
or as an isolated linear DNA sequence. The transfected DNA can
become a chromosomally integrated recombinant DNA sequence, which
is composed of sequence encoding the siRNA.
[0173] To confirm the presence of the recombinant DNA sequence in
the host cell, a variety of assays may be performed. Such assays
include, for example, "molecular biological" assays well known to
those of skill in the art, such as Southern and Northern blotting,
RT-PCR and PCR; "biochemical" assays, such as detecting the
presence or absence of a particular peptide, e.g., by immunological
means (ELISAs and Western blots) or by assays described herein to
identify agents falling within the scope of the invention.
[0174] To detect and quantitate RNA produced from introduced
recombinant DNA segments, RT-PCR may be employed. In this
application of PCR, it is first necessary to reverse transcribe RNA
into DNA, using enzymes such as reverse transcriptase, and then
through the use of conventional PCR techniques amplify the DNA. In
most instances PCR techniques, while useful, will not demonstrate
integrity of the RNA product. Further information about the nature
of the RNA product may be obtained by Northern blotting. This
technique demonstrates the presence of an RNA species and gives
information about the integrity of that RNA. The presence or
absence of an RNA species can also be determined using dot or slot
blot Northern hybridizations. These techniques are modifications of
Northern blotting and only demonstrate the presence or absence of
an RNA species.
[0175] While Southern blotting and PCR may be used to detect the
recombinant DNA segment in question, they do not provide
information as to whether the preselected DNA segment is being
expressed. Expression may be evaluated by specifically identifying
the peptide products of the introduced recombinant DNA sequences or
evaluating the phenotypic changes brought about by the expression
of the introduced recombinant DNA segment in the host cell.
[0176] The instant invention provides a cell expression system for
expressing exogenous nucleic acid material in a mammalian
recipient. The expression system, also referred to as a
"genetically modified cell", comprises a cell and an expression
vector for expressing the exogenous nucleic acid material. The
genetically modified cells are suitable for administration to a
mammalian recipient, where they replace the endogenous cells of the
recipient. Thus, the preferred genetically modified cells are
non-immortalized and are non-tumorigenic.
[0177] According to one embodiment, the cells are transfected or
otherwise genetically modified ex vivo. The cells are isolated from
a mammal (preferably a human), nucleic acid introduced (i.e.,
transduced or transfected in vitro) with a vector for expressing a
heterologous (e.g., recombinant) gene encoding the therapeutic
agent, and then administered to a mammalian recipient for delivery
of the therapeutic agent in situ. The mammalian recipient may be a
human and the cells to be modified are autologous cells, i.e., the
cells are isolated from the mammalian recipient.
[0178] According to another embodiment, the cells are transfected
or transduced or otherwise genetically modified in vivo. The cells
from the mammalian recipient are transduced or transfected in vivo
with a vector containing exogenous nucleic acid material for
expressing a heterologous (e.g., recombinant) gene encoding a
therapeutic agent and the therapeutic agent is delivered in
situ.
[0179] As used herein, "exogenous nucleic acid material" refers to
a nucleic acid or an oligonucleotide, either natural or synthetic,
which is not naturally found in the cells; or if it is naturally
found in the cells, is modified from its original or native form.
Thus, "exogenous nucleic acid material" includes, for example, a
non-naturally occurring nucleic acid that can be transcribed into
an anti-sense RNA, a siRNA, as well as a "heterologous gene" (i.e.,
a gene encoding a protein that is not expressed or is expressed at
biologically insignificant levels in a naturally-occurring cell of
the same type). To illustrate, a synthetic or natural gene encoding
human erythropoietin (EPO) would be considered "exogenous nucleic
acid material" with respect to human peritoneal mesothelial cells
since the latter cells do not naturally express EPO. Still another
example of "exogenous nucleic acid material" is the introduction of
only part of a gene to create a recombinant gene, such as combining
an regulatable promoter with an endogenous coding sequence via
homologous recombination.
[0180] IV. Promoters of the Invention
[0181] As described herein, an expression cassette of the invention
contains, inter alia, a promoter. Such promoters include the CMV
promoter, as well as the RSV promoter, SV40 late promoter and
retroviral LTRs (long terminal repeat elements), or brain cell
specific promoters, although many other promoter elements well
known to the art, such as tissue specific promoters or regulatable
promoters may be employed in the practice of the invention.
[0182] In one embodiment of the present invention, an expression
cassette may contain a pol II promoter that is operably linked to a
nucleic acid sequence encoding a siRNA. Thus, the pol II promoter,
i.e., a RNA polymerase II dependent promoter, initiates the
transcription of the siRNA. In another embodiment, the pol II
promoter is regulatable.
[0183] Three RNA polymerases transcribe nuclear genes in
eukaryotes. RNA polymerase II (pol II) synthesizes mRNA, i.e., pol
II transcribes the genes that encode proteins. In contrast, RNA
polymerase I (pol 1) and RNA polymerase III (pol III) transcribe
only a limited set of transcripts, synthesizing RNAs that have
structural or catalytic roles. RNA polymerase I makes the large
ribosomal RNAs (rRNA), which are under the control of pol I
promoters. RNA polymerase III makes a variety of small, stable
RNAs, including the small 5S rRNA and transfer RNAs (tRNA), the
transcription of which is under the control of pol III
promoters.
[0184] As described herein, the inventors unexpectedly discovered
that pol II promoters are useful to direct transcription of the
siRNA. This was surprising because, as discussed above, pol II
promoters are thought to be responsible for transcription of
messenger RNA, i.e., relatively long RNAs as compared to RNAs of 30
bases or less.
[0185] A pol II promoter may be used in its entirety, or a portion
or fragment of the promoter sequence may be used in which the
portion maintains the promoter activity. As discussed herein, pol
II promoters are known to a skilled person in the art and include
the promoter of any protein-encoding gene, e.g., an endogenously
regulated gene or a constitutively expressed gene. For example, the
promoters of genes regulated by cellular physiological events,
e.g., heat shock, oxygen levels and/or carbon monoxide levels,
e.g., in hypoxia, may be used in the expression cassettes of the
invention. In addition, the promoter of any gene regulated by the
presence of a pharmacological agent, e.g., tetracycline and
derivatives thereof, as well as heavy metal ions and hormones may
be employed in the expression cassettes of the invention. In an
embodiment of the invention, the pol II promoter can be the CMV
promoter or the RSV promoter. In another embodiment, the pol II
promoter is the CMV promoter.
[0186] As discussed above, a pol II promoter of the invention may
be one naturally associated with an endogenously regulated gene or
sequence, as may be obtained by isolating the 5' non-coding
sequences located upstream of the coding segment and/or exon. The
pol II promoter of the expression cassette can be, for example, the
same pol II promoter driving expression of the targeted gene of
interest. Alternatively, the nucleic acid sequence encoding the
siRNA may be placed under the control of a recombinant or
heterologous pol II promoter, which refers to a promoter that is
not normally associated with the targeted gene's natural
environment. Such promoters include promoters isolated from any
eukaryotic cell, and promoters not "naturally occurring," i.e.,
containing different elements of different transcriptional
regulatory regions, and/or mutations that alter expression. In
addition to producing nucleic acid sequences of promoters
synthetically, sequences may be produced using recombinant cloning
and/or nucleic acid amplification technology, including PCR.TM., in
connection with the compositions disclosed herein (see U.S. Pat.
No. 4,683,202, U.S. Pat. No. 5,928,906, each incorporated herein by
reference).
[0187] In one embodiment, a pol II promoter that effectively
directs the expression of the siRNA in the cell type, organelle,
and organism chosen for expression will be employed. Those of
ordinary skill in the art of molecular biology generally know the
use of promoters for protein expression, for example, see Sambrook
and Russell (2001), incorporated herein by reference. The promoters
employed may be constitutive, tissue-specific, inducible, and/or
useful under the appropriate conditions to direct high level
expression of the introduced DNA segment, such as is advantageous
in the large-scale production of recombinant proteins and/or
peptides. The identity of tissue-specific promoters, as well as
assays to characterize their activity, is well known to those of
ordinary skill in the art.
[0188] V. Methods for Introducing the Expression Cassettes of the
Invention into Cells
[0189] The condition amenable to gene inhibition therapy may be a
prophylactic process, i.e., a process for preventing disease or an
undesired medical condition. Thus, the instant invention embraces a
system for delivering siRNA that has a prophylactic function (i.e.,
a prophylactic agent) to the mammalian recipient.
[0190] The inhibitory nucleic acid material (e.g., an expression
cassette encoding siRNA directed to a gene of interest) can be
introduced into the cell ex vivo or in vivo by genetic transfer
methods, such as transfection or transduction, to provide a
genetically modified cell. Various expression vectors (i.e.,
vehicles for facilitating delivery of exogenous nucleic acid into a
target cell) are known to one of ordinary skill in the art.
[0191] As used herein, "transfection of cells" refers to the
acquisition by a cell of new nucleic acid material by incorporation
of added DNA. Thus, transfection refers to the insertion of nucleic
acid into a cell using physical or chemical methods. Several
transfection techniques are known to those of ordinary skill in the
art including: calcium phosphate DNA co-precipitation (Methods in
Molecular Biology (1991)); DEAE-dextran (supra); electroporation
(supra); cationic liposome-mediated transfection (supra); and
tungsten particle-facilitated microparticle bombardment (Johnston
(1990)). Strontium phosphate DNA co-precipitation (Brash et al.
(1987)) is also a transfection method.
[0192] In contrast, "transduction of cells" refers to the process
of transferring nucleic acid into a cell using a DNA or RNA virus.
A RNA virus (i.e., a retrovirus) for transferring a nucleic acid
into a cell is referred to herein as a transducing chimeric
retrovirus. Exogenous nucleic acid material contained within the
retrovirus is incorporated into the genome of the transduced cell.
A cell that has been transduced with a chimeric DNA virus (e.g., an
adenovirus carrying a cDNA encoding a therapeutic agent), will not
have the exogenous nucleic acid material incorporated into its
genome but will be capable of expressing the exogenous nucleic acid
material that is retained extrachromosomally within the cell.
[0193] The exogenous nucleic acid material can include the nucleic
acid encoding the siRNA together with a promoter to control
transcription. The promoter characteristically has a specific
nucleotide sequence necessary to initiate transcription. The
exogenous nucleic acid material may further include additional
sequences (i.e., enhancers) required to obtain the desired gene
transcription activity. For the purpose of this discussion an
"enhancer" is simply any non-translated DNA sequence that works
with the coding sequence (in cis) to change the basal transcription
level dictated by the promoter. The exogenous nucleic acid material
may be introduced into the cell genome immediately downstream from
the promoter so that the promoter and coding sequence are
operatively linked so as to permit transcription of the coding
sequence. An expression vector can include an exogenous promoter
element to control transcription of the inserted exogenous gene.
Such exogenous promoters include both constitutive and regulatable
promoters.
[0194] Naturally-occurring constitutive promoters control the
expression of essential cell functions. As a result, a nucleic acid
sequence under the control of a constitutive promoter is expressed
under all conditions of cell growth. Constitutive promoters include
the promoters for the following genes which encode certain
constitutive or "housekeeping" functions: hypoxanthine
phosphoribosyl transferase (HPRT), dihydrofolate reductase (DHFR)
(Scharfmann et al. (1991)), adenosine deaminase, phosphoglycerol
kinase (PGK), pyruvate kinase, phosphoglycerol mutase, the
beta-actin promoter (Lai et al. (1989)), and other constitutive
promoters known to those of skill in the art. In addition, many
viral promoters function constitutively in eukaryotic cells. These
include: the early and late promoters of SV40; the long terminal
repeats (LTRs) of Moloney Leukemia Virus and other retroviruses;
and the thymidine kinase promoter of Herpes Simplex Virus, among
many others.
[0195] Nucleic acid sequences that are under the control of
regulatable promoters are expressed only or to a greater or lesser
degree in the presence of an inducing or repressing agent, (e.g.,
transcription under control of the metallothionein promoter is
greatly increased in presence of certain metal ions). Regulatable
promoters include responsive elements (REs) that stimulate
transcription when their inducing factors are bound. For example,
there are REs for serum factors, steroid hormones, retinoic acid,
cyclic AMP, and tetracycline and doxycycline. Promoters containing
a particular RE can be chosen in order to obtain an regulatable
response and in some cases, the RE itself may be attached to a
different promoter, thereby conferring regulatability to the
encoded nucleic acid sequence. Thus, by selecting the appropriate
promoter (constitutive versus regulatable; strong versus weak), it
is possible to control both the existence and level of expression
of a nucleic acid sequence in the genetically modified cell. If the
nucleic acid sequence is under the control of an regulatable
promoter, delivery of the therapeutic agent in situ is triggered by
exposing the genetically modified cell in situ to conditions for
permitting transcription of the nucleic acid sequence, e.g., by
intraperitoneal injection of specific inducers of the regulatable
promoters which control transcription of the agent. For example, in
situ expression of a nucleic acid sequence under the control of the
metallothionein promoter in genetically modified cells is enhanced
by contacting the genetically modified cells with a solution
containing the appropriate (i.e., inducing) metal ions in situ.
[0196] Accordingly, the amount of siRNA generated in situ is
regulated by controlling such factors as the nature of the promoter
used to direct transcription of the nucleic acid sequence, (i.e.,
whether the promoter is constitutive or regulatable, strong or
weak) and the number of copies of the exogenous nucleic acid
sequence encoding a siRNA sequence that are in the cell.
[0197] In addition to at least one promoter and at least one
heterologous nucleic acid sequence encoding the siRNA, the
expression vector may include a selection gene, for example, a
neomycin resistance gene, for facilitating selection of cells that
have been transfected or transduced with the expression vector.
[0198] Cells can also be transfected with two or more expression
vectors, at least one vector containing the nucleic acid
sequence(s) encoding the siRNA(s), the other vector containing a
selection gene. The selection of a suitable promoter, enhancer,
selection gene and/or signal sequence is deemed to be within the
scope of one of ordinary skill in the art without undue
experimentation.
[0199] The following discussion is directed to various utilities of
the instant invention. For example, the instant invention has
utility as an expression system suitable for silencing the
expression of gene(s) of interest.
[0200] The instant invention also provides various methods for
making and using the above-described genetically-modified
cells.
[0201] The instant invention also provides methods for genetically
modifying cells of a mammalian recipient in vivo. According to one
embodiment, the method comprises introducing an expression vector
for expressing a siRNA sequence in cells of the mammalian recipient
in situ by, for example, injecting the vector into the
recipient.
[0202] VI. Delivery Vehicles for the Expression Cassettes of the
Invention
[0203] Delivery of compounds into tissues and across the
blood-brain barrier can be limited by the size and biochemical
properties of the compounds. Currently, efficient delivery of
compounds into cells in vivo can be achieved only when the
molecules are small (usually less than 600 Daltons). Gene transfer
for the correction of inborn errors of metabolism and
neurodegenerative diseases of the central nervous system (CNS), and
for the treatment of cancer has been accomplished with recombinant
adenoviral vectors.
[0204] The selection and optimization of a particular expression
vector for expressing a specific siRNA in a cell can be
accomplished by obtaining the nucleic acid sequence of the siRNA,
possibly with one or more appropriate control regions (e.g.,
promoter, insertion sequence); preparing a vector construct
comprising the vector into which is inserted the nucleic acid
sequence encoding the siRNA; transfecting or transducing cultured
cells in vitro with the vector construct; and determining whether
the siRNA is present in the cultured cells.
[0205] Vectors for cell gene therapy include viruses, such as
replication-deficient viruses (described in detail below).
Exemplary viral vectors are derived from Harvey Sarcoma virus, ROUS
Sarcoma virus, (MPSV), Moloney murine leukemia virus and DNA
viruses (e.g., adenovirus) (Ternin (1986)).
[0206] Replication-deficient retroviruses are capable of directing
synthesis of all virion proteins, but are incapable of making
infectious particles. Accordingly, these genetically altered
retroviral expression vectors have general utility for
high-efficiency transduction of nucleic acid sequences in cultured
cells, and specific utility for use in the method of the present
invention. Such retroviruses further have utility for the efficient
transduction of nucleic acid sequences into cells in vivo.
Retroviruses have been used extensively for transferring nucleic
acid material into cells. Standard protocols for producing
replication-deficient retroviruses (including the steps of
incorporation of exogenous nucleic acid material into a plasmid,
transfection of a packaging cell line with plasmid, production of
recombinant retroviruses by the packaging cell line, collection of
viral particles from tissue culture media, and infection of the
target cells with the viral particles) are provided in Kriegler
(1990) and Murray (1991).
[0207] An advantage of using retroviruses for gene therapy is that
the viruses insert the nucleic acid sequence encoding the siRNA
into the host cell genome, thereby permitting the nucleic acid
sequence encoding the siRNA to be passed on to the progeny of the
cell when it divides. Promoter sequences in the LTR region have
been reported to enhance expression of an inserted coding sequence
in a variety of cell types (see e.g., Hilberg et al. (1987);
Holland et al. (1987); Valerio et al. (1989). Some disadvantages of
using a retrovirus expression vector are (1) insertional
mutagenesis, i.e., the insertion of the nucleic acid sequence
encoding the siRNA into an undesirable position in the target cell
genome which, for example, leads to unregulated cell growth and (2)
the need for target cell proliferation in order for the nucleic
acid sequence encoding the siRNA carried by the vector to be
integrated into the target genome (Miller et al. (1990)).
[0208] Another viral candidate useful as an expression vector for
transformation of cells is the adenovirus, a double-stranded DNA
virus. The adenovirus is infective in a wide range of cell types,
including, for example, muscle and endothelial cells (Larrick and
Burck (1991)). The adenovirus also has been used as an expression
vector in muscle cells in vivo (Quantin et al. (1992)).
[0209] Adenoviruses (Ad) are double-stranded linear DNA viruses
with a 36 kb genome. Several features of adenovirus have made them
useful as transgene delivery vehicles for therapeutic applications,
such as facilitating in vivo gene delivery. Recombinant adenovirus
vectors have been shown to be capable of efficient in situ gene
transfer to parenchymal cells of various organs, including the
lung, brain, pancreas, gallbladder, and liver. This has allowed the
use of these vectors in methods for treating inherited genetic
diseases, such as cystic fibrosis, where vectors may be delivered
to a target organ. In addition, the ability of the adenovirus
vector to accomplish in situ tumor transduction has allowed the
development of a variety of anticancer gene therapy methods for
non-disseminated disease. In these methods, vector containment
favors tumor cell-specific transduction.
[0210] Like the retrovirus, the adenovirus genome is adaptable for
use as an expression vector for gene therapy, i.e., by removing the
genetic information that controls production of the virus itself
(Rosenfeld et al. (1991)). Because the adenovirus functions in an
extrachromosomal fashion, the recombinant adenovirus does not have
the theoretical problem of insertional mutagenesis.
[0211] Several approaches traditionally have been used to generate
the recombinant adenoviruses. One approach involves direct ligation
of restriction endonuclease fragments containing a nucleic acid
sequence of interest to portions of the adenoviral genome.
Alternatively, the nucleic acid sequence of interest may be
inserted into a defective adenovirus by homologous recombination
results. The desired recombinants are identified by screening
individual plaques generated in a lawn of complementation
cells.
[0212] Most adenovirus vectors are based on the adenovirus type 5
(Ad5) backbone in which an expression cassette containing the
nucleic acid sequence of interest has been introduced in place of
the early region 1 (E1) or early region 3 (E3). Viruses in which E1
has been deleted are defective for replication and are propagated
in human complementation cells (e.g., 293 or 911 cells), which
supply the missing gene E1 and pIX in trans.
[0213] In one embodiment of the present invention, one will desire
to generate siRNA in a brain cell or brain tissue. A suitable
vector for this application is an FIV vector (Brooks et al. (2002);
Alisky et al. (2000a)) or an AAV vector. For example, one may use
AAV5 (Davidson et al. (2000); Alisky et al. (2000a)). Also, one may
apply poliovirus (Bledsoe et al. (2000)) or HSV vectors (Alisky et
al. (2000b)).
[0214] Thus, as will be apparent to one of ordinary skill in the
art, a variety of suitable viral expression vectors are available
for transferring exogenous nucleic acid material into cells. The
selection of an appropriate expression vector to express a
therapeutic agent for a particular condition amenable to gene
silencing therapy and the optimization of the conditions for
insertion of the selected expression vector into the cell, are
within the scope of one of ordinary skill in the art without the
need for undue experimentation.
[0215] In another embodiment, the expression vector is in the form
of a plasmid, which is transferred into the target cells by one of
a variety of methods: physical (e.g., microinjection (Capecchi
(1980)), electroporation (Andreason and Evans (1988), scrape
loading, microparticle bombardment (Johnston (1990)) or by cellular
uptake as a chemical complex (e.g., calcium or strontium
co-precipitation, complexation with lipid, complexation with
ligand) (Methods in Molecular Biology (1991)). Several commercial
products are available for cationic liposome complexation including
Lipofectin.TM. (Gibco-BRL, Gaithersburg, Md.) (Felgner et al.
(1987)) and Transfectam.TM. (ProMega, Madison, Wis.) (Behr et al.
(1989); Loeffler et al. (1990)). However, the efficiency of
transfection by these methods is highly dependent on the nature of
the target cell and accordingly, the conditions for optimal
transfection of nucleic acids into cells using the above-mentioned
procedures must be optimized. Such optimization is within the scope
of one of ordinary skill in the art without the need for undue
experimentation.
[0216] VII. Diseases and Conditions Amendable to the Methods of the
Invention
[0217] In the certain embodiments of the present invention, a
mammalian recipient to an expression cassette of the invention has
a condition that is amenable to gene silencing therapy. As used
herein, "gene silencing therapy" refers to administration to the
recipient exogenous nucleic acid material encoding a therapeutic
siRNA and subsequent expression of the administered nucleic acid
material in situ. Thus, the phrase "condition amenable to siRNA
therapy" embraces conditions such as genetic diseases (i.e., a
disease condition that is attributable to one or more gene
defects), acquired pathologies (i.e., a pathological condition that
is not attributable to an inborn defect), cancers,
neurodegenerative diseases, e.g., trinucleotide repeat disorders,
and prophylactic processes (i.e., prevention of a disease or of an
undesired medical condition). A gene "associated with a condition"
is a gene that is either the cause, or is part of the cause, of the
condition to be treated. Examples of such genes include genes
associated with a neurodegenerative disease (e.g., a
trinucleotide-repeat disease such as a disease associated with
polyglutamine repeats, Huntington's disease, and several
spinocerebellar ataxias), and genes encoding ligands for chemokines
involved in the migration of a cancer cells, or chemokine receptor.
Also siRNA expressed from viral vectors may be used for in vivo
antiviral therapy using the vector systems described.
[0218] Accordingly, as used herein, the term "therapeutic siRNA"
refers to any siRNA that has a beneficial effect on the recipient.
Thus, "therapeutic siRNA" embraces both therapeutic and
prophylactic siRNA.
[0219] Differences between alleles that are amenable to targeting
by siRNA include disease-causing mutations as well as polymorphisms
that are not themselves mutations, but may be linked to a mutation
or associated with a predisposition to a disease state. Examples of
targetable disease mutations include tau mutations that cause
frontotemporal dementia and the GAG deletion in the TOR1A gene that
causes DYT1 dystonia. An example of a targetable polymorphism that
is not itself a mutation is the C/G single nucleotide polymorphism
(G987C) in the MJD1 gene immediately downstream of the mutation
that causes spinocerebellar ataxia type 3 and the polymorphism in
exon 58 associated with Huntington's disease.
[0220] Single nucleotide polymorphisms comprise most of the genetic
diversity between humans. Many disease genes, including the HD gene
in Huntington's disease, contain numerous single nucleotide or
multiple nucleotide polymorphisms that could be separately targeted
in one allele vs. the other, as shown in FIG. 15. The major risk
factor for developing Alzheimer's disease is the presence of a
particular polymorphism in the apolipoprotein E gene.
[0221] A. Gene Defects
[0222] A number of diseases caused by gene defects have been
identified. For example, this strategy can be applied to a major
class of disabling neurological disorders. For example this
strategy can be applied to the polyglutamine diseases, as is
demonstrated by the reduction of polyglutamine aggregation in cells
following application of the strategy. The neurodegenerative
disease may be a trinucleotide-repeat disease, such as a disease
associated with polyglutamine repeats, including Huntington's
disease, and several spinocerebellar ataxias. Additionally, this
strategy can be applied to a non-degenerative neurological
disorder, such as DYTI dystonia.
[0223] B. Acquired Pathologies
[0224] As used herein, "acquired pathology" refers to a disease or
syndrome manifested by an abnormal physiological, biochemical,
cellular, structural, or molecular biological state. For example,
the disease could be a viral disease, such as hepatitis or
AIDS.
[0225] C. Cancers
[0226] The condition amenable to gene silencing therapy
alternatively can be a genetic disorder or an acquired pathology
that is manifested by abnormal cell proliferation, e.g., cancer.
According to this embodiment, the instant invention is useful for
silencing a gene involved in neoplastic activity. The present
invention can also be used to inhibit overexpression of one or
several genes. The present invention can be used to treat
neuroblastoma, medulloblastoma, or glioblastoma.
[0227] VIII. Dosages, Formulations and Routes of Administration of
the Agents of the Invention
[0228] The agents of the invention are preferably administered so
as to result in a reduction in at least one symptom associated with
a disease. The amount administered will vary depending on various
factors including, but not limited to, the composition chosen, the
particular disease, the weight, the physical condition, and the age
of the mammal, and whether prevention or treatment is to be
achieved. Such factors can be readily determined by the clinician
employing animal models or other test systems which are well known
to the art.
[0229] Administration of siRNA may be accomplished through the
administration of the nucleic acid molecule encoding the siRNA
(see, for example, Felgner et al., U.S. Pat. No. 5,580,859, Pardoll
et al. 1995; Stevenson et al. 1995; Molling 1997; Donnelly et al.
1995; Yang et al. II; Abdallah et al. 1995). Pharmaceutical
formulations, dosages and routes of administration for nucleic
acids are generally disclosed, for example, in Felgner et al.,
supra.
[0230] The present invention envisions treating a disease, for
example, a neurodegenerative disease, in a mammal by the
administration of an agent, e.g., a nucleic acid composition, an
expression vector, or a viral particle of the invention.
Administration of the therapeutic agents in accordance with the
present invention may be continuous or intermittent, depending, for
example, upon the recipient's physiological condition, whether the
purpose of the administration is therapeutic or prophylactic, and
other factors known to skilled practitioners. The administration of
the agents of the invention may be essentially continuous over a
preselected period of time or may be in a series of spaced doses.
Both local and systemic administration is contemplated.
[0231] One or more suitable unit dosage forms having the
therapeutic agent(s) of the invention, which, as discussed below,
may optionally be formulated for sustained release (for example
using microencapsulation, see WO 94/07529, and U.S. Pat. No.
4,962,091 the disclosures of which are incorporated by reference
herein), can be administered by a variety of routes including
parenteral, including by intravenous and intramuscular routes, as
well as by direct injection into the diseased tissue. For example,
the therapeutic agent may be directly injected into the brain.
Alternatively the therapeutic agent may be introduced intrathecally
for brain and spinal cord conditions. In another example, the
therapeutic agent may be introduced intramuscularly for viruses
that traffic back to affected neurons from muscle, such as AAV,
lentivirus and adenovirus. The formulations may, where appropriate,
be conveniently presented in discrete unit dosage forms and may be
prepared by any of the methods well known to pharmacy. Such methods
may include the step of bringing into association the therapeutic
agent with liquid carriers, solid matrices, semi-solid carriers,
finely divided solid carriers or combinations thereof, and then, if
necessary, introducing or shaping the product into the desired
delivery system.
[0232] When the therapeutic agents of the invention are prepared
for administration, they are preferably combined with a
pharmaceutically acceptable carrier, diluent or excipient to form a
pharmaceutical formulation, or unit dosage form. The total active
ingredients in such formulations include from 0.1 to 99.9% by
weight of the formulation. A "pharmaceutically acceptable" is a
carrier, diluent, excipient, and/or salt that is compatible with
the other ingredients of the formulation, and not deleterious to
the recipient thereof. The active ingredient for administration may
be present as a powder or as granules; as a solution, a suspension
or an emulsion.
[0233] Pharmaceutical formulations containing the therapeutic
agents of the invention can be prepared by procedures known in the
art using well known and readily available ingredients. The
therapeutic agents of the invention can also be formulated as
solutions appropriate for parenteral administration, for instance
by intramuscular, subcutaneous or intravenous routes.
[0234] The pharmaceutical formulations of the therapeutic agents of
the invention can also take the form of an aqueous or anhydrous
solution or dispersion, or alternatively the form of an emulsion or
suspension.
[0235] Thus, the therapeutic agent may be formulated for parenteral
administration (e.g., by injection, for example, bolus injection or
continuous infusion) and may be presented in unit dose form in
ampules, pre-filled syringes, small volume infusion containers or
in multi-dose containers with an added preservative. The active
ingredients may take such forms as suspensions, solutions, or
emulsions in oily or aqueous vehicles, and may contain formulatory
agents such as suspending, stabilizing and/or dispersing agents.
Alternatively, the active ingredients may be in powder form,
obtained by aseptic isolation of sterile solid or by lyophilization
from solution, for constitution with a suitable vehicle, e.g.,
sterile, pyrogen-free water, before use.
[0236] It will be appreciated that the unit content of active
ingredient or ingredients contained in an individual aerosol dose
of each dosage form need not in itself constitute an effective
amount for treating the particular indication or disease since the
necessary effective amount can be reached by administration of a
plurality of dosage units. Moreover, the effective amount may be
achieved using less than the dose in the dosage form, either
individually, or in a series of administrations.
[0237] The pharmaceutical formulations of the present invention may
include, as optional ingredients, pharmaceutically acceptable
carriers, diluents, solubilizing or emulsifying agents, and salts
of the type that are well-known in the art. Specific non-limiting
examples of the carriers and/or diluents that are useful in the
pharmaceutical formulations of the present invention include water
and physiologically acceptable buffered saline solutions, such as
phosphate buffered saline solutions pH 7.0-8.0.
[0238] The invention will now be illustrated by the following
non-limiting
EXAMPLE
Example 1
siRNA-Mediated Silencing of Genes Using Viral Vectors
[0239] In this Example, it is shown that genes can be silenced in
an allele-specific manner. It is also demonstrated that
viral-mediated delivery of siRNA can specifically reduce expression
of targeted genes in various cell types, both in vitro and in vivo.
This strategy was then applied to reduce expression of a neurotoxic
polyglutamine disease protein. The ability of viral vectors to
transduce cells efficiently in vivo, coupled with the efficacy of
virally expressed siRNA shown here, extends the application of
siRNA to viral-based therapies and in vivo targeting experiments
that aim to define the function of specific genes.
[0240] Experimental Protocols
[0241] Generation of the expression cassettes and viral vectors.
The modified CMV (mCMV) promoter was made by PCR amplification of
CMV by primers 5'-AAGGTACCAGATCTTAGTTATTAATAGTAATCAATTACGG-3' (SEQ
ID NO:1) and 5'-GAATCGATGCATGCCTCGAGACGGTTCACTAAACCAGCTCTGC-3' (SEQ
ID NO:2) with peGFPN1 plasmid (purchased from Clontech, Inc) as
template. The mCMV product was cloned into the KpnI and ClaI sites
of the adenoviral shuttle vector pAd5KnpA, and was named pmCMVknpA.
To construct the minimal polyA cassette, the oligonucleotides,
5'-CTAGAACTAGTAATAAAGGATCCTTTATTTTCATTGGA- TCCGTGTGTTGG
TTTTTTGTGTGCGGCCGCG-3' (SEQ ID NO:3) and
5'-TCGACGCGGCCGCACACAAAAAACCAACACACGGATCC
AATGAAAATAAAGGATCCTTTATTACTAGTT- -3' (SEQ ID NO:4), were used. The
oligonucleotides contain SpeI and SalI sites at the 5' and 3' ends,
respectively. The synthesized polyA cassette was ligated into SpeI,
SalI digested pmCMVKnpA. The resultant shuttle plasmid, pmCMVmpA
was used for construction of head-to-head 21 bp hairpins of eGFP
(bp 418 to 438), human .beta.-glucuronidase (bp 649 to 669), mouse
.beta.-glucuronidase (bp 646 to 666) or E. coli
.beta.-galactosidase (bp 1152-1172). The eGFP hairpins were also
cloned into the Ad shuttle plasmid containing the commercially
available CMV promoter and polyA cassette from SV40 large T antigen
(pCMVsiGFPx). Shuttle plasmids were co-transfected into HEK293
cells along with the adenovirus backbones for generation of
full-length Ad genomes. Viruses were harvested 6-10 days after
transfection and amplified and purified as described (Anderson, R.
D., et al., Gene Ther. 7:1034-1038 (2000)).
[0242] Northern blotting. Total RNA was isolated from HEK293 cells
transfected by plasmids or infected by adenoviruses using
TRIZOL.RTM. Reagent (Invitrogen Life Technologies, Carlsbad,
Calif.) according to the manufacturer's instruction. RNAs (30
.mu.g) were separated by electrophoresis on 15% (wt/vol)
polyacrylamide-urea gels to detect transcripts, or on 1%
agarose-formaldehyde gel for target mRNAs analysis. RNAs were
transferred by electroblotting onto hybond N+ membrane (Amersham
Pharmacia Biotech). Blots were probed with .sup.32P-labeled sense
(5'-CACAAGCTGGAGTACAACTAC-3' (SEQ ID NO:5)) or antisense
(5'-GTACTTGTACTCCAGCTTTGTG-3' (SEQ ID NO:6)) oligonucleotides at
37.degree. C. for 3 h for evaluation of siRNA transcripts, or
probed for target mRNAs at 42.degree. C. overnight. Blots were
washed using standard methods and exposed to film overnight. In
vitro studies were performed in triplicate with a minimum of two
repeats.
[0243] In vivo studies and tissue analyses. All animal procedures
were approved by the University of Iowa Committee on the Care and
Use of Animals. Mice were injected into the tail vein (n=10 per
group) or into the brain (n=6 per group) as described previously
(Stein, C. S., et al., J. Virol. 73:3424-3429 (1999)) with the
virus doses indicated. Animals were sacrificed at the noted times
and tissues harvested and sections or tissue lysates evaluated for
.beta.-glucuronidase expression, eGFP fluorescence, or
.beta.-galactosidase activity using established methods (Xia, H. et
al., Nat. Biotechnol. 19:640-644 (2001)). Total RNA was harvested
from transduced liver using the methods described above.
[0244] Cell Lines. PC12 tet off cell lines (Clontech Inc., Palo
Alto, Calif.) were stably transfected with a tetracycline
regulatable plasmid into which was cloned GFPQ19 or GFPQ80 (Chai,
Y. et al., J. Neurosci. 19:10338-10347 (1999)). For GFP-Q80, clones
were selected and clone 29 chosen for regulatable properties and
inclusion formation. For GFP-Q19 clone 15 was selected for
uniformity of GFP expression following gene expression induction.
In all studies 1.5 .mu.g/ml dox was used to repress transcription.
All experiments were done in triplicate and were repeated 4
times.
[0245] Results and Discussion
[0246] To accomplish intracellular expression of siRNA, a 21-bp
hairpin representing sequences directed against eGFP was
constructed, and its ability to reduce target gene expression in
mammalian cells using two distinct constructs was tested.
Initially, the siRNA hairpin targeted against eGFP was placed under
the control of the CMV promoter and contained a full-length SV-40
polyadenylation (polyA) cassette (pCMVsiGFPx). In the second
construct, the hairpin was juxtaposed almost immediate to the CMV
transcription start site (within 6 bp) and was followed by a
synthetic, minimal polyA cassette (FIG. 1A, pmCMVsiGFPmpA)
(Experimental Protocols), because we reasoned that functional siRNA
would require minimal to no overhangs (Caplan, N. J., et al., Proc.
Natl. Acad. Sci. U.S.A. 98:9742-9747 (2001); Nykanen, A., et al.,
Cell 107:309-321 (2001)). Co-transfection of pmCMVsiGFPmpA with
pEGFPN1 (Clontech Inc) into HEK293 cells markedly reduced eGFP
fluorescence (FIG. 1C). pmCMVsiGFPmpA transfection led to the
production of an approximately 63 bp RNA specific for eGFP (FIG.
1D), consistent with the predicted size of the siGFP
hairpin-containing transcript. Reduction of target mRNA and eGFP
protein expression was noted in pmCMVsiGFPmpA-transfected cells
only (FIG. 1E, F). In contrast, eGFP RNA, protein and fluorescence
levels remained unchanged in cells transfected with pEGFPN1 and
pCMVsiGFPx (FIG. 1E, G), pEGFPN1 and pCMVsi.beta.glucmpA (FIG. 1E,
F, H), or pEGFPN1 and pCMVsi.beta.galmpA, the latter expressing
siRNA against E. coli 13-galactosidase (FIG. 1E). These data
demonstrate the specificity of the expressed siRNAs.
[0247] Constructs identical to pmCMVsiGFPmpA, except that a spacer
of 9, 12 and 21 nucleotides was present between the transcription
start site and the 21 bp hairpin, were also tested. In each case,
there was no silencing of eGFP expression (data not shown).
Together the results indicate that the spacing of the hairpin
immediate to the promoter can be important for functional target
reduction, a fact supported by recent studies in MCF-7 cells
(Brummelkamp, T. R., et al., Science 296:550-553 (2002)).
[0248] Recombinant adenoviruses were generated from the siGFP
(pmCMVsiGFPmpA) and si.beta.gluc (pmCMVsi.beta.glucmpA) plasmids
(Xia, H., et al., Nat. Biotechnol. 19:640-644 (2001); Anderson, R.
D., et al., Gene Ther. 7:1034-1038 (2000)) to test the hypothesis
that virally expressed siRNA allows for diminished gene expression
of endogenous targets in vitro and in vivo. HeLa cells are of human
origin and contain moderate levels of the soluble lysosomal enzyme
.beta.-glucuronidase. Infection of HeLa cells with viruses
expressing si.beta.gluc caused a specific reduction in human
13-glucuronidase mRNA (FIG. 1I) leading to a 60% decrease in
.beta.-glucuronidase activity relative to siGFP or control cells
(FIG. 13). Optimization of siRNA sequences using methods to refine
target mRNA accessible sequences (Lee, N. S., et al., Nat.
Biotechnol. 19:500-505 (2002)) could improve further the diminution
of 13-glucuronidase transcript and protein levels.
[0249] The results in FIG. 1 are consistent with earlier work
demonstrating the ability of synthetic 21-bp double stranded RNAs
to reduce expression of target genes in mammalian cells following
transfection, with the important difference that in the present
studies the siRNA was synthesized intracellularly from readily
available promoter constructs. The data support the utility of
regulatable, tissue or cell-specific promoters for expression of
siRNA when suitably modified for close juxtaposition of the hairpin
to the transcriptional start site and inclusion of the minimal
polyA sequence containing cassette (see, Methods above).
[0250] To evaluate the ability of virally expressed siRNA to
diminish target-gene expression in adult mouse tissues in vivo,
transgenic mice expressing eGFP (Okabe, M. et al., FEBS Lett.
407:313-319 (1997)) were injected into the striatal region of the
brain with 1.times.10.sup.7 infectious units of recombinant
adenovirus vectors expressing siGFP or control si.beta.gluc.
Viruses also contained a dsRed expression cassette in a distant
region of the virus for unequivocal localization of the injection
site. Brain sections evaluated 5 days after injection by
fluorescence (FIG. 2A) or western blot assay (FIG. 2B) demonstrated
reduced eGFP expression. Decreased eGFP expression was confined to
the injected hemisphere (FIG. 2B). The in vivo reduction is
promising, particularly since transgenically expressed eGFP is a
stable protein, making complete reduction in this short time frame
unlikely. Moreover, evaluation of eGFP levels was done 5 days after
injection, when inflammatory changes induced by the adenovirus
vector likely enhance transgenic eGFP expression from the CMV
enhancer (Ooboshi, H., et al., Arterioscler. Thromb. Vasc. Biol.
17:1786-1792 (1997)).
[0251] It was next tested whether virus mediated siRNA could
decrease expression from endogenous alleles in vivo. Its ability to
decrease .beta.-glucuronidase activity in the murine liver, where
endogenous levels of this relatively stable protein are high, was
evaluated. Mice were injected via the tail vein with a construct
expressing murine-specific si.beta.gluc (AdsiMu.beta.gluc), or the
control viruses Adsi.beta.gluc (specific for human
.beta.-glucuronidase) or Adsi.beta.gal. Adenoviruses injected into
the tail vein transduced hepatocytes as shown previously (Stein, C.
S., et al., J. Virol. 73:3424-3429 (1999)). Liver tissue harvested
3 days later showed specific reduction of target 13-glucuronidase
RNA in AdsiMu.beta.gluc treated mice only (FIG. 2C). Fluorometric
enzyme assay of liver lysates confirmed these results, with a 12%
decrease in activity from liver harvested from AdsiMu.beta.gluc
injected mice relative to Adsi.beta.gal and Adsi.beta.gluc treated
ones (p<0.01; n=10). Interestingly, sequence differences between
the murine and human siRNA constructs are limited, with 14 of 21 bp
being identical. These results confirm the specificity of virus
mediated siRNA, and indicate that allele-specific applications are
possible. Together, the data are the first to demonstrate the
utility of siRNA to diminish target gene expression in brain and
liver tissue in vivo, and establish that allele-specific silencing
in vivo is possible with siRNA.
[0252] One powerful therapeutic application of siRNA is to reduce
expression of toxic gene products in dominantly inherited diseases
such as the polyglutamine (polyQ) neurodegenerative disorders
(Margolis, R. L. & Ross, C. A. Trends Mol. Med. 7:479-482
(2001)). The molecular basis of polyQ diseases is a novel toxic
property conferred upon the mutant protein by polyQ expansion. This
toxic property is associated with disease protein aggregation. The
ability of virally expressed siRNA to diminish expanded polyQ
protein expression in neural PC-12 clonal cell lines was evaluated.
Lines were developed that express tetracycline-repressible
eGFP-polyglutamine fusion proteins with normal or expanded
glutamine of 19 (eGFP-Q19) and 80 (eGFP-Q80) repeats, respectively.
Differentiated, eGFP-Q19-expressing PC12 neural cells infected with
recombinant adenovirus expressing siGFP demonstrated a specific and
dose-dependent decrease in eGFP-Q19 fluorescence (FIG. 3A, C) and
protein levels (FIG. 3B). Application of Adsi.beta.gluc as a
control had no effect (FIG. 3A-C). Quantitative image analysis of
eGFP fluorescence demonstrated that siGFP reduced GFPQ19 expression
by greater than 96% and 93% for 100 and 50 MOI respectively,
relative to control siRNA (FIG. 3C). The multiplicity of infection
(MOI) of 100 required to achieve maximal inhibition of eGFP-Q19
expression results largely from the inability of PCl.sub.2 cells to
be infected by adenovirus-based vectors. This barrier can be
overcome using AAV- or lentivirus-based expression systems
(Davidson, B. L., et al., Proc. Natl. Acad. Sci. U.S. A.
97:3428-3432 (2000); Brooks, A. I., et al, Proc. Natl. Acad. Sci.
U.S.A. 99:6216-6221 (2002)).
[0253] To test the impact of siRNA on the size and number of
aggregates formed in eGFP-Q80 expressing cells, differentiated
PC-12/eGFP-Q80 neural cells were infected with AdsiGFP or
Adsi.beta.gluc 3 days after doxycycline removal to induce GFP-Q80
expression. Cells were evaluated 3 days later. In mock-infected
control cells (FIG. 4A), aggregates were very large 6 days after
induction as reported by others (Chai, Y., et al., J. Neurosci.
19:10338-10347 (1999; Moulder, K. L., et al., J. Neurosci.
19:705-715 (1999)). Large aggregates were also seen in cells
infected with Adsi.beta.gluc (FIG. 4B), AdsiGFPx, (FIG. 4C, siRNA
expressed from the normal CMV promoter and containing the SV40
large T antigen polyadenylation cassette), or Adsi.beta.gal (FIG.
4D). In contrast, polyQ aggregate formation was significantly
reduced in AdsiGFP infected cells (FIG. 4E), with fewer and smaller
inclusions and more diffuse eGFP fluorescence. AdsiGFP-mediated
reduction in aggregated and monomeric GFP-Q80 was verified by
Western blot analysis (FIG. 4F), and quantitation of cellular
fluorescence (FIG. 4G). AdsiGFP caused a dramatic and specific,
dose-dependent reduction in eGFP-Q80 expression (FIG. 4F, G).
[0254] It was found that transcripts expressed from the modified
CMV promoter and containing the minimal polyA cassette were capable
of reducing gene expression in both plasmid and viral vector
systems (FIGS. 1-4). The placement of the hairpin immediate to the
transcription start site and use of the minimal polyadenylation
cassette was of critical importance. In plants and Drosophila, RNA
interference is initiated by the ATP-dependent, processive cleavage
of long dsRNA into 21-25 bp double-stranded siRNA, followed by
incorporation of siRNA into a RNA-induced silencing complex that
recognizes and cleaves the target (Nykanen, A., et al., Cell
107:309-321 (2001); Zamore, P D., et al., Cell 101:25-33 (2000);
Bernstein, E., et al., Nature 409:363-366 (2001); Hamilton, A. J.
& Baulcombe, D. C. Science 286:950-952 (1999); Hammond, S. M.
et al., Nature 404:293-296 (2000)). Viral vectors expressing siRNA
are useful in determining if similar mechanisms are involved in
target RNA cleavage in mammalian cells in vivo.
[0255] In summary, these data demonstrate that siRNA expressed from
viral vectors in vitro and in vivo specifically reduce expression
of stably expressed plasmids in cells, and endogenous transgenic
targets in mice. Importantly, the application of virally expressed
siRNA to various target alleles in different cells and tissues in
vitro and in vivo was demonstrated. Finally, the results show that
it is possible to reduce polyglutamine protein levels in neurons,
which is the cause of at least nine inherited neurodegenerative
diseases, with a corresponding decrease in disease protein
aggregation. The ability of viral vectors based on adeno-associated
virus (Davidson, B. L., et al., Proc. Natl. Acad. Sci. U.S.A.
97:3428-3432 (2000)) and lentiviruses (Brooks, A. I., et al., Proc.
Natl. Acad. Sci. U.S.A. 99:6216-6221 (2002)) to efficiently
transduce cells in the CNS, coupled with the effectiveness of
virally-expressed siRNA demonstrated here, extends the application
of siRNA to viral-based therapies and to basic research, including
inhibiting novel ESTs to define gene function.
Example 2
siRNA Suppression of Genes Involved in MJD/SCA3 and FTDP-17
[0256] Modulation of gene expression by endogenous, noncoding RNAs
is increasingly appreciated to play a role in eukaryotic
development, maintenance of chromatin structure and genomic
integrity. Recently, techniques have been developed to trigger RNA
interference (RNAi) against specific targets in mammalian cells by
introducing exogenously produced or intracellularly expressed
siRNAs. These methods have proven to be quick, inexpensive and
effective for knockdown experiments in vitro and in vivo. The
ability to accomplish selective gene silencing has led to the
hypothesis that siRNAs might be employed to suppress gene
expression for therapeutic benefit.
[0257] Dominantly inherited diseases are ideal candidates for
siRNA-based therapy. To explore the utility of siRNA in inherited
human disorders, the inventors employed cellular models to test
whether we could target mutant alleles causing two classes of
dominantly inherited, untreatable neurodegenerative diseases:
polyglutamine (polyQ) neurodegeneration in MJD/SCA3 and
frontotemporal dementia with parkinsonism linked to chromosome 17
(FTDP-17). The polyQ neurodegenerative disorders consist of at
least nine diseases caused by CAG repeat expansions that encode
polyQ in the disease protein. PolyQ expansion confers a dominant
toxic property on the mutant protein that is associated with
aberrant accumulation of the disease protein in neurons. In
FTDP-17, Tau mutations lead to the formation of neurofibrillary
tangles accompanied by neuronal dysfunction and degeneration. The
precise mechanisms by which these mutant proteins cause neuronal
injury are unknown, but considerable evidence suggests that the
abnormal proteins themselves initiate the pathogenic process.
Accordingly, eliminating expression of the mutant protein by siRNA
or other means should, in principle, slow or even prevent disease.
However, because many dominant disease genes may also encode
essential proteins, the inventors sought to develop siRNA-mediated
approaches that selectively inactivate mutant alleles while
allowing continued expression of the wild type protein.
[0258] Methods
[0259] siRNA Synthesis. In vitro siRNA synthesis was previously
described (Donze 2000). Reactions were performed with desalted DNA
oligonucleotides (IDT Coralville, Iowa) and the AmpliScribeT7 High
Yield Transcription Kit (Epicentre Madison, Wis.). Yield was
determined by absorbance at 260 nm. Annealed siRNAs were assessed
for double stranded character by agarose gel (1% w/v)
electrophoresis and ethidium bromide staining. Note that for all
siRNAs generated in this study the most 5' nucleotide in the
targeted cDNA sequence is referred to as position 1 and each
subsequent nucleotide is numbered in ascending order from 5' to
3'.
[0260] Plasmid Construction. The human ataxin-3 cDNA was expanded
to 166 CAG's by PCR (Laccone 1999). PCR products were digested at
BamHI and KpnI sites introduced during PCR and ligated into BglII
and KpnI sites of pEGFP-N1 (Clontech) resulting in full-length
expanded ataxin-3 fused to the N-terminus of EGFP. Untagged
Ataxin-3-Q166 was constructed by ligating a PpuMI-NotI ataxin-3
fragment (3' of the CAG repeat) into Ataxin-3-Q166-GFP cut with
PpuMI and NotI to remove EGFP and replace the normal ataxin-3 stop
codon. Ataxin-3-Q28-GFP was generated as above from
pcDNA3.1-ataxin-3-Q28. Constructs were sequence verified to ensure
that no PCR mutations were present. Expression was verified by
Western blot with anti-ataxin-3 (Paulson 1997) and GFP antibodies
(MBL). The construct encoding a flag tagged, 352 residue tau
isoform was previously described (Leger 1994). The pEGFP-tau
plasmid was constructed by ligating the human tau cDNA into
pEGFP-C2 (Clontech) and encodes tau with EGFP fused to the amino
terminus. The pEGFP-tauV337M plasmid was derived using
site-directed mutagenesis (QuikChange Kit, Stratagene) of the
pEFGP-tau plasmid.
[0261] Cell Culture and Transfections. Culture of Cos-7 and HeLa
cells has been described (Chai 1999b). Transfections with plasmids
and siRNA were performed using Lipofectamine Plus
(LifeTechnologies) according to the manufacturer's instructions.
For ataxin-3 expression 1.5 .mu.g plasmid was transfected with 5
.mu.g in vitro synthesized siRNAs. For Tau experiments 1 .mu.g
plasmid was transfected with 2.5 .mu.g siRNA. For expression of
hairpin siRNA from the phU6 constructs, 1 .mu.g ataxin-3 expression
plasmid was transfected with 4 .mu.g phU6-siC10i or phU6-siG10i.
Cos-7 cells infected with siRNA-expressing adenovirus were
transfected with 0.5 .mu.g of each expression plasmid.
[0262] Stably transfected, doxycycline-inducible cell lines were
generated in a subclone of PC12 cells, PC6-3, because of its strong
neural differentiation properties (Pittman 19938). A PC6-3 clone
stably expressing Tet repressor plasmid (provided by S. Strack,
Univ. of Iowa), was transfected with pcDNA5/TO-ataxin-3(Q28) or
pcDNA5/TO-ataxin-3(Q166) (Invitrogen). After selection in
hygromycin, clones were characterized by Western blot and
immunofluorescence. Two clones, PC6-3-ataxin3(Q28)#33 and
PC6-3-ataxin3(Q166)#41, were chosen because of their tightly
inducible, robust expression of ataxin-3.
[0263] siRNA Plasmid and Viral Production. Plasmids expressing
ataxin-3 shRNAs were generated by insertion of head-to-head 21 bp
hairpins in phU6 that corresponded to siC10 and siG10 (Xia
2002).
[0264] Recombinant adenovirus expressing ataxin-3 specific shRNA
were generated from phU6-C10i (encoding C10 hairpin siRNA) and
phU6si-G10i (encoding G10 hairpin siRNA) as previously described
(Xia 2002, Anderson 2000).
[0265] Western Blotting and Immunofluorescence. Cos-7 cells
expressing ataxin-3 were harvested 24-48 hours after transfection
(Chai 1999b). Stably transfected, inducible cell lines were
harvested 72 hours after infection with adenovirus. Lysates were
assessed for ataxin-3 expression by Western blot analysis as
previously described (Chai 1999b), using polyclonal rabbit
anti-ataxin-3 antisera at a 1:15,000 dilution or 1C2 antibody
specific for expanded polyQ tracts (Trottier 1995) at a 1:2,500
dilution. Cells expressing Tau were harvested 24 hours after
transfection. Protein was detected with an affinity purified
polyclonal antibody to a human tau peptide (residues 12-24) at a
1:500 dilution. Anti-alpha-tubulin mouse monoclonal antibody (Sigma
St. Louis, Mo.) was used at a 1:10,000 dilution and GAPDH mouse
monoclonal antibody (Sigma St. Louis, Mo.) was used at a 1:1,000
dilution.
[0266] Immunofluorescence for ataxin-3 (Chai 1999b) was carried out
using 1C2 antibody (Chemicon International Temecula, Calif.) at
1:1,000 dilution 48 hours after transfection. Flag-tagged, wild
type tau was detected using mouse monoclonal antibody (Sigma St.
Louis, Mo.) at 1:1,000 dilution 24 hours after transfection. Both
proteins were detected with rhodamine conjugated secondary antibody
at a 1:1,000 dilution.
[0267] Fluorescent Imaging and Quantification. Fixed samples were
observed with a Zeiss Axioplan fluorescence microscope. Digital
images were collected on separate red, green and blue fluorescence
channels using a SPOT digital camera. Images were assembled and
overlaid using Adobe Photoshop 6.0. Live cell images were collected
with a Kodak MDS 290 digital camera mounted to an Olympus (Tokyo,
Japan) CK40 inverted microscope. Fluorescence was quantitated by
collecting 3 non-overlapping images per well at low power
(10.times.). Pixel count and intensity for each image was
determined using Bioquant Nova Prime software (BIOQUANT Image
Analysis Corporation). Background was subtracted by quantitation of
images from cells of equivalent density under identical fluorescent
illumination. Mock transfected cells were used to assess background
fluorescence for all experiments and were stained with appropriate
primary and secondary antibodies for simulated heterozygous
experiments. Average fluorescence is reported from 2 to 3
independent experiments. The mean of 2 to 3 independent experiments
for cells transfected with the indicated expression plasmid and
siMiss was set at one. Errors bars depict variation between
experiments as standard error of the mean. In simulated
heterozygous experiments, a blinded observer scored cells with a
positive fluorescence signal for expression of wild type, mutant or
both proteins in random fields at high power for two independent
experiments. More than 100 cells were scored in each experiment and
reported as number of cells with co-expression divided by total
number of transfected cells.
[0268] Results
[0269] Direct Silencing of Expanded Alleles. The inventors first
attempted suppression of mutant polyQ expression using siRNA
complementary to the CAG repeat and immediately adjacent sequences
to determine if the expanded repeat differentially altered the
susceptibility of the mutant allele to siRNA inhibition (FIG. 6).
HeLa cells were transfected with various in vitro synthesized
siRNAs (Danze 2002) and plasmids encoding normal or expanded polyQ
fused to red or green fluorescent protein, respectively (Q19-RFP
and Q80-GFP) (FIG. 5a). In negative control cells transfected with
Q80-GFP, Q19-RFP and a mistargeted siRNA (siMiss), Q80-GFP formed
aggregates (Onodera 1997) which recruited the normally diffuse
Q19-RFP (FIG. 5a). When the experiment was performed with siRNA
targeted to GFP as a positive control for allele specific
silencing, Q80-GFP expression was nearly abolished while Q19-RFP
continued to be expressed as a diffusely distributed protein (FIG.
5a). When Q19-RFP and Q80-GFP were co-transfected with siRNA
directly targeting the CAG repeat (siCAG) (FIG. 5a) or an
immediately adjacent 5' region (data not shown), expression of both
proteins was efficiently suppressed.
[0270] To test whether siRNA could selectively silence expression
of a full-length polyQ disease protein, siRNAs were designed that
target the transcript encoding ataxin-3, the disease protein in
Machado-Joseph Disease, also known as Spinocerebellar Ataxia Type 3
(MJD/SCA3) (Zoghbi 2000) (FIG. 5b). In transfected cells, siRNA
directed against three separate regions--the CAG repeat, a distant
5' site, or a site just 5' to the CAG repeat (siN'CAG)--resulted in
efficient, but not allele-specific, suppression of ataxin-3
containing normal or expanded repeats (data not shown). Consistent
with an earlier study using longer dsRNA (Caplen 2002) the present
results show that expanded CAG repeats and adjacent sequences,
while accessible to RNAi, may not be preferential targets for
silencing.
[0271] Allele-specific Silencing of the Mutant PolyQ Gene in
MJD/SCA3. In further efforts to selectively inactivate the mutant
allele the inventors took advantage of a SNP in the MJD1 gene, a G
to C transition immediately 3' to the CAG repeat (G987C) (FIG. 5b).
This SNP is in linkage disequilibrium with the disease-causing
expansion, in most families segregating perfectly with the disease
allele. Worldwide, 70% of disease chromosomes carry the C variant
(Gaspar 2001). The present ataxin-3 expression cassettes, which
were generated from patients (Paulson 1997), contain the C variant
in all expanded ataxin-3 constructs and the G variant in all normal
ataxin-3 constructs. To test whether this G-C mismatch could be
distinguished by siRNA, siRNAs were designed that included the last
2 CAG triplets of the repeat followed by the C variant at position
7 (siC7) (FIG. 6 and FIG. 5b), resulting in a perfect match only
for expanded alleles. Despite the presence of a single mismatch to
the wild type allele, siC7 strongly inhibited expression of both
alleles (FIG. 5c,d). A second G-C mismatch was then introduced at
position 8 such that the siRNA contained two mismatches as compared
to wild type and only one mismatch as compared to mutant alleles
(siC7/8). The siC7/8 siRNA effectively suppressed mutant ataxin-3
expression, reducing total fluorescence to an average 8.6% of
control levels, with only modest effects on wild type ataxin-3
(average 75.2% of control). siC7/8 also nearly eliminated the
accumulation of aggregated mutant ataxin-3, a pathological hallmark
of disease (Chan 2000) (FIG. 5d).
[0272] To optimize differential suppression, siRNAs were designed
containing a more centrally placed mismatch. Because the center of
the antisense strand directs cleavage of target mRNA in the RNA
Induced Silencing Complex (RISC) complex (Elbashir 2001c), it was
reasoned that central mismatches might more efficiently
discriminate between wild type and mutant alleles. siRNAs were
designed that place the C of the SNP at position 10 (siC10),
preceded by the final three triplets in the CAG repeat (FIG. 6 and
FIG. 5b). In transfected cells, siC10 caused allele-specific
suppression of the mutant protein (FIG. 5c,d). Fluorescence from
expanded Atx-3-Q166-GFP was dramatically reduced (7.4% of control
levels), while fluorescence of Atx-3-Q28-GFP showed minimal change
(93.6% of control; FIG. 5c,d). Conversely, siRNA engineered to
suppress only the wild type allele (siG10) inhibited wild type
expression with little effect on expression of the mutant allele
(FIG. 5c,d). Inclusion of three CAG repeats at the 5' end of the
siRNA did not inhibit expression of Q19-GFP, Q80-GFP, or
full-length ataxin-1-Q30 proteins that are each encoded by CAG
repeat containing transcripts (FIG. 7).
[0273] In the disease state, normal and mutant alleles are
simultaneously expressed. In plants and worms, activation of RNAi
against one transcript results in the spread of silencing signals
to other targets due to RNA-dependent RNA polymerase (RDRP)
activity primed by the introduced RNA (Fire 1998, Tang 2003).
Although spreading has not been detected in mammalian cells and
RDRP activity is not required for effective siRNA inhibition (Chiu
2002, Schwarz 2002, Martinez 2002), most studies have used
cell-free systems in which a mammalian RDRP could have been
inactivated. If triggering the mammalian RNAi pathway against one
allele activates cellular mechanisms that also silence the other
allele, then siRNA applications might be limited to non-essential
genes. To test this possibility, the heterozygous state was
simulated by co-transfecting Atx-3-Q28-GFP and Atx-3-Q166 and
analyzing suppression by Western blot. As shown in FIG. 5e each
siRNA retained the specificity observed in separate transfections:
siC7 inhibited both alleles, siG10 inhibited only the wild type
allele, and siC7/8 and siC10 inhibited only mutant allele
expression.
[0274] Effective siRNA therapy for late onset disease will likely
require sustained intracellular expression of the siRNA.
Accordingly, the present experiments were extended to two
intracellular methods of siRNA production and delivery: expression
plasmids and recombinant virus (Brummelkamp 2002, Xia 2002).
Plasmids were constructed expressing siG10 or siC10 siRNA from the
human U6 promoter as a hairpin transcript that is processed
intracellularly to produce siRNA (Brummelkamp 2002, Xia 2002). When
co-transfected with ataxin-3-GFP expression plasmids, phU6-G10i and
phU6-C10i-siRNA plasmids specifically suppressed wild type or
mutant ataxin-3 expression, respectively (FIG. 5f).
[0275] This result encouraged the inventors to engineer recombinant
adenoviral vectors expressing allele-specific siRNA (Xia 2002).
Viral-mediated suppression was tested in Cos-7 cells transiently
transfected with both Atx-3-Q28-GFP and Atx-3-Q166 to simulate the
heterozygous state. Cos-7 cells infected with adenovirus encoding
siG10, siC10 or negative control siRNA (Ad-G10i, Ad-C10i, and
Ad-LacZi respectively) exhibited allele-specific silencing of wild
type ataxin-3 expression with Ad-G10i and of mutant ataxin-3 with
Ad-C10i (FIG. 8a,b,c). Quantitation of fluorescence (FIG. 8b)
showed that Ad-G10i reduced wild type ataxin-3 to 5.4% of control
levels while mutant ataxin-3 expression remained unchanged.
Conversely, Ad-C10i reduced mutant ataxin-3 fluorescence levels to
8.8% of control and retained 97.4% of wild type signal. These
results were confirmed by Western blot where it was further
observed that Ad-G10i virus decreased endogenous (primate) ataxin-3
while Ad-C10i did not (FIG. 8c).
[0276] Viral mediated suppression was also assessed in
differentiated PC12 neural cell lines that inducibly express normal
(Q28) or expanded (Q166) mutant ataxin-3. Following infection with
Ad-G10i, Ad-C10i, or Ad-LacZi, differentiated neural cells were
placed in doxycycline for three days to induce maximal expression
of ataxin-3. Western blot analysis of cell lysates confirmed that
the Ad-G10i virus suppressed only wild type ataxin-3, Ad-C10i virus
suppressed only mutant ataxin-3, and Ad-LacZi had no effect on
either normal or mutant ataxin-3 expression (FIG. 8d). Thus, siRNA
retains its efficacy and selectivity across different modes of
production and delivery to achieve allele-specific silencing of
ataxin-3.
[0277] Allele-Specific Silencing of a Missense Tau Mutation. The
preceding results indicate that, for DNA repeat mutations in which
the repeat itself does not present an effective target, an
associated SNP can be exploited to achieve allele-specific
silencing. To test whether siRNA works equally well to silence
disease-causing mutations directly, the inventors targeted missense
Tau mutations that cause FTDP-17 (Poorkaj 1998, Hutton 1998). A
series of 21-24 nt siRNAs were generated in vitro against four
missense FTDP-17 mutations: G272V, P301L, V337M, and R406W (FIG. 6
and FIG. 9a). In each case the point mutation was placed centrally,
near the likely cleavage site in the RISC complex (position 9, 10
or 11) (Laccone 1999). A fifth siRNA designed to target a 5'
sequence in all Tau transcripts was also tested. To screen for
siRNA-mediated suppression, the inventors co-transfected GFP
fusions of mutant and wild type Tau isoforms together with siRNA
into Cos-7 cells. Of the five targeted sites, the inventors
obtained robust suppression with siRNA corresponding to V337M (FIG.
6 and FIG. 9A) (Poorkaj 1998, Hutton 1998), and thus focused
further analysis on this mutation. The V337M mutation is a G to A
base change in the first position of the codon (GTG to ATG), and
the corresponding V337M siRNA contains the A missense change at
position 9 (siA9). This intended V337M-specific siRNA
preferentially silenced the mutant allele but also caused
significant suppression of wild type Tau (FIG. 9b,c).
[0278] Based on the success of this approach with ataxin-3, the
inventors designed two additional siRNAs that contained the V337M
(G to A) mutation at position 9 as well as a second introduced G-C
mismatch immediately 5' to the mutation (siA9/C8) or three
nucleotides 3' to the mutation (siA9/C12), such that the siRNA now
contained two mismatches to the wild type but only one to the
mutant allele. This strategy resulted in further preferential
inactivation of the mutant allele. One siRNA, siA9/C12, showed
strong selectivity for the mutant tau allele, reducing fluorescence
to 12.7% of control levels without detectable loss of wild type Tau
(FIG. 9b,c). Next, we simulated the heterozygous state by
co-transfecting V337M-GFP and flag-tagged WT-Tau expression
plasmids (FIG. 10). In co-transfected HeLa cells, siA9/C12 silenced
the mutant allele (16.7% of control levels) with minimal alteration
of wild type expression assessed by fluorescence (FIG. 10a) and
Western blot (FIG. 10b). In addition, siA9 and siA9/C8 displayed
better allele discrimination than we had observed in separate
transfections, but continued to suppress both wild type and mutant
tau expression (FIG. 10a,b,c).
[0279] Discussion
[0280] Despite the rapidly growing siRNA literature, questions
remain concerning the design and application of siRNA both as a
research tool and a therapeutic strategy. The present study,
demonstrating allele-specific silencing of dominant disease genes,
sheds light on important aspects of both applications.
[0281] Because many disease genes encode essential proteins,
development of strategies to exclusively inactivate mutant alleles
is important for the general application of siRNA to dominant
diseases. The present results for two unrelated disease genes
demonstrate that in mammalian cells it is possible to silence a
single disease allele without activating pathways analogous to
those found in plants and worms that result in the spread of
silencing signals (Fire 1998, Tang 2003).
[0282] In summary, siRNA can be engineered to silence expression of
disease alleles differing from wild type alleles by as little as a
single nucleotide. This approach can directly target missense
mutations, as in frontotemporal dementia, or associated SNPs, as in
MJD/SCA3. The present stepwise strategy for optimizing
allele-specific targeting extends the utility of siRNA to a wide
range of dominant diseases in which the disease gene normally plays
an important or essential role. One such example is the
polyglutamine disease, Huntington disease (HD), in which normal HD
protein levels are developmentally essential (Nasir 1995). The
availability of mouse models for many dominant disorders, including
MJD/SCA3 (Cemal 2002), HD (Lin 2001), and FTDP-17 (Tanemura 2002),
allows for the in vivo testing of siRNA-based therapy for these and
other human diseases.
Example 3
Therapy for DYT1 Dystonia: Allele-Specific Silencing of Mutant
TorsinA
[0283] DYT1 dystonia is the most common cause of primary
generalized dystonia. A dominantly inherited disorder, DYT1 usually
presents in childhood as focal dystonia and progresses to severe
generalized disease. With one possible exception, all cases of DYT1
result from a common GAG deletion in TOR1A, eliminating one of two
adjacent glutamic acids near the C-terminus of the protein TorsinA
(TA). Although the precise cellular function of TA is unknown, it
seems clear that mutant TA (TAmut) acts through a dominant-negative
or dominant-toxic mechanism. The dominant nature of the genetic
defect in DYT1 dystonia suggests that efforts to silence expression
of TAmut should have potential therapeutic benefit.
[0284] Several characteristics of DYT1 make it an ideal disease in
which to explore siRNA-mediated gene silencing as potential
therapy. Of greatest importance, the dominant nature of the disease
suggests that a reduction in mutant TA, whatever the precise
pathogenic mechanism proves to be, will be helpful. Moreover, the
existence of a single common mutation that deletes a full three
nucleotides suggests it may be feasible to design siRNA that will
specifically target the mutant allele and will be applicable to all
affected persons. Finally, there is no effective therapy for DYT1,
a relentless and disabling disease. Thus, any therapeutic approach
with promise needs to be explored. Because TAwt may be an essential
protein, however, it is critically important that efforts be made
to silence only the mutant allele.
[0285] In the studies reported here , the inventors explored the
utility of siRNA for DYT1. As outlined in the strategy in FIG. 11,
the inventors sought to develop siRNA that would specifically
eliminate production of protein from the mutant allele. By
exploiting the three base pair difference between wild type and
mutant alleles, the inventors successfully silenced expression of
TAmut without interfering with expression of the wild type protein
(TAwt).
[0286] Methods
[0287] siRNA design and synthesis Small-interfering RNA duplexes
were synthesized in vitro according to a previously described
protocol (Donze 2002), using AmpliScribeT7 High Yield Transcription
Kit (Epicentre Technologies) and desalted DNA oligonucleotides
(IDT). siRNAs were designed to target different regions of human TA
transcript: 1) an upstream sequence common to both TAwt and TAmut
(com-siRNA); 2) the area corresponding to the mutation with either
the wild type sequence (wt-siRNA) or the mutant sequence positioned
at three different places (mutA-siRNA, mutB-siRNA, mutC-siRNA); and
3) a negative control siRNA containing an irrelevant sequence that
does not target any region of TA (mis-siRNA). The design of the
primers and targeted sequences are shown schematically in FIG. 12.
After in vitro synthesis, the double stranded structure of the
resultant RNA was confirmed in 1.5% agarose gels and RNA
concentration determined with a SmartSpect 3000 UV
Spectrophotometer (BiORad).
[0288] Plasmids pcDNA3 containing TAwt or TAmut cDNA were kindly
provided by Xandra Breakefield (Mass General Hospital, Boston,
Mass.). This construct was produced by cloning the entire coding
sequences of human TorsinA (1-332), both wild-type and mutant (GAG
deleted), into the mammalian expression vector, pcDNA3 (Clontech,
Palo Alto, Calif.). Using PCR based strategies, an N-terminal
hemagglutinin (HA) epitope tag was inserted into both constructs.
pEGFP-C3-TAwt was kindly provided by Pullanipally Shashidharan (Mt
Sinai Medical School, N.Y.). This construct was made by inserting
the full-length coding sequence of wild-type TorsinA into the EcORI
and BamHI restriction sites of the vector pEGFP-C3 (Clontech). This
resulted in a fusion protein including eGFP, three "stuffer" amino
acids and the 331 amino acids of TorsinA. HA-tagged TAmut was
inserted into the ApaI and SalI restriction sites of pEGFP-C1
vector (Clontech), resulting in a GFP-HA-TAmut construct.
[0289] Cell culture and transfections Methods for cell culture of
Cos-7 have been described previously (Chai 1999b). Transfections
with DNA plasmids and siRNA were performed using Lipofectamine Plus
(LifeTechnologies) according to the manufacturer's instructions in
six or 12 well plates with cells at 70-90% confluence. For single
plasmid transfection, 1 .mu.g of plasmid was transfected with 5
.mu.g of siRNA. For double plasmid transfection, 0.75 .mu.g of each
plasmid was transfected with 3.75 .mu.g of siRNA.
[0290] Western Blotting and Fluorescence Microscopy. Cells were
harvested 36 to 48 hours after transfection and lysates were
assessed for TA expression by Western Blot analysis (WB) as
previously described (Chai 1999b). The antibody used to detect TA
was polyclonal rabbit antiserum generated against a TA-maltose
binding protein fusion protein (kindly provided by Xandra
Breakefield) at a 1:500 dilution. Additional antibodies used in the
experiments described here are the anti-HA mouse monoclonal
antibody 12CA5 (Roche) at 1:1,000 dilution, monoclonal mouse
anti-GFP antibody (MBL) at 1:1,000 dilution, and for loading
controls, anti .alpha.-tubulin mouse monoclonal antibody (Sigma) at
1:20,000 dilution.
[0291] Fluorescence visualization of fixed cells expressing
GFP-tagged TA was performed with a Zeiss Axioplan fluorescence
microscope. Nuclei were visualized by staining with 5 .mu.g/ml DAPI
at room temperature for 10 minutes. Digital images were collected
on separate red, green and blue fluorescence channels using a
Diagnostics SPOT digital camera. Live cell images were collected
with a Kodak MDS 290 digital camera mounted on an Olympus CK40
inverted microscope equipped for GFP fluorescence and phase
contrast microscopy. Digitized images were assembled using Adobe
Photoshop 6.0.
[0292] Western Blot and Fluorescence Quantification. For
quantification of WB signal, blots were scanned with a Hewlett
Packard ScanJet 510.degree. C. scanner. The pixel count and
intensity of bands corresponding to TA and .alpha.-tubulin were
measured and the background signal subtracted using Scion Image
software (Scion Corporation). Using the .alpha.-tubulin signal from
control lanes as an internal reference, the TA signals were
normalized based on the amount of protein loaded per lane and the
result was expressed as percentage of TA signal in the control
lane. Fluorescence quantification was determined by collecting
three non-overlapping images per well at low power (10.times.), and
assessing the pixel count and intensity for each image with
Bioquant Nova Prime software (BIOQUANT Image Analysis Corporation).
Background fluorescence, which was subtracted from experimental
images, was determined by quantification of fluorescence images of
untransfected cells at equivalent confluence, taken under identical
illumination and exposure settings.
[0293] Results
[0294] Expression of tagged TorsinA constructs. To test whether
allele-specific silencing could be applied to DYT1, a way to
differentiate TAwt and TAmut proteins needed to be developed.
Because TAwt and TAmut display identical mobility on gels and no
isoform-specific antibodies are available, amino-terminal
epitope-tagged TA constructs and GFP-TA fusion proteins were
generated that would allow distinguishing TAwt and TAmut. The use
of GFP-TA fusion proteins also facilitated the ability to screen
siRNA suppression because it allowed visualization of TA levels in
living cells over time.
[0295] In transfected Cos-7 cells, epitope-tagged TA and GFP-TA
fusion protein expression was confirmed by using the appropriate
anti-epitope and anti-TA antibodies. Fluorescence microscopy in
living cells showed that GFP-TAwt and GFP-TAmut fusion proteins
were expressed diffusely in the cell, primarily in the cytoplasm,
although perinuclear inclusions were also seen. It is important to
note that these construct were designed to express reporter
proteins in order to assess allele-specific RNA interference rather
than to study TA function. The N-terminal epitope and GFP domains
likely disrupt the normal signal peptide-mediated translocation of
TA into the lumen of the endoplasmic reticulum, where TA is thought
to function. Thus, while these constructs facilitated expression
analysis in the studies described here, they are of limited utility
for studying TA function.
[0296] Silencing TorsinA with siRNA. Various siRNAs were designed
to test the hypothesis that siRNA-mediated suppression of TA
expression could be achieved in an allele-specific manner (FIG.
12). Because siRNA can display exquisite sequence specificity, the
three base pair difference between mutant and wild type TOR1A
alleles might be sufficient to permit the design of siRNA that
preferentially recognizes mRNA derived from the mutant allele. Two
siRNAs were initially designed to target TAmut (mutA-siRNA and
mutB-siRNA) and one to target TAwt (wt-siRNA). In addition, a
positive control siRNA was designed to silence both alleles
(com-siRNA) and a negative control siRNA of irrelevant sequence
(mis-siRNA) was designed. Cos-7 cells were first cotransfected with
siRNA and plasmids encoding either GFP-TAwt or untagged TAwt at a
siRNA to plasmid ratio of 5:1. With wt-siRNA, potent silencing of
TAwt expression was observed to less than 1% of control levels,
based on western blot analysis of cell lysates (FIGS. 13A and 13C).
With com-siRNA, TAwt expression was suppressed to .about.30% of
control levels. In contrast, mutA-siRNA did not suppress TAwt and
mutB-siRNA suppressed TAwt expression only modestly. These results
demonstrate robust suppression of TAwt expression by wild
type-specific siRNA but not mutant-specific siRNA.
[0297] To assess suppression of TAmut, the same siRNAs were
cotransfected with plasmids encoding untagged or HA-tagged TAmut.
With mutA-siRNA or mutB-siRNA, marked, though somewhat variable,
suppression of TAmut expression was observed as assessed by western
blot analysis of protein levels (FIGS. 13B and 13C). With
com-siRNA, suppression of TAmut expression was observed similar to
what was observed with TAwt expression. In contrast, wt-siRNA did
not suppress expression of TAmut. Thus differential suppression of
TAmut expression was observed by allele-specific siRNA in precisely
the manner anticipated by the inventors.
[0298] To achieve even more robust silencing of TAmut, a third
siRNA was engineered to target TAmut (mutC-siRNA, FIG. 12).
MutC-siRNA places the GAG deletion more centrally in the siRNA
duplex. Because the central portion of the antisense strand of
siRNA guides mRNA cleavage, it was reasoned that placing the GAG
deletion more centrally might enhance specific suppression of
TAmut. As shown in FIG. 13, mutC-siRNA suppressed TAmut expression
more specifically and robustly than the other mut-siRNAs tested. In
transfected cells, mutC-siRNA suppressed TAmut to less than 0.5% of
control levels, and had no effect on the expression of TAwt.
[0299] To confirm allele-specific suppression by wt-siRNA and
mutC-siRNA, respectively, the inventors cotransfected cells with
GFP-TAwt or GFP-TAmut together with mis-siRNA, wt-siRNA or
mutC-siRNA. Levels of TA expression were assessed 24 and 48 hours
later by GFP fluorescence, and quantified the fluorescence signal
from multiple images was quantified. The results (FIGS. 13D and
13E) confirmed the earlier western blots results in showing potent,
specific silencing of TAwt and TAmut by wt-siRNA and mutC-siRNA,
respectively, in cultured mammalian cells.
[0300] Allele-specific silencing in simulated heterozygous state.
In DYTI, both the mutant and wild type alleles are expressed. Once
the efficacy of siRNA silencing was established, the inventors
sought to confirm siRNA specificity for the targeted allele in
cells that mimic the heterozygous state of DYT1. In plants and
Caenorhabditis elegans, RNA-dependent RNA polymerase activity
primed by introduction of exogenous RNA can result in the spread of
silencing signals along the entire length of the targeted mRNA
(Fire 1998, Tang 2003). No evidence for such a mechanism has been
discovered in mammalian cells (Schwarz 2002, Chiu 2002).
Nonetheless it remained possible that silencing of the mutant
allele might activate cellular processes that would also inhibit
expression from the wild type allele. To address this possibility,
Cos-7 cells were cotransfected with both GFP-TAwt and HA-TAmut, and
suppression by mis-siRNA, wt-siRNA or mutC-siRNA was assessed. As
shown in FIG. 14, potent and specific silencing of the targeted
allele (either TAmut or TAwt) to levels less than 1% of controls
was observed, with only slight suppression in the levels of the
non-targeted protein. Thus, in cells expressing mutant and wild
type forms of the protein, siRNA can suppress TAmut while sparing
expression of TAwt.
[0301] Discussion
[0302] In this study the inventors succeeded in generating siRNA
that specifically and robustly suppresses mutant TA, the defective
protein responsible for the most common form of primary generalized
dystonia. The results have several implications for the treatment
of DYT1 dystonia. First and foremost, the suppression achieved was
remarkably allele-specific, even in cells simulating the
heterozygous state. In other words, efficient suppression of mutant
TA occurred without significant reduction in wild type TA.
Homozygous TA knockout mice die shortly after birth, while the
heterozygous mice are normal (Goodchild 2002), suggesting an
essential function for TA. Thus, therapy for DYTI needs to
eliminate the dominant negative or dominant toxic properties of the
mutant protein while sustaining expression of the normal allele in
order to prevent the deleterious consequences of loss of TA
function. Selective siRNA-mediated suppression of the mutant allele
fulfills these criteria without requiring detailed knowledge of the
pathogenic mechanism.
[0303] An appealing feature of the present siRNA therapy is
applicable to all individuals afflicted with DYT1. Except for one
unusual case (Leung 2001, Doheny 2002, Klein 2002b), all persons
with DYTI have the same (GAG) deletion mutation (Ozelius 1997,
Ozelius 1999). This obviates the need to design individually
tailored siRNAs. In addition, the fact that the DYT1 mutation
results in a full three base pair difference from the wild type
allele suggests that siRNA easily distinguishes mRNA derived from
normal and mutant TOR1A alleles.
[0304] It is important to recognize that DYT1 is not a fully
penetrant disease (Fahn 1998, Klein 2002a). Even when expressed
maximally, mutant TA causes significant neurological dysfunction
less than 50% of the time. Thus, even partial reduction of mutant
TA levels might be sufficient to lower its pathological brain
activity below a clinically detectable threshold. In addition, the
DYT1 mutation almost always manifests before age 25, suggesting
that TAmut expression during a critical developmental window is
required for symptom onset. This raises the possibility that
suppressing TAmut expression during development might be sufficient
to prevent symptoms throughout life. Finally, unlike many other
inherited movement disorders DYT1 is not characterized by
progressive neurodegeneration. The clinical phenotype must result
primarily from neuronal dysfunction rather than neuronal cell death
(Homykiewicz 1986, Walker 2002, Augood 2002, Augood 1999). This
suggests the potential reversibility of DYT1 by suppressing TAmut
expression in overtly symptomatic persons.
Example 4
siRNA Specific for Huntington's Disease
[0305] The present inventors have developed huntingtin siRNA
focused on two targets. One is non-allele specific (siHDexon2), the
other is targeted to the exon 58 codon deletion, the only known
common intragenic polymorphism in linkage dysequilibirum with the
disease mutation (Ambrose et al, 1994). Specifically, 92% of wild
type huntingtin alleles have four GAGs in exon 58, while 38% of HD
patients have 3 GAGs in exon 58. To assess a siRNA targeted to the
intragenic polymorphism, PC6-3 cells were transfected with a
full-length huntingtin containing the exon 58 deletion.
Specifically, PC6-3 rat pheochromocytoma cells were co-transfected
with CMV-human Htt (37Qs) and U6 siRNA hairpin plasmids. Cell
extracts were harvested 24 hours later and western blots were
performed using 15 .mu.g total protein extract. Primary antibody
was an anti-huntingtin monoclonal antibody (MAB2166, Chemicon) that
reacts with human, monkey, rat and mouse Htt proteins.
[0306] As seen in FIG. 15, the siRNA lead to silencing of the
disease allele. As a positive control, a non-allele specific siRNA
targeted to exon 2 of the huntingtin gene was used. siRNA directed
against GFP was used as a negative control. Note that only siEx58#
2 is functional.
Example 5
Targeting Alzheimer's Disease Genes with RNA Interference
[0307] Introduction
[0308] RNA interference (RNAi) plays an important role in diverse
aspects of biology (McManus et al., 2002). Techniques that exploit
the power of RNAi to suppress target genes have already become
indispensable tools in research and are therapeutically useful
(McManus et al., 2002; Song et al., 2003). In particular, the
production of small interfering RNAs (siRNAs) that silence specific
disease-related genes have wide-ranging therapeutic
applications.
[0309] One promising therapeutic role for siRNA is the silencing of
genes that cause dominantly inherited disease. The present
inventors and others recently established the feasibility of this
approach, and demonstrated that it is possible to engineer siRNAs
that selectively silence mutant alleles while retaining expression
of normal alleles (Miller et al., 2003; Gonzalez-Alegre et al.,
2003; Ding et al., 2003; Abdelgany et al., 2003; Martinez et al.
2002a). Such allele-specific suppression is important for disorders
in which the defective gene normally plays an important or
essential role.
[0310] Generating effective siRNAs for target genes is not always
straightforward, however, particularly when designing siRNAs that
selectively target mutant alleles (Miller et al. 2003; Ding et al.
2003). Here the present inventors describe a simple, novel approach
for producing siRNAs that should facilitate the development of gene
and allele-specific siRNAs. Using this strategy, the inventors then
created allele-specific siRNA for mutations in two important
neurodegenerative disease genes, the genes encoding amyloid
precursor protein (APP) and tau.
[0311] Recently the inventors demonstrated allele-specific
silencing for tau and two other dominant neurogenetic disease genes
(see examples above; Miller et al., 2003; Gonzalez-Alegre et al.,
2003). But due to constraints imposed by the method of siRNA
production, the inventors could not systematically analyze the
effect of positioning mutations at each point along the antisense
guide strand that mediates siRNA silencing. Here, the inventors
have developed an efficient strategy to produce and screen siRNAs.
Using this approach with APP and tau as model target genes, the
inventors demonstrate that allele specificity of siRNA targeting is
optimal when mutations are placed centrally within the
21-nucleotide siRNA.
[0312] Materials and Methods:
[0313] siRNA Synthesis. In vitro synthesis of siRNA was done using
a previously described protocol (Miller et al., 2003; Donze et al.,
2002). Desalted DNA oligonucleotides (Integrated DNA Technologies,
Coralville, Iowa) encoding sense and antisense target sequences
were used with the AmpliScribeT7 high-yield transcription kit
(Epicentre Technologies, Madison, Wis.) to generate siRNA duplexes
(FIG. 16). After measuring reaction yields through absorbance at
260 nm, double-stranded nature was confirmed by agarose gel (1%
wt/vol) electrophoresis and ethidium bromide staining. Note that
for all siRNAs used in this study the most 5' nucleotide in the
targeted cDNA sequence is referred to as position 1 and each
subsequent nucleotide is numbered in ascending order from 5' to
3'.
[0314] Plasmids. The plasmid used for GFP expression was pEGFP-C1
(BD Biosciences Clontech, Palo Alto, Calif.). Gloria Lee
(University of Iowa, Iowa City, Iowa) kindly provided the
constructs encoding human flag-tagged tau and V337M-GFP tau (Miller
et al., 2003). Constructs encoding APP and APPsw mutant proteins
were kindly provided by R. Scott Turner (University of Michigan,
Ann Arbor, Mich.). shRNA Plasmid Construction. The tRNA-valine
vector was constructed by annealing two primers, (forward
5'-CAGGACTAGTCTTTTAGGTCAAAAAGAAGAAGCTTTGTAACCGTTGGTT TCCGTAGTGTA-3'
(SEQ ID NO:56) and reverse 5'-CTTCGAACCGGGGACCTTTCGCGTGTTA-
GGCGAACGTGATAACCACTAC ACTACGGAAACCAAC-3' (SEQ ID NO:57)), extending
the primers with PCR, and cloning them into pCR 2.1-TOPO vector
using the TOPO TA Cloning Kit (Invitrogen Life Technologies,
Carlsbad, Calif.) (Koseki et al., 1999; Kawasaki et al., 2003).
Head-to-head 21 bp shRNA fragments were PCR amplified using as a
template the resulting tRNA-valine vector, the forward primer
above, and the reverse primers below. Each shRNA fragment was
subsequently cloned into pCR 2.1-TOPO vector. Reverse primers used
for generation of tRNA-valine driven shRNA are as follows:
3 tau: tvTau: AAAAAAGTGGCCAGGTGGAAGTAAAATCCAAGCTTC (SEQ ID NO:58)
GATTTTACTTCCACCTGGCCACCTTCGAACCGGGGA CCTTTCG tvA10:
AAAAAAGGTGGCCAGATGGAAGTAA- ACCAAGCTTCG (SEQ ID NO:59)
TTTACTTCCATCTGGCCACCCTTCGAACCGG- GGACC TTTCG APP: tvAPP
AAAAAATGAAGTGAAGATGGATGCAGCCAAGCTTCG (SEQ ID NO:60)
CTGCATCCATCTTCACTTCACTTCGAACCGGGGACC TTTCG tvT10/C11
AAAAAATGAAGTGAATCTGGATGCAGCCAAGCTTCG (SEQ ID NO:61)
CTGCATCCAGATTCACTTCACTTCGAACCGGGGACC TTTCG
[0315] Cell Culture and Transfections. Methods for culturing Cos-7
and HeLa cells have been described previously (Chai et al., 1999b).
Plasmids and siRNAs were transiently transfected with Lipofectamine
Plus (Invitrogen) in 12-well plates with cells plated at 70-90%
confluency. For siRNA experiments, a 5:1 ratio of siRNA to
expression plasmid was transfected into cells, while for
tRNA-valine shRNA experiments, a 10:1 ratio of shRNA plasmid to
expression plasmid was transfected into cells (Miller et al.,
2003).
[0316] Western Blot Analysis. Lysates from Cos-7 cells expressing
GFP and tau constructs were harvested 24 h after transfection,
while APP and APPsw expressing cell lysates were harvested at 48 h.
Lysates from HeLa cells expressing endogenous lamin were harvested
at 72 h after transfection of anti-lamin siRNA. Lysates were
analyzed by Western blot as reported previously (Chai et al.,
1999b). GFP and lamin were detected with anti-GFP mouse monoclonal
antibody (1:1000 dilution; Medical and Biological Laboratories,
Naka-ku Nagoya, Japan) and anti-lamin goat polyclonal antibody
(1:25 dilution; Santa Cruz Biotechnology, Santa Cruz, Calif.)
respectively. Additional antibodies used in this study include
anti-tau mouse monoclonal antibody at 1:500 dilution (Calbiochem,
San Diego, Calif.), 22C11 anti-APP mouse monoclonal antibody at
1:500 dilution (Chemicon International, Temecula, Calif.), and as a
loading control, mouse monoclonal antibody to .alpha.-tubulin at
1:20,000 dilution (Sigma, St. Louis, Mo.). Secondary antibodies
were peroxidase-conjugated donkey anti-goat or
peroxidase-conjugated donkey anti-mouse (Jackson ImmunOResearch
Laboratories, West Grove, Pa.) at 1:15,000 dilution.
[0317] Immunofluorescence. 48 hours after transfection, Cos-7 cells
were fixed with 4% paraformaldehyde/PBS. APP and APPsw expression
were detected with 22C11 at 1:1000 dilution, followed by
fluorescein (FITC)-conjugated donkey anti-mouse secondary antibody
(Jackson Labs) at 1:2,000 dilution. Nuclei were stained with 5
.mu.g/ml 4',6-diamidine-2-phenylindole HCl (DAPI) at room
temperature for 10 minutes. Fluorescence was visualized with a
Zeiss (Thornwood, N.Y.) Axioplan fluorescence microscope. All
images were captured digitally with a Zeiss MRM AxioCam camera and
assembled in Photoshop 6.0 (Adobe Systems, Mountain View,
Calif.).
[0318] Results
[0319] An approach to in vitro transcription of siRNA that
eliminates priming constraints of T7 RNA polymerase.
[0320] An efficient way to create siRNAs against a gene of interest
is to produce short RNA duplexes complementary to the target gene
in in vitro transcription reactions employing T7 RNA polymerase.
However, the priming requirements for T7 polymerase dictate that a
G be the priming nucleotide initiating transcription (Kato et al.,
2001). This limits the nucleotide positions in a target gene to
which corresponding in vitro transcribed RNA duplexes can be
generated. To overcome this restriction imposed by T7 RNA
polymerase, siRNAs were designed that contained a noncomplementary
G nucleotide at the 5' ends. The resulting siRNA contains 20
complementary nucleotides on the antisense strand with a single 5'
mismatch to the target (FIG. 16 and FIG. 17A). This incorporation
of an initiating G allows dsRNAs to be generated in vitro against
any twenty nucleotide segment of a targeted gene.
[0321] To determine whether adding this noncomplementary G still
produced effective siRNAs, the inventors compared the silencing
capability of this novel "+G" configuration to in vitro synthesized
siRNA that was perfectly complementary to the target. The inventors
assessed suppression of a reporter gene product, green fluorescent
protein (GFP), and of an endogenous gene product, lamin (FIG. 17B,
17C, 17D). Cos-7 cells were co-transfected with a plasmid encoding
GFP and siRNAs containing either a perfect match to the GFP mRNA or
the single 5' G mismatch. siRNAs containing multiple mismatches
were used as negative controls for any non-specific effects of the
transfection or siRNA. As assessed by fluorescence microscopy and
Western blot (FIGS. 17B, 17C), the 5' mismatched siRNA displayed
silencing efficiency similar to that of the perfectly matched siRNA
targeted to the same region of the GFP mRNA.
[0322] The inventors next investigated the ability of these novel
siRNAs to inhibit expression of an endogenous gene product, lamin.
The inventors transfected HeLa cells with a negative control siRNA
(siMiss) or a siRNA directed against endogenous lamin (Elbashir et
al., 2001), and assessed expression 72 hr after transfection. Lamin
expression was markedly reduced in cells transfected with
siLamin+G, but remained robust in cells transfected with siMiss+G
(FIG. 17D). Thus, "+G" siRNA remains an effective trigger of RNA
interference.
[0323] Optimizing Allele-Specific Inhibition of Mutant Tau
[0324] In a previous study of the FTDP-17 tau mutant (V337M) (see
Example 2 above), the inventors succeeded in engineering siRNA
duplexes that preferentially silenced the mutant allele (Miller et
al., 2003). Placing the mismatch near the center of the siRNA was
most effective for allele discrimination, but due to the
constraints imposed by T7 polymerase the inventors could not place
the mutation precisely at the center of the siRNA. To enhance
allele specificity in this earlier study, it was thus needed to
introduce additional mismatches into the siRNA such that it
contained two mismatches versus wild type alleles but only a single
mismatch versus the mutant tau allele (Miller et al., 2003).
Although this improved preferential suppression of the mutant
allele, recent data suggest that siRNAs with multiple internal
mismatches may act by inhibiting translation (via a microRNA-like
mechanism) rather than by cleaving the targeted mRNA (Zeng et al.,
2003; Doench et al., 2003). Accordingly, the inventors took
advantage of the new siRNA synthesis strategy in an effort to
improve allele-specific silencing with the single mismatch.
[0325] The inventors systematically tested the effect of placing
the single nucleotide mismatch at each position near the predicted
RISC cleavage site. Through this, it was hoped to identify siRNAs
that would maximize allele specificity for V337M tau. The inventors
co-transfected Cos-7 cells with flag epitope-tagged wild type tau,
GFP-tagged mutant tau (V337M) and siRNAs in which the mutation had
been placed at positions 9 through 12 of the targeted sequence.
When the mismatch was placed at position 10 (siA10), the mutant
allele was strongly suppressed (FIG. 18A). In contrast, placement
of the mismatch more toward the 5' or 3' end of the target sequence
resulted in siRNAs that poorly discriminated between alleles (FIG.
18A). It is important to note that although silencing of the mutant
allele was strongly preferred with more centrally located
mismatches, no siRNA was completely inactive against the wild type
allele. Even with the mismatch optimally placed at position 10,
some residual activity was still observed against the wild type
allele. These results support the inventors' previous work (Miller
et al., 2003; Gonzalez-Alegre et al., 2003) and results from other
laboratories (Ding et al., 2003; Abdelgany et al., 2003; Martinez
et al., 2002a) indicating that central mismatches at or near the
RISC cleavage site are best at discriminating between alleles.
However, specificity will also be determined in part by the precise
nucleotide change (Ding et al., 2003). For some mutations,
introducing additional mismatches at other sites in the siRNA may
be required to obtain optimal specificity.
[0326] Therapeutic applications of siRNA to neurodegenerative
diseases may require sustained intracellular production of siRNA.
Accordingly, the inventors next constructed and tested shRNA
expression plasmids against tau that were based on the inventors'
most effective in vitro synthesized duplexes. Expression was driven
by the tRNA-valine promoter (Kawasaki et al., 2003). The inventors
again co-transfected flag-WT-tau and V337M-GFP mutant tau together
with shRNA plasmids designed to target either wild type or mutant
tau. The tvA10 plasmid, based on the siA10 siRNA, showed strong
silencing of the mutant allele with only slight inhibition of wild
type expression. An shRNA directed against the wild type allele
silenced wild type tau expression but also produced some
suppression of the mutant allele (FIG. 18B).
[0327] Thus, multiple siRNA designs can rapidly be generated and
screened by the method described here in order to identify the best
target sequence with which to create successful shRNA expression
vectors. Once validated, these shRNAs can be incorporated into
recombinant viral vectors for in vivo testing (Miller et al., 2003;
Xia et al., 2002).
[0328] Allele-Specific Silencing of APP
[0329] Next the inventors chose to test this approach with a second
gene implicated in age-related dementia, the APP gene. Many
mutations have been identified in APP that cause early onset,
dominantly inherited AD (Alzheimer Disease Mutations Database:
http://molgen-www.uia.ac.be/ADMuta- tions/ and references therein).
The inventors sought to suppress expression of wild type APP and
the Swedish double APP mutation (K670N/M671L), or APPsw, a tandem
nucleotide missense mutation that is widely employed in mouse
models of AD (Mullan et al., 1992; Lewis et al., 2001; Oddo et al.,
2003). The inventors systematically placed the tandem mismatch at
each point in the central region of the siRNA duplexes to define
the optimal placement for allele-specific suppression. APP
silencing was assessed in Cos-7 cells cotransfected with constructs
encoding wild type APP and APPsw together with the in vitro
synthesized siRNAs. Similar to the results with tau, allelic
discrimination was conferred only when the mismatches were placed
centrally, as shown by APP immunofluorescence 48 hr after
transfection (FIG. 19A). The inventors confirmed these results by
Western blot analysis, which revealed highly specific silencing of
APPsw with siT10/C11, the siRNA in which the double mismatch is
placed immediately across from the presumed RISC cleavage site
(FIG. 19B, lanes 5-10). The corresponding wild type-specific siRNA
led to robust suppression of wild type APP (FIG. 19B, lanes
2-3).
[0330] Next, the inventors engineered plasmids expressing anti-APP
shRNAs based on our most effective in vitro duplex sequences. As
shown in FIG. 18C, shRNA designed to target the wild type sequence
silenced only wild type APP expression, whereas shRNA designed to
target APPsw specifically suppressed the mutant allele. These
results describe novel and important reagents for functional
studies of APP.
[0331] Discussion: Efficient siRNA Design for any Target
Sequence
[0332] RNAi holds promise as a potential therapy for human
diseases. Yet a limitation to successfully developing gene-specific
or allele-specific siRNAs is the selection and design of siRNAs
with the desired silencing characteristics. Individual siRNAs
targeted to different regions of a transcript often display
striking differences in efficacy and specificity (Miller et al.,
2003; Ding et al., 2003). Typically, several target sites and
designs need to be tested before optimal silencing is achieved
(Miller et al., 2003). Here the inventors have described a simple
method that not only circumvents the time and cost disadvantages of
chemically synthesizing siRNA duplexes but also removes the
sequence restrictions imposed by in vitro transcription with T7
polymerase.
[0333] The insertion of a single G mismatch at the 5' of the siRNA
duplex permitted efficient priming by T7 polymerase without
compromising the silencing efficacy of the resultant siRNA. Such
"+G" siRNAs can rapidly be generated to essentially any point in a
targeted gene and tested for efficacy. This approach to siRNA
design facilitates the in vitro generation of effective siRNAs. As
demonstrated here for two important disease targets, tau and APP,
these in vitro transcribed duplexes can then serve as guides for
producing shRNA plasmids that retain silencing capability and
allele specificity. This approach represents an improved, stepwise
method for optimized silencing of essentially any gene of
interest.
[0334] Indeed, based on new insights into RISC assembly,
manipulating the 5' terminal nucleotide of the guide strand in this
way may be highly advantageous. Schwarz et al. (Schwarz et al.,
2003) recently discovered marked asymmetry in the rate at which
each strand of an RNA duplex enters the RISC complex. Preferential
entry of the guide, or antisense, strand into RISC can be achieved
by introducing 5' mismatches in the antisense strand while
maintaining perfect base pairing at the 5' terminus of the sense
strand. This maximizes entry of the antisense strand into the RISC
complex, while also reducing potential off-target inhibition by the
sense strand. The "+G" approach to siRNA design is perfectly suited
to engineering dsRNAs based on this principle that should display
preferred RISC entry of the guide strand.
[0335] Central Placement of Mismatches are Required for Allelic
Discrimination
[0336] Using the present approach to in vitro siRNA production, the
inventors were able to systematically test the effect of placing
mismatches at each point along the guide strand of the siRNA. For
tau and APP, central placement of mismatches resulted in optimal
allele-specific silencing of mutant alleles. With the APPsw double
mutation, for example, the inventors found that placing the two
mismatches immediately across from the predicted RISC cleavage site
resulted in highly specific allele discrimination. These results
demonstrate the importance of central placement of mutations for
successful allele-specific silencing.
[0337] For tau, however, siRNAs with centrally placed mismatches
still retained some activity against the wild type allele. This
suggests that both the position of the mismatch along the guide
strand and the chemical nature of the mismatch are important for
determining whether RISC associated nucleases will cleave a given
mRNA. For example, in RNAi studies targeting a single nucleotide
change in the polyglutamine disease gene MJD1, a G-G clash between
the antisense strand of the siRNA and the target mRNA resulted in a
complete inability to silence the wild type allele while the mutant
allele was strongly suppressed (Miller et al., 2003). In contrast,
even with the tau (V337M) mutation optimally placed centrally in
the siRNA, some silencing of wild type tau was observed (Miller et
al., 2003). This suggests that the less disruptive G-U clash in the
case of the tau mutation does not allow for complete allelic
discrimination by siRNA. In such cases additional mismatches may
need to be incorporated into the siRNA.
[0338] Experimental and Therapeutic Implications
[0339] The RNAi reagents developed here against tau and APP
constitute an experimental and potential therapeutic advance for AD
and related dementias. Although abnormal deposition of tau and the
APP cleavage product A.beta. are central to AD pathogenesis, the
precise roles of these proteins in the brain remain to be
elucidated (hardy et al., 2002; Lee et al., 2001). These siRNA
reagents, which can be used to selectively silence expression of
mutant or wild type tau and APP, should facilitate loss of function
experiments aimed at identifying the neuronal functions of these
proteins.
[0340] For potential therapeutic applications of siRNA, the
inventors have established expression vectors that silence mutant
or wild type forms of tau and APP. For individuals with dominantly
inherited AD or tauopathy, selective removal of the mutant protein
might ameliorate or even prevent disease. The demonstration of
specific silencing of mutant alleles extends the potential utility
of the approach to genes with important or essential functions. For
APP, specific silencing of either the widely studied Swedish double
mutant or wild type APP was achieved. Reagents that suppress APPsw
are useful in testing RNAi therapy in mouse models of AD, and
reduction of wild type APP also has therapeutic potential for the
common, sporadic form of AD. Based on the amyloid cascade
hypothesis of AD, the most selective intervention would be a
reagent that suppresses APP protein production with minimal effects
on unintended targets (Hardy et al., 2002). A.beta. production
requires cleavage of APP by two proteases, the P site APP-cleaving
enzyme BACE and the .gamma.-secretase complex, which contains
presenilin (Sisodia et al., 2002). Thus, additional gene targets in
AD include BACE and, for most familial AD, dominantly acting
presenilin mutations.
[0341] A major challenge in applying siRNA therapy to the nervous
system is achieving sustained, effective delivery of siRNA to the
correct target cells in the brain. These data, combined with in
vivo results from other groups (Xia et al., 2002; Rubinson et al.,
2003), suggest that siRNA will effectively suppress expression of
the targeted gene, provided that it can be delivered efficiently to
the appropriate neurons. Hope is offered by the observation here
and elsewhere that sustained intracellular production of siRNA can
be achieved with expression plasmids. These plasmids retain their
silencing characteristics when incorporated into viral vectors that
are known to transduce CNS neurons (Davidson et al., 2003).
[0342] All publications, patents and patent applications are
incorporated herein by reference. While in the foregoing
specification this invention has been described in relation to
certain preferred embodiments thereof, and many details have been
set forth for purposes of illustration, it will be apparent to
those skilled in the art that the invention is susceptible to
additional embodiments and that certain of the details described
herein may be varied considerably without departing from the basic
principles of the invention.
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Sequence CWU 1
1
90 1 40 DNA Artificial Sequence A synthetic primer 1 aaggtaccag
atcttagtta ttaatagtaa tcaattacgg 40 2 43 DNA Artificial Sequence A
synthetic primer 2 gaatcgatgc atgcctcgag acggttcact aaaccagctc tgc
43 3 69 DNA Artificial Sequence A synthetic oligonucleotide used
with SEQ ID NO4 to form a minimal polyA 3 ctagaactag taataaagga
tcctttattt tcattggatc cgtgtgttgg ttttttgtgt 60 gcggccgcg 69 4 69
DNA Artificial Sequence A synthetic oligonucleotide used with SEQ
ID NO3 to form a minimal polyA 4 tcgacgcggc cgcacacaaa aaaccaacac
acggatccaa tgaaaataaa ggatccttta 60 ttactagtt 69 5 21 DNA
Artificial Sequence A synthetic P32 labeled sense oligonucleotide
used to probe a blot 5 cacaagctgg agtacaacta c 21 6 22 DNA
Artificial Sequence A synthetic P32 labeled antisense
oligonucleotide used to probe a blot 6 gtacttgtac tccagctttg tg 22
7 28 DNA Homo sapiens 7 cagcagcagc agggggacct atcaggac 28 8 28 DNA
Homo sapiens 8 cagcagcagc agcgggacct atcaggac 28 9 17 DNA
Artificial Sequence A synthetic T7 promoter sequence 9 tatagtgagt
cgtatta 17 10 18 DNA Artificial Sequence A synthetic primer
annealed to all oligos to synthesize siRNAs 10 taatacgact cactatag
18 11 22 DNA Homo sapiens 11 cggcaagctg cgcatgaagt tc 22 12 22 DNA
Homo sapiens 12 atgaacttca tgctcagctt gc 22 13 22 DNA Homo sapiens
13 atgaacttca gggtcagctt gc 22 14 22 DNA Homo sapiens 14 cggcaagctg
accctgaagt tc 22 15 22 DNA Homo sapiens 15 cagcagcggg acctatcagg ac
22 16 22 DNA Homo sapiens 16 ctgtcctgat aggtcccgct gc 22 17 20 DNA
Homo sapiens 17 cagcagcagg gggacctatc 20 18 20 DNA Homo sapiens 18
ctgataggtc cccctgctgc 20 19 22 DNA Homo sapiens 19 cagcagccgg
acctatcagg ac 22 20 22 DNA Homo sapiens 20 ctgtcctgat aggtccggct gc
22 21 20 DNA Homo sapiens 21 cagcagcagc gggacctatc 20 22 20 DNA
Homo sapiens 22 ctgataggtc ccgctgctgc 20 23 21 DNA Homo sapiens 23
ttgaaaaaca gcagcaaaag c 21 24 21 DNA Homo sapiens 24 ctgcttttgc
tgctgttttt c 21 25 22 DNA Homo sapiens 25 cagcagcagc agcagcagca gc
22 26 22 DNA Homo sapiens 26 ctgctgctgc tgctgctgct gc 22 27 22 DNA
Homo sapiens 27 tcgaagtgat ggaagatcac gc 22 28 22 DNA Homo sapiens
28 cagcgtgatc ttccatcact tc 22 29 22 DNA Homo sapiens 29 cagccgggag
tcgggaaggt gc 22 30 22 DNA Homo sapiens 30 ctgcaccttc ccgactcccg gc
22 31 24 DNA Homo sapiens 31 acgtcctcgg cggcggcagt gtgc 24 32 24
DNA Homo sapiens 32 ttgcacactg ccgcctccgc ggac 24 33 21 DNA Homo
sapiens 33 acgtctccat ggcatctcag c 21 34 21 DNA Homo sapiens 34
ttgctgagat gccatggaga c 21 35 22 DNA Homo sapiens 35 gtggccagat
ggaagtaaaa tc 22 36 22 DNA Homo sapiens 36 cagattttac ttccatctgg cc
22 37 22 DNA Homo sapiens 37 gtggccacat ggaagtaaaa tc 22 38 22 DNA
Homo sapiens 38 cagattttac ttccatgtgg cc 22 39 22 DNA Homo sapiens
39 gtggccagat gcaagtaaaa tc 22 40 22 DNA Homo sapiens 40 cagattttac
ttgcatctgg cc 22 41 22 DNA Homo sapiens 41 gtggccaggt ggaagtaaaa tc
22 42 22 DNA Homo sapiens 42 atgaacttca tgctcagctt gc 22 43 22 DNA
Homo sapiens 43 cggcaagctg agcatgaagt tc 22 44 22 DNA Homo sapiens
44 cagtggcttc tggcacagca gc 22 45 22 DNA Homo sapiens 45 aagctgctgt
gccagaagcc ac 22 46 42 DNA Homo sapiens 46 gtaagcagag tggctgagga
gatgacattt ttccccaaag ag 42 47 21 DNA Homo sapiens 47 cagagtggct
gaggagatga c 21 48 21 DNA Homo sapiens 48 gtgtcatctc ctcagccact c
21 49 18 DNA Homo sapiens 49 cagagtggct gagatgac 18 50 18 DNA Homo
sapiens 50 atgtcatctc agccactc 18 51 20 DNA Homo sapiens 51
ctgagatgac atttttcccc 20 52 20 DNA Homo sapiens 52 ttggggaaaa
atgtcatctc 20 53 23 DNA Homo sapiens 53 gagtggctga gatgacattt ttc
23 54 23 DNA Homo sapiens 54 gggaaaaatg tcatctcagc cac 23 55 39 DNA
Homo sapiens 55 gtaagcagag tggctgagat gacatttttc cccaaagag 39 56 60
DNA Artificial Sequence A synthetic primer 56 caggactagt cttttaggtc
aaaaagaaga agctttgtaa ccgttggttt ccgtagtgta 60 57 64 DNA Artificial
Sequence A synthetic primer 57 cttcgaaccg gggacctttc gcgtgttagg
cgaacgtgat aaccactaca ctacggaaac 60 caac 64 58 79 DNA Artificial
Sequence A synthetic primer 58 aaaaaagtgg ccaggtggaa gtaaaatcca
agcttcgatt ttacttccac ctggccacct 60 tcgaaccggg gacctttcg 79 59 77
DNA Artificial Sequence A synthetic primer 59 aaaaaaggtg gccagatgga
agtaaaccaa gcttcgttta cttccatctg gccacccttc 60 gaaccgggga cctttcg
77 60 77 DNA Artificial Sequence A synthetic primer 60 aaaaaatgaa
gtgaagatgg atgcagccaa gcttcgctgc atccatcttc acttcacttc 60
gaaccgggga cctttcg 77 61 77 DNA Artificial Sequence A synthetic
primer 61 aaaaaatgaa gtgaatctgg atgcagccaa gcttcgctgc atccagattc
acttcacttc 60 gaaccgggga cctttcg 77 62 18 DNA Artificial Sequence A
synthetic primer 62 ctatagtgag tcgtatta 18 63 20 DNA Artificial
Sequence A synthetic oligonucleotide 63 ggtggccaga tggaagtaaa 20 64
20 DNA Artificial Sequence A synthetic oligonucleotide 64
tgaagtgaat ctggatgcag 20 65 20 DNA Artificial Sequence A synthetic
primer 65 aacttcaccc tgagcttgcc 20 66 20 DNA Artificial Sequence A
synthetic primer 66 cggcaagctc agggtgaagt 20 67 20 DNA Artificial
Sequence A synthetic primer 67 aacttcaggg tcagcttgcc 20 68 20 DNA
Artificial Sequence A synthetic primer 68 cggcaagctg accctgaagt 20
69 20 DNA Artificial Sequence A synthetic primer 69 aactggactt
ccagaagaac 20 70 20 DNA Artificial Sequence A synthetic primer 70
tgttcttctg gaagtccagt 20 71 20 DNA Artificial Sequence A synthetic
primer 71 gtggccagat ggaagtaaaa 20 72 20 DNA Artificial Sequence A
synthetic primer 72 attttacttc catctggcca 20 73 20 DNA Artificial
Sequence A synthetic primer 73 ttttacttcc atctggccac 20 74 20 DNA
Artificial Sequence A synthetic primer 74 aggtggccag atggaagtaa 20
75 20 DNA Artificial Sequence A synthetic primer 75 tttacttcca
tctggccacc 20 76 20 DNA Artificial Sequence A synthetic primer 76
gaggtggcca gatggaagta 20 77 20 DNA Artificial Sequence A synthetic
primer 77 ttacttccat ctggccacct 20 78 23 DNA Artificial Sequence A
synthetic primer 78 aagtgaagat ggatgcagaa ttc 23 79 23 DNA
Artificial Sequence A synthetic primer 79 cggaattctg catccatctt cac
23 80 20 DNA Artificial Sequence A synthetic primer 80 tgaagtgaag
atggatgcag 20 81 20 DNA Artificial Sequence A synthetic primer 81
tctgcatcca tcttcacttc 20 82 20 DNA Artificial Sequence A synthetic
primer 82 aagtgaatct ggatgcagaa 20 83 20 DNA Artificial Sequence A
synthetic primer 83 attctgcatc cagattcact 20 84 20 DNA Artificial
Sequence A synthetic primer 84 gaagtgaatc tggatgcaga 20 85 20 DNA
Artificial Sequence A synthetic primer 85 ttctgcatcc agattcactt 20
86 20 DNA Artificial Sequence A synthetic primer 86 tctgcatcca
gattcacttc 20 87 20 DNA Artificial Sequence A synthetic primer 87
ctgaagtgaa tctggatgca 20 88 20 DNA Artificial Sequence A synthetic
primer 88 ctgcatccag attcacttca 20 89 20 DNA Artificial Sequence A
synthetic primer 89 tctgaagtga atctggatgc 20 90 20 DNA Artificial
Sequence A synthetic primer 90 tgcatccaga ttcacttcag 20
* * * * *
References