U.S. patent application number 14/709951 was filed with the patent office on 2015-12-03 for methods for treating viral infections.
This patent application is currently assigned to PTC THERAPEUTICS, INC.. The applicant listed for this patent is PTC THERAPEUTICS, INC.. Invention is credited to Nikolai A. Naryshkin, Sergey V. Paushkin, Ellen Welch.
Application Number | 20150344877 14/709951 |
Document ID | / |
Family ID | 41131843 |
Filed Date | 2015-12-03 |
United States Patent
Application |
20150344877 |
Kind Code |
A1 |
Paushkin; Sergey V. ; et
al. |
December 3, 2015 |
METHODS FOR TREATING VIRAL INFECTIONS
Abstract
The present invention relates to compounds that modulate
ribosomal frameshifting and nucleic acid constructs for use in
methods for identifying or validation such compounds. In
particular, the present invention relates to the use of nucleic
acid constructs to identify or validate compounds capable of
modulating the efficiency of programmed ribosomal frameshifting and
the use of said compounds to inhibit the replication or infectivity
of viruses that employ programmed ribosomal frameshifting.
Inventors: |
Paushkin; Sergey V.; (Belle
Mead, NJ) ; Naryshkin; Nikolai A.; (East Brunswick,
NJ) ; Welch; Ellen; (Califon, NJ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
PTC THERAPEUTICS, INC. |
South Plainfield |
NJ |
US |
|
|
Assignee: |
PTC THERAPEUTICS, INC.
South Plainfield
NJ
|
Family ID: |
41131843 |
Appl. No.: |
14/709951 |
Filed: |
May 12, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14569212 |
Dec 12, 2014 |
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14709951 |
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13058613 |
Feb 11, 2011 |
8932818 |
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PCT/US2009/004636 |
Aug 13, 2009 |
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14569212 |
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61088649 |
Aug 13, 2008 |
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61156429 |
Feb 27, 2009 |
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Current U.S.
Class: |
514/44A ;
435/238; 435/320.1; 435/8; 536/23.1 |
Current CPC
Class: |
C12N 2740/14063
20130101; C12N 2770/20063 20130101; C12N 2770/38063 20130101; C12N
2770/10063 20130101; C12N 2740/16063 20130101; C12N 2770/12063
20130101; C12N 2795/10263 20130101; C12N 2710/16663 20130101; C12N
2795/10363 20130101; A61P 43/00 20180101; A61P 31/12 20180101; C12N
2720/00063 20130101; C12Q 1/6897 20130101; C12N 2740/11063
20130101; C12Q 1/66 20130101; C12N 2740/12063 20130101; C12N 15/11
20130101; C12N 2770/00063 20130101; C12N 2740/15063 20130101; C12N
7/00 20130101; A61K 31/4035 20130101 |
International
Class: |
C12N 15/11 20060101
C12N015/11; C12N 7/00 20060101 C12N007/00; C12Q 1/66 20060101
C12Q001/66 |
Claims
1.-14. (canceled)
15. A nucleic acid construct comprising, in 5' to 3' order: (a) a
start codon; (b) a minimum of one nucleotide; (c) a fragment of the
nucleic acid residues of exon 7 of SMN, wherein the fragment of the
nucleic acid residues of exon 7 of SMN comprises a minimum of the
first six nucleotides from the 3' end of exon 7 of SMN and wherein
a single guanine residue is inserted into the fragment of the
nucleic acid residues of exon 7 of SMN at the location that
corresponds to the location in exon 7 of SMN that is after the
48.sup.th nucleotide from the 5' end of exon 7 of SMN; (d) the
nucleic acid residues of intron 7 of SMN or a fragment thereof,
wherein the fragment of the nucleic acid residues of intron 7 of
SMN comprises any number of nucleotides of intron 7 required for a
functional, minimum intron; (e) a fragment of the nucleic acid
residues of exon 8 of SMN; and (f) a reporter gene coding sequence
lacking a start codon, wherein: (i) the reporter gene coding
sequence is fused to the fragment of the nucleic acid residues of
exon 8 of SMN such that the first codon of the reporter gene coding
sequence and the first codon of the fragment are out of frame with
each other in the mRNA transcript transcribed from the nucleic acid
construct; and (ii) the production of the mRNA transcript generates
a stop codon upstream from the reporter gene coding sequence in the
region of the mRNA transcript that corresponds to the fragment of
the nucleic acid residues of exon 8 of SMN; and (iii) the first
start codon and the stop codon upstream from the reporter gene
coding sequence in the mRNA transcript are in the same contiguous
open reading frame without any interruption.
16. (canceled)
17. The nucleic acid construct of claim 15, wherein the nucleic
acid construct comprises the nucleic acid residues of exon 6 of SMN
or a fragment thereof downstream (3') to the start codon and
upstream (5') of the nucleic acid residues of exon 7 of SMN,
wherein the fragment of the nucleic acid residues of exon 6 of SMN
comprises any number of nucleotides of exon 6 of SMN so long as in
the mRNA transcript the first start codon and the stop codon
upstream of the reporter gene coding sequence are maintained in the
same contiguous open reading frame without any interruption.
18. The nucleic acid construct of claim 17, wherein the nucleic
acid construct comprises the nucleic acid residues of intron 6 of
SMN or a fragment thereof downstream (3') of the nucleic acid
residues of exon 6 of SMN or a fragment thereof and upstream (5')
of the nucleic acid residues of exon 7 of SMN, wherein the fragment
of the nucleic acid residues of intron 6 of SMN comprises any
number of nucleotides of intron 6 of SMN required for a functional,
minimum intron.
19. (canceled)
20. (canceled)
21. An mRNA transcript encoded by the nucleic acid construct of
claim 15.
22. A method for the identification or validation of a compound
that increases ribosomal frameshifting comprising: (a) contacting a
compound with either a host cell containing an mRNA transcript
encoded by the nucleic acid construct of claim 15, or a composition
comprising a cell-free extract and an mRNA transcript transcribed
from a nucleic acid construct of claim 15; and (b) detecting the
activity or amount of a fusion protein translated from the mRNA
transcript, wherein an increase in the activity or amount of the
fusion protein translated from the mRNA transcript in the presence
of a compound when compared to (i) a previously determined
reference range for a negative control, (ii) the activity or amount
of the fusion protein translated from the mRNA transcript in the
absence of the compound, or (iii) the activity or amount of the
fusion protein translated from the mRNA transcript in the presence
of a negative control indicates that the compound modulates
ribosomal frameshifting.
23. A method for the identification or validation of a compound
that modulates the efficiency of programmed ribosomal frameshifting
comprising: (a) contacting a compound with either a host cell
containing an mRNA transcript encoded by the nucleic acid construct
of claim 15, or a composition comprising a cell-free extract and an
mRNA transcript transcribed from a nucleic acid construct of claim
15; and (b) detecting the activity or amount of a fusion protein
translated from the mRNA transcript, wherein an increase in the
activity or amount of the fusion protein translated from the mRNA
transcript in the presence of a compound when compared to (i) a
previously determined reference range for a negative control, (ii)
the activity or amount of the fusion protein translated from the
mRNA transcript in the absence of the compound, or (iii) the
activity or amount of the fusion protein translated from the mRNA
transcript in the presence of a negative control indicates that the
compound modulates the efficiency of programmed ribosomal
frameshifting.
24. A method for reducing or inhibiting a viral infection,
comprising contacting a cell containing a virus or provirus that
employs programmed ribosomal frameshifting with a compound, wherein
the compound in vitro or in cells increases the amount or activity
of a fusion protein encoded by the nucleic acid construct of claim
15 or translated from a RNA transcript transcribed from the nucleic
acid construct of claim 15.
25. A method for reducing or inhibiting a viral infection,
comprising contacting a compound with a mixture of a cell or a
population of cells and a virus that employs programmed ribosomal
frameshifting, wherein the compound in vitro or in cells increases
the amount or activity of a fusion protein encoded by the nucleic
acid construct of claim 15 or translated from a RNA transcript
transcribed from the nucleic acid construct of claim 15.
26. A method for inhibiting or reducing viral replication or
infectivity in a subject, comprising administering to a subject in
need thereof an effective amount of a compound or pharmaceutical
composition thereof, wherein the compound modulates the efficiency
of programmed ribosomal frameshifting as measured in vitro or in
cells by an increase in the amount or activity of a fusion protein
encoded by the nucleic acid construct of claim 15 or translated
from a RNA transcript transcribed from the nucleic acid construct
of claim 15.
27. A method for reducing viral titers in a subject, comprising
administering to a subject in need thereof an effective amount of a
compound or pharmaceutical composition thereof, wherein the
compound modulates the efficiency of programmed ribosomal
frameshifting as measured in vitro or in cells by an increase in
the amount or activity of a fusion protein encoded by the nucleic
acid construct of claim 15 or translated from a RNA transcript
transcribed from the nucleic acid construct of claim 15.
28. A method for treating a viral infection in a subject,
comprising administering to a subject in need thereof an effective
amount of a compound or pharmaceutical composition thereof, wherein
the compound modulates the efficiency of programmed ribosomal
frameshifting as measured in vitro or in cells by an increase in
the amount or activity of a fusion protein encoded by the nucleic
acid construct of claim 15 or translated from a RNA transcript
transcribed from the nucleic acid construct of claim 15.
29. A method for preventing or treating a viral disease in a
subject, comprising administering to a subject in need thereof an
effective amount of a compound or pharmaceutical composition
thereof, wherein the compound modulates the efficiency of
programmed ribosomal frameshifting as measured in vitro or in cells
by an increase in the amount or activity of a fusion protein
encoded by the nucleic acid construct of claim 15 or translated
from a RNA transcript transcribed from the nucleic acid construct
of claim 15.
30. The method of claim 23, wherein the compound is selected from
compounds of Formula (I) or a form thereof or Formula (II) or a
form thereof, wherein Formula (I) and Formula (II) have the
following structures: ##STR00004## wherein, W is selected from the
group consisting of C(O), C(S), and CH.sub.2; B is CH.sub.2 or
CH(C.sub.nH.sub.2n+1), wherein n is an integer from 1 to 8; Ring C
is selected from the group consisting of a fused thienyl ring, a
fused pyridinyl ring, and a fused cyclohexyl ring, any of which can
be saturated or contain, one or two non-conjugated double bonds;
R.sub.1 and R.sub.2 are independently selected from the group
consisting of H and C.sub.1-C.sub.3 alkyl, or R.sub.1 and R.sub.2
may be taken together with the carbon atom to which they are
attached to form a C.sub.3-C.sub.6 cycloalkyl ring or a carbonyl
group; R.sub.3 is selected from the group consisting of H, halogen,
C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 alkoxy, C.sub.1-C.sub.4
haloalkyl, CN, NO.sub.2, heteroaryl, and phenyl optionally
substituted with any combination of one to five halogen, NO.sub.2,
CN, C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 haloalkyl, or
C.sub.1-C.sub.4 alkoxy substituents; R.sub.4, R.sub.5, R.sub.6 and
R.sub.7 are independently selected from the group consisting of H,
hydroxyl, halogen, CN, NO.sub.2, sulfonamide, C.sub.1-C.sub.8
alkyl, C.sub.3-C.sub.6 cycloalkyl, cycloalkyloxy, C.sub.1-C.sub.6
alkoxy, C.sub.1-C.sub.6 haloalkoxy, C.sub.1-C.sub.4 haloalkyl,
C.sub.2-C.sub.8 alkenyl, amino, C.sub.1-C.sub.4 alkylamino,
C.sub.1-C.sub.4 dialkylamino, C.sub.3-C.sub.6 cycloalkylamino,
morpholinyl, heteroaryl, arylamino, arylalkylamino, phenyl, C(O)R',
NR'(COR''), NR'SO.sub.2R'' and NR'(CONR''R'''), wherein R', R'' and
R''' are independently H, C.sub.1-C.sub.6 alkyl, phenyl, or
substituted phenyl, and wherein C.sub.1-C.sub.8 alkyl is optionally
substituted with one or more substituents selected from the group
consisting of C.sub.1-C.sub.4 alkoxy, C.sub.1-C.sub.4 haloalkyl,
C.sub.1-C.sub.6 dialkylamino, C.sub.1-C.sub.6 alkylamino,
cycloalkylamino, and morpholinyl, and the phenyl is optionally
substituted with one or more substituents selected from the group
consisting of halogen, NO.sub.2, CN, C.sub.1-C.sub.4 alkyl,
C.sub.1-C.sub.4 haloalkyl, and C.sub.1-C.sub.4 alkoxy, or R.sub.4
and R.sub.5, R.sub.5 and R.sub.6, or R.sub.6 and R.sub.7, taken
together with the carbon to which they are attached, form a ring; X
is selected from the group consisting of H; CN; C(O)OR.sub.8,
wherein R.sub.8 is H or C.sub.1-C.sub.8 alkyl, and C.sub.1-C.sub.8
alkyl optionally is substituted with one or more substituents
selected from the group consisting of C.sub.1-C.sub.4 alkoxy,
C.sub.1-C.sub.4 haloalkyl, C.sub.1-C.sub.6 dialkylamino,
C.sub.1-C.sub.6 alkylamino, cycloalkylamino, phenyl, and
morpholinyl; C(O)NR.sub.9R.sub.10 or CH.sub.2NR.sub.9R.sub.10,
wherein R.sub.9 and R.sub.10 are independently selected from the
group consisting of H and C.sub.1-C.sub.6 alkyl, or R.sub.9 and
R.sub.10 together with the nitrogen to which they are attached form
a heterocyclyl ring; CH.sub.2OR.sub.11, wherein R.sub.11 is H,
C.sub.1-C.sub.8 alkyl, or C.sub.3-C.sub.6 cycloalkyl, wherein
C.sub.1-C.sub.8 alkyl is optionally substituted with one or more
substituents selected from the group consisting of C.sub.1-C.sub.4
alkoxy, C.sub.1-C.sub.4 haloalkyl, C.sub.1-C.sub.6 dialkylamino,
C.sub.1-C.sub.6 alkylamino, cycloalkylamino, and morpholinyl;
CH.sub.2Z, wherein Z is halogen; C(O)NHOH; C(O)NHCN;
C(O)N(R.sub.1)SO.sub.2R.sub.13, wherein R.sub.13 is C.sub.1-C.sub.4
alkyl, phenyl, or substituted phenyl; C.sub.1-C.sub.8 alkyl,
optionally substituted with one or more substituents selected from
the group consisting of C.sub.1-C.sub.4 alkoxy, C.sub.1-C.sub.4
haloalkyl, C.sub.1-C.sub.6 dialkylamino, and C.sub.1-C.sub.6
alkylamino; and C.sub.2-C.sub.8 alkenyl, optionally substituted
with one or more substituents selected from the group consisting of
C.sub.1-C.sub.4 alkoxy, C.sub.1-C.sub.4 haloalkyl, C.sub.1-C.sub.6
dialkylamino, and C.sub.1-C.sub.6 alkylamino.
31. The method of claim 23, wherein the compound is selected from
compounds of Formula (Ia) or a form thereof or Formula (IIa) or a
form thereof, wherein Formula (Ia) and Formula (IIa) have the
following structures: ##STR00005## wherein, W.sub.1 is selected
from the group consisting of C(O), C(S), and CH.sub.2; B.sub.1 is
CH.sub.2 or CH(C.sub.mH.sub.2m+1), wherein m is an integer from 1
to 8; Ring C.sub.1 is selected from the group consisting of a
thienyl ring, a pyridinyl ring, a cyclohexyl ring, a
benzo[d][1,3]dioxolyl ring and a 2,3-dihydrobenzo[b][1,4]dioxinyl
ring, each of said rings fused to the moiety of Formula (IIa),
wherein benzo[d][1,3]dioxolyl and 2,3-dihydrobenzo[b][1,4]dioxinyl,
each having a benzo ring portion, are fused via said benzoportion,
and wherein any of the foregoing rings may optionally be fully or
partially saturated; R.sub.20 and R.sub.21 are independently
selected from the group consisting of H and C.sub.1-C.sub.3 alkyl,
or R.sub.20 and R.sub.21 may be taken together with the carbon atom
to which they are attached to form a C.sub.3-C.sub.6 cycloalkyl
ring or a carbonyl group; R.sub.22 is selected from the group
consisting of H, halogen, C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4
alkoxy, C.sub.1-C.sub.4 haloalkyl, cyano, nitro, heteroaryl, and
phenyl optionally substituted with any combination of one to five
halogen, nitro, cyano, C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4
haloalkyl or C.sub.1-C.sub.4 alkoxy substituents; R.sub.23,
R.sub.24, R.sub.25 and R.sub.26 are independently selected from the
group consisting of H, hydroxyl, halogen, cyano, nitro,
sulfonamide, C.sub.1-C.sub.8 alkyl, C.sub.1-C.sub.6 alkoxy,
C.sub.1-C.sub.6 alkoxyalkoxy, C.sub.1-C.sub.6 alkoxyalkyl,
C.sub.1-C.sub.6 haloalkoxy, C.sub.1-C.sub.4 haloalkyl,
C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.4 haloalkenyl, formyl,
C.sub.1-C.sub.6 alkylcarbonyl, amino, C.sub.1-C.sub.4 alkylamino,
C.sub.1-C.sub.4 dialkylamino, C.sub.1-C.sub.4 aminoalkyl,
C.sub.1-C.sub.4 alkylaminoalkyl, C.sub.1-C.sub.4 dialkylaminoalkyl,
phenyl, C.sub.3-C.sub.6 cycloalkyl, C.sub.3-C.sub.6
cycloalkylalkyl, C.sub.3-C.sub.6 cycloalkylalkoxy, cycloalkyloxy,
heterocyclyl, heterocyclylalkyl, heteroaryl, and phenylcarbonyl,
wherein amino is optionally disubstituted with one substituent
selected from hydrogen, C.sub.1-C.sub.6 alkyl or phenyl and the
other is selected from formyl, phenyl, C.sub.3-C.sub.6 cycloalkyl,
C.sub.1-C.sub.6 alkylcarbonyl, aminocarbonyl, C.sub.1-C.sub.6
alkylaminocarbonyl, C.sub.1-C.sub.6 dialkylaminocarbonyl,
phenylcarbonyl, phenylaminocarbonyl, N-phenyl-N--C.sub.1-C.sub.6
alkyl-aminocarbonyl, C.sub.1-C.sub.6 alkylsulfonyl, aminosulfonyl,
C.sub.1-C.sub.6 alkylaminosulfonyl, C.sub.1-C.sub.6
dialkylaminosulfonyl or phenylsulfonyl, wherein each instance of
C.sub.1-C.sub.6 alkylcarbonyl is optionally substituted on the
alkyl portion with one or more substituents selected from the group
consisting of halogen, C.sub.1-C.sub.4 alkoxy, C.sub.1-C.sub.6
alkylamino, C.sub.1-C.sub.6 dialkylamino, cycloalkylamino and
heterocyclyl, wherein each instance of phenyl is optionally
substituted with one or more substituents selected from the group
consisting of halogen, nitro, cyano, C.sub.1-C.sub.4 alkyl,
C.sub.1-C.sub.4 haloalkyl and C.sub.1-C.sub.4 alkoxy, and
alternatively, R.sub.23 and R.sub.24, R.sub.24 and R.sub.25 or
R.sub.25 and R.sub.26 may be taken together with the carbon to
which they are attached to form a C.sub.3-C.sub.6 cycloalkyl ring
fused to the moiety of Formula (Ia); X.sub.1 is absent or is
selected from the group consisting of H, cyano, C.sub.1-C.sub.8
alkyl, C.sub.1-C.sub.4 alkoxy, amino, C.sub.1-C.sub.4 alkylamino,
C.sub.1-C.sub.4 dialkylamino, carboxy, C.sub.1-C.sub.8
alkoxycarbonyl, aminocarbonyl, C.sub.1-C.sub.8 alkylaminocarbonyl,
C.sub.1-C.sub.8 dialkylaminocarbonyl, hydroxylaminocarbonyl,
cyanoaminocarbonyl, phenylaminocarbonyl,
aminosulfonylaminocarbonyl, C.sub.1-C.sub.8
alkylaminosulfonylaminocarbonyl, C.sub.1-C.sub.8
dialkylaminosulfonylaminocarbonyl, C.sub.1-C.sub.8
alkylsulfonylaminocarbonyl, phenylsulfonylaminocarbonyl and
heterocyclylcarbonyl, wherein C.sub.1-C.sub.4 alkoxy and the
C.sub.1-C.sub.8 alkoxy portion of C.sub.1-C.sub.8 alkoxycarbonyl is
optionally substituted with one or more substituents selected from
the group consisting of halogen, C.sub.1-C.sub.4 alkoxy,
C.sub.1-C.sub.4 haloalkyl, amino, C.sub.1-C.sub.6 alkylamino,
C.sub.1-C.sub.6 dialkylamino, cycloalkylamino, phenyl and
heterocyclyl, wherein C.sub.1-C.sub.8 alkyl is optionally
substituted with one or more substituents selected from the group
consisting of halogen, hydroxyl, C.sub.1-C.sub.4 haloalkyl,
C.sub.2-C.sub.8 alkenyl, C.sub.1-C.sub.6 alkoxy, C.sub.1-C.sub.4
alkoxyalkoxy, C.sub.3-C.sub.6 cycloalkyloxy, amino, C.sub.1-C.sub.6
alkylamino, C.sub.1-C.sub.6 dialkylamino, cycloalkylamino,
aminocarbonyl, C.sub.1-C.sub.6 alkylaminocarbonyl, C.sub.1-C.sub.6
dialkylaminocarbonyl, hydroxylaminocarbonyl, cyanoaminocarbonyl,
C.sub.1-C.sub.6 alkylsulfonylaminocarbonyl,
phenylsulfonylaminocarbonyl and heterocyclyl, wherein
C.sub.1-C.sub.4 alkoxy or C.sub.2-C.sub.8 alkenyl are each further
optionally substituted with one or more substituents selected from
the group consisting of C.sub.1-C.sub.4 alkoxy, C.sub.1-C.sub.4
haloalkyl, amino, C.sub.1-C.sub.6 alkylamino and C.sub.1-C.sub.6
dialkylamino.
32. The method of claim 31, wherein m is an integer selected from
1, 2 or 3; and, wherein Ring C.sub.1 is selected from the group
consisting of a thienyl ring, a pyridinyl ring, a cyclohexyl ring,
a cyclohexenyl ring, a cyclohexa-1,4-dienyl ring, a
benzo[d][1,3]dioxolyl ring and a 2,3-dihydrobenzo[b][1,4]dioxinyl
ring, each of said rings fused to the moiety of Formula (IIa),
wherein benzo[d][1,3]dioxolyl and 2,3-dihydrobenzo[b][1,4]dioxinyl,
each having a benzo ring portion, are fused via said
benzoportion.
33. The method of claim 31, wherein R.sub.20 and R.sub.21 are each
H; alternatively, R.sub.20 and R.sub.21 are each C.sub.1-C.sub.3
alkyl; or wherein R.sub.22 is selected from the group consisting of
H, halogen, C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 alkoxy,
C.sub.1-C.sub.4 haloalkyl, cyano, thienyl, furanyl, pyridinyl,
pyrimidinyl and phenyl, wherein phenyl is optionally substituted
with one or two halogen, C.sub.1-C.sub.4 alkyl or C.sub.1-C.sub.4
alkoxy substituents.
34. (canceled)
35. The method of claim 31, wherein, when one, two or three of
R.sub.23, R.sub.24, R.sub.25 and R.sub.26 are each H, then three,
two or one of R.sub.23, R.sub.24, R.sub.25 and R.sub.26,
respectively, are each selected from hydroxyl, halogen, cyano,
nitro, C.sub.1-C.sub.8 alkyl, C.sub.1-C.sub.6 alkoxy,
C.sub.1-C.sub.6 alkoxyalkoxy, C.sub.1-C.sub.6 alkoxyalkyl,
C.sub.1-C.sub.6 difluoroalkoxy, C.sub.1-C.sub.6 trifluoroalkoxy,
C.sub.1-C.sub.4 trifluoroalkyl, C.sub.2-C.sub.6 alkenyl,
C.sub.2-C.sub.4 trifluoroalkenyl, amino, C.sub.1-C.sub.4
alkylamino, C.sub.1-C.sub.4 dialkylamino, C.sub.1-C.sub.4
aminoalkyl, C.sub.1-C.sub.4 alkylaminoalkyl or C.sub.1-C.sub.4
dialkylaminoalkyl; or wherein when three of R.sub.23, R.sub.24,
R.sub.25 and R.sub.26 are each H, then one of R.sub.23, R.sub.24,
R.sub.25 and R.sub.26 is selected from phenyl, cyclopentyl,
cyclopropyl, benzyloxy, C.sub.1-C.sub.4 cyclopentylalkoxy,
C.sub.1-C.sub.4 cyclobutylalkoxy, cyclopentyloxy, pyrrolidinyl,
piperidinyl, morpholinyl, C.sub.1-C.sub.4 morpholinylalkyl,
thienyl, pyridinyl, pyrimidinyl, or amino, wherein amino is
optionally disubstituted with one substituent selected from
hydrogen or C.sub.1-C.sub.6 alkyl and the other is selected from
phenyl, C.sub.1-C.sub.4 alkylcarbonyl, aminocarbonyl,
C.sub.1-C.sub.4 alkylaminocarbonyl, C.sub.1-C.sub.4
dialkylaminocarbonyl, phenylcarbonyl, phenylaminocarbonyl,
N-phenyl-N--C.sub.1-C.sub.4 alkyl-aminocarbonyl, C.sub.1-C.sub.6
alkylsulfonyl or phenylsulfonyl, and wherein each instance of
phenyl is optionally substituted with one or two substituents
selected from halogen, C.sub.1-C.sub.4 alkyl or C.sub.1-C.sub.4
alkoxy.
36. (canceled)
37. The method of claim 31, wherein X.sub.1 is absent or is
selected from the group consisting of H, cyano, C.sub.1-C.sub.4
alkyl, C.sub.1-C.sub.4 alkoxy, C.sub.1-C.sub.4 haloalkyl,
C.sub.1-C.sub.6 hydroxylalkyl, C.sub.1-C.sub.6 alkoxyalkyl,
C.sub.1-C.sub.4 morpholinylalkyl, amino, C.sub.1-C.sub.4
alkylamino, C.sub.1-C.sub.4 dialkylamino, C.sub.1-C.sub.4
aminoalkyl, C.sub.1-C.sub.4 alkylaminoalkyl, C.sub.1-C.sub.4
dialkylaminoalkyl, carboxy, C.sub.1-C.sub.6 alkoxycarbonyl,
benzyloxycarbonyl, aminocarbonyl, C.sub.1-C.sub.8
alkylaminocarbonyl, C.sub.1-C.sub.8 dialkylaminocarbonyl,
hydroxylaminocarbonyl, cyanoaminocarbonyl, phenylaminocarbonyl,
aminosulfonylaminocarbonyl, C.sub.1-C.sub.8
alkylaminosulfonylaminocarbonyl C.sub.1-C.sub.8 dialkylamino
sulfonylaminocarbonyl, C.sub.1-C.sub.8 alkylsulfonylaminocarbonyl,
phenylsulfonylaminocarbonyl, morpholinylcarbonyl and
piperidinylcarbonyl.
38. The method of claim 30, wherein the compound is
2-(4-isopropylphenyl)-6-methoxyisoindolin-1-one.
39. The method of claim 23, wherein the virus is human
immunodeficiency virus type I, human immunodeficiency virus type
II, feline immunodeficiency virus, rous sarcoma virus, mouse
mammary tumor virus, simian retrovirus type 1, human T cell
leukemia virus type I, human T cell leukemia virus type II,
infectious bronchitis virus, human coronavirus, transmissible
gastroenteritis virus, berne virus, equine arteritis virus, human
astrovirus serotype-1, Giardia lamblia virus, Saccharomyces
cerevisiae dsRNA virus L-A, S. cerevisiae dsRNA virus L1,
bacteriophage T7, bacteriophage lambda, barley yellow dwarf virus,
beet western yellows virus, potato leaf roll virus, severe acute
respiratory syndrome coronavirus, herpes simplex virus, or red
clover necrotic mosaic virus.
40. (canceled)
Description
[0001] This application is a continuation of U.S. patent
application Ser. No. 14/569,212, filed Dec. 12, 2014, which is a
divisional of U.S. patent application Ser. No. 13/058,613, filed
Feb. 11, 2011, now U.S. Pat. No. 8,932,818, which is a national
stage application of International Application No.
PCT/US2009/004636, filed Aug. 13, 2009, which claims priority
benefit of U.S. Provisional Application No. 61/088,649, filed Aug.
13, 2008, and U.S. Provisional Application No. 61/156,429, filed
Feb. 27, 2009, each of which is incorporated herein by reference in
its entirety.
INTRODUCTION
[0002] The present invention relates to compounds that modulate
ribosomal frameshifting and nucleic acid constructs for use in
methods for identifying or validation such compounds. In
particular, the present invention relates to the use of nucleic
acid constructs to identify or validate compounds capable of
modulating the efficiency of programmed ribosomal frameshifting and
the use of said compounds to inhibit the replication or infectivity
of viruses that employ programmed ribosomal frameshifting.
BACKGROUND
[0003] Programmed ribosomal frameshifting is a specific mode of
gene regulation designed to increase the informational content of
small and limited viral genomes. Programmed ribosomal frameshifting
in all viruses occurs by the same mechanism (Brierley, 1995, J.
Gen. Virol., 76:1885-1892; Farabaugh, 1997, "Programmed alternative
reading of the genetic code," R.G. Landes Company, Austin, Tex.;
Jacks, 1990, Curr. Top. Microbiol. Immunol. 157:93-124). Two basic
RNA elements, a slippery site and downstream stem-loop structure,
have been identified which are generally required to generate and
regulate -1 ribosomal frameshifting (Brierley, 1995, J. Gen.
Virol., 76:1885-1892; Farabaugh, 1997, "Programmed alternative
reading of the genetic code," R.G. Landes Company, Austin, Tex.;
Bekaert, et al., 2003, Bioinformatics, 19:327-335; Dulude, et al.,
2002, Nucleic Acids Res., 30:5094-5102; Gaudin, et al., 2005, J.
Mol. Biol., 349:1024-1035; Staple, et al., 2005, J. Mol. Biol.
349:1011-1023). The slippery site consists of a stretch of seven
nucleotides that do not have a uniform sequence but span three
amino acid codons and must conform to the sequence X XXY YYZ (the
gag ORF is indicated by spaces) where X is any nucleotide, Y is an
A or U, and Z is A, U, or C (Brierley, et al., 1992, J. Mol. Biol.
227:463-479; Dinman, et al., 1991, Proc. Natl. Acad. Sci. USA,
88:174-178; Dinman, et al., 1992, J. Virol. 66:3669-3676; Jacks, et
al., 1988, Cell, 55:447-458). Earlier studies indicate that the
downstream sequence forms a pseudoknot (Brierley, 1995, J. Gen.
Virol., 76:1885-1892; Farabaugh, 1997, "Programmed alternative
reading of the genetic code," R.G. Landes Company, Austin, Tex.;
Bekaert, et al., 2003, Bioinformatics, 19:327-335). However, recent
data indicate that the downstream sequence forms a stem-loop
structure (Dulude, et al., 2002, Nucleic Acids Res., 30:5094-5102;
Gaudin, et al., 2005, J. Mol. Biol., 349:1024-1035; Staple, et al.,
2005, J. Mol. Biol. 349:1011-1023). The stem-loop is a sequence
that forms a defined RNA secondary structure and is thought to
regulate production of the Gag-Pol polyprotein (Brierley, et al.,
1989, Cell, 57:537-547; Dinman, et al., 1991, Proc. Natl. Acad.
Sci. USA, 88:174-178; Dulude, et al., 2002, Nucleic Acids Res.,
30:5094-5102; Morikawa, et al., 1992, Virology, 186:389-397; Plant,
et al., 2003, RNA, 9:168-174; Somogyi, et al., 1993, Mol. Cell.
Biol. 13:6931-6940; Tu, et al., 1992, Proc. Natl. Acad. Sci. USA,
89:8636-8640). The importance of the stem loop in programmed
ribosomal frameshifting is evidenced by the fact that human
immunodeficiency virus-1 (HIV-1) genomes with mutations in the stem
loop have shown a reduction in frameshift activity and have been
found to be profoundly defective in viral replication (Telenti, et
al., 2002, J. Virol. 76:7868-7873).
[0004] The rate of programmed ribosomal frameshifting is strictly
regulated. Small changes in either or both the slippery site and
stem-loop have been shown to have profound effects on the
efficiency of ribosomal frameshifting (Baril, et al., 2003, RNA,
9:1246-1253; Brierley, 1995, J. Gen. Virol., 76:1885-1892; Dinman,
1995, Yeast, 11:1115-1127; Farabaugh, 1997, "Programmed alternative
reading of the genetic code," R.G. Landes Company, Austin, Tex.).
The sequences of the RNA elements may affect the secondary
structure and thermodynamic stability of the stem-loop.
Furthermore, these sequences can affect the relative position of
the RNA stem-loop in relation to the slippery site, in turn
affecting the ability of the ribosome-bound tRNAs to unpair from
the 0-frame codon, and thus the ability of these tRNAs to then pair
with the -1 frame codon (Brierley, et al., 1989, Cell, 57:537-547;
Brierley, et al., 1992, J. Mol. Biol. 227:463-479; Brierley, et
al., 1991, J. Mol. Biol. 220:889-902; Dinman, et al., 1991, Proc.
Natl. Acad. Sci. USA, 88:174-178; Dinman, et al., 1992, J. Virol.
66:3669-3676; Honda, et al., 1995, Biochem. Biophys. Res. Commun.
213:575-582; Jacks, et al., 1988, Cell, 55:447-458; Jacks, et al.,
1988, Nature, 331:280-283; Morikawa, et al., 1992, Virology,
186:389-397; Namy, et al., 2006, Nature, 441:244-247).
[0005] Several viruses have been shown to utilize -1 programmed
ribosomal frameshifting to produce the Gag-Pol polyprotein.
Frameshifting ensures that the correct ratio of viral structural
proteins (Gag) to non-structural proteins (Pol) is maintained in
the cytoplasm (Brierley, 1995, J. Gen. Virol., 76:1885-1892). The
maintenance of a precise ratio of Gag to Gag-Pol synthesis for
viral propagation has been demonstrated for a number of
retroviruses as well as for endogenous viruses of the yeast
Saccharomyces cerevisiae (Dinman, et al., 1998, Trends Biotech.
16(4):3669-3676; Telenti, et al., 2002, J. Virol. 76:7868-7873).
The ratio of HIV-1 Gag to Gag-Pol synthesized as a consequence of
-1 programmed ribosomal frameshifting varies within a narrow window
of approximately 10:1 to 20:1 (Farabaugh, 1997, "Programmed
alternative reading of the genetic code," R.G. Landes Company,
Austin, Tex.; Jacks, et al., 1988, Nature, 331:280-283; Telenti, et
al., 2002, J. Virol. 76:7868-7873). Changing the ratio of Gag to
Gag-Pol proteins in retroviruses like HIV-1 or Moloney murine
leukemia virus interferes with particle formation and replication
(Farabaugh, 1997, "Programmed alternative reading of the genetic
code," R.G. Landes Company, Austin, Tex.; Felsenstein, et al.,
1988, J. Virol. 62(6):2179-2182; Karacostas, et al., 1993,
Virology, 193:661-671; Park, et al., 1991, J.
Virol.65(9):5111-5117). For example, overexpression of the Gag-Pol
precursor protein results in the inefficient processing of the
polyprotein and consequently inhibition of virus production. It has
been shown that maintaining the proper ratio of Gag to Gag-Pol is
necessary for proteolytic processing as well as for RNA
dimerization (essential for the packaging of the viral RNA genome)
and viral infectivity (Hill, et al., 2002, J. Virol.
76:11245-11253; Shehu-Xhilaga, et al., 2001, J. Virol.
75(4):1834-1841).
[0006] Clearly, alterations in programmed ribosomal frameshifting
efficiencies can have a pronounced negative effect on viral
production. Thus, ribosomal frameshifting is a compelling,
unexploited target for novel antiviral agents (Dinman, et al.,
1998, Trends Biotechnol. 16:190-196).
[0007] Accordingly, the present invention provides compounds
capable of modulating the efficiency of programmed ribosomal
frameshifting, methods by which such compounds capable of
modulating the efficiency of viral programmed ribosomal
frameshifting may be identified or validated and methods for
treating viral infections using such compounds.
SUMMARY OF THE INVENTION
[0008] The present invention relates to compounds that modulate
ribosomal frameshifting and nucleic acid constructs for use in
methods for identifying or validation such compounds. In
particular, the present invention relates to antiviral compounds
capable of modulating the efficiency of viral programmed ribosomal
frameshifting, nucleic acid constructs for use in methods for
identifying or validating such compounds and methods for treating
viral infections using such compounds.
[0009] The invention is based, in part, on the Applicants'
discovery that a cryptic splice site is created by a single base
change when a guanine nucleotide is inserted after nucleic acid
residue 48 of exon 7 of SMN (Survival Motor Neuron) in a nucleic
acid construct comprising, in 5' to 3' order: (i) the nucleic acid
residues of exon 6 of SMN; (ii) the nucleic acid residues of intron
6 of SMN; (iii) the nucleic acid residues of exon 7 of SMN, wherein
a single guanine is inserted after the 48th nucleotide residue from
the 5' end of exon 7 of SMN (i.e., before the 6th nucleotide from
the 3' end of exon 7 of SMN); (iv) the nucleic acid residues of
intron 7 of SMN; (v) a fragment of the nucleic acid residues of
exon 8 of SMN, wherein the fragment is composed of the first 23
nucleotides from the 5' end of exon 8 of SMN; and (vi) a reporter
gene coding sequence lacking a start codon, wherein the reporter
gene coding sequence is fused to the fragment of the nucleic acid
residues of exon 8 of SMN such that the first codon of the reporter
gene coding sequence and the first codon of the fragment are out of
frame with each other in the mRNA transcript transcribed from the
nucleic acid construct, and wherein the production of the mRNA
transcript generates a stop codon in the region of the mRNA
transcript that corresponds to the fragment of the nucleic acid
residues of exon 8 of SMN.
[0010] The cryptic splice site results in a deletion of the last
seven nucleotides of exon 7 and creates a spliced mRNA in which (i)
the open reading frame defined by the first start codon on the SMN
open reading frame is frameshifted relative to the open reading
frame of the reporter gene and (ii) the open reading frame defined
by the first start codon in the SMN open reading frame contains an
aberrant stop codon upstream from the reporter gene coding
sequence. Without being limited by theory, the presence of the
aberrant stop codon generated by the 5' cryptic splice site,
possibly, but not necessarily, in combination with a secondary
structure of the downstream RNA, may cause the ribosome to pause
and thus affect the efficiency of programmed ribosomal
frameshifting. The Applicants have also demonstrated that a
compound identified using a similar nucleic acid construct (i.e.,
the SMN2-linked reporter gene construct described in Zhang, et al.,
2001, Gene Therapy, 8:1532-1538) modulates the efficiency of
programmed ribosomal frameshifting. Thus, the nucleic acid
constructs of the present invention may be used to identify or
validate compounds that modulate the efficiency of programmed
ribosomal frameshifting, and which may be of therapeutic benefit
for treating viral infections.
[0011] Certain nucleic acid constructs described in the present
invention for use in methods for identifying or validating
compounds that modulate the efficiency of viral programmed
ribosomal frameshifting have been disclosed in co-pending U.S.
patent application Ser. No. 12/473,116, filed May 27, 2009.
[0012] In an aspect of the invention, the present invention
provides nucleic acid constructs for use in cell-based and
cell-free screening assays for the identification or validation of
compounds that modulate (e.g., increases/causes or decreases)
ribosomal frameshifting. In another aspect of the invention, the
invention provides nucleic acid constructs for use in cell-based
and cell-free screening assays for the identification or validation
of compounds that modulate the efficiency of programmed ribosomal
frameshifting. In one embodiment, a nucleic acid construct
comprises, in 5' to 3' order: (i) a fragment of the nucleic acid
residues of exon 8 of SMN; and (ii) a reporter gene coding sequence
lacking a start codon, wherein the reporter gene coding sequence is
fused to the fragment of the nucleic acid residues of exon 8 of SMN
such that the open reading frames of the reporter gene coding
sequence and the fragment are out of frame with each other in the
mRNA transcript transcribed from the nucleic acid construct and a
stop codon is upstream of the reporter gene in the mRNA transcript.
In certain embodiments, the fragment of the nucleic acid residues
of exon 8 of SMN consists of the first 3, 5, 7, or 9 nucleotides
from the 5' end of exon 8 of SMN. In other embodiments, the
fragment of the nucleic acid residues of exon 8 of SMN consists of
the first 11, 13, 15, 17, or 19 nucleotides from the 5' end of exon
8 of SMN. In a specific embodiment, the fragment of the nucleic
acid residues of exon 8 of SMN consists of the first 21 or 23
nucleotides from the 5' end of exon 8 of SMN. In certain
embodiments, the nucleic acid construct comprises a start codon
upstream (5') to the fragment of the nucleic acid residues of exon
8 of SMN. In accordance with such embodiments, the first start
codon and the stop codon upstream of the reporter gene coding
sequence are in the same contiguous open reading frame without any
interruption by, e.g., a stop codon.
[0013] In another embodiment, a nucleic acid construct comprises,
in 5' to 3' order: (a) a fragment of the nucleic acid residues of
exon 7 of SMN; (b) a fragment of the nucleic acid residues of exon
8 of SMN; and (c) a reporter gene coding sequence lacking a start
codon, wherein: (i) in the mRNA transcript transcribed from the
nucleic acid construct, the region of the mRNA transcript
corresponding to the fragment of the nucleic acid residues of exon
7 of SMN does not contain a stop codon; (ii) the fragment of the
nucleic acid residues of exon 7 of SMN comprises any number of
nucleotides of exon 7 of SMN so long as in the mRNA transcript
transcribed from the nucleic acid construct the open reading frames
of the fragment of the nucleic acid residues of exon 7 of SMN and
the fragment of the nucleic acid residues of exon 8 of SMN are in
frame with each other; and (iii) the reporter gene coding sequence
is fused to the fragment of the nucleic acid residues of exon 8 of
SMN such that the open reading frames of the reporter gene coding
sequence and the fragment are out of frame with each other in the
mRNA transcript transcribed from the nucleic acid construct and
there is a stop codon in the region of the mRNA transcript
corresponding to the fragment of the nucleic acid residues of exon
8 of SMN (i.e., upstream of the reporter gene coding sequence). In
a specific embodiment, the fragment of the nucleic acid residues of
exon 8 of SMN consists of the first 21 or 23 nucleotides from the
5' end of exon 8 of SMN. In another embodiment, the fragment of the
nucleic acid residues of SMN consists of the first 23 nucleotides
from the 5' end of exon 8 of SMN. In certain embodiments, the
nucleic acid construct comprises a start codon upstream (5') to the
fragment of the nucleic acid residues of exon 7 of SMN. In
accordance with such embodiments, the first start codon and the
stop codon upstream of the reporter gene coding sequence in the
mRNA transcript are in the same contiguous open reading frame
without any interruption by, e.g., a stop codon.
[0014] In another embodiment, a nucleic acid construct comprises,
in 5' to 3' order: (a) a start codon, (b) a fragment of the nucleic
acid residues of exon 7 of SMN; (c) a fragment of the nucleic acid
residues of exon 8 of SMN; and (d) a reporter gene coding sequence
lacking a start codon, wherein: (i) the reporter gene coding
sequence is fused to the fragment of the nucleic acid residues of
exon 8 of SMN such that the first codon of the reporter gene coding
sequence and the first codon of the fragment are out of frame with
each other in the mRNA transcript transcribed from the nucleic acid
construct and there is a stop codon in the region of the mRNA
transcript corresponding to the fragment of the nucleic acid
residues of exon 8 of SMN (i.e., upstream of the reporter gene
coding sequence); and (ii) the fragment of the nucleic acid
residues of exon 7 of SMN comprises any number of nucleotides of
exon 7 of SMN so long as in the mRNA transcript transcribed from
the nucleic acid construct the first start codon and the stop codon
upstream of the reporter gene coding sequence are maintained in the
same contiguous open reading frame without any interruption by,
e.g., stop codon.
[0015] In another embodiment, a nucleic acid construct comprises,
in 5' to 3' order: (a) the nucleic acid residues of exon 6 of SMN
or a fragment thereof; (b) a fragment of the nucleic acid residues
of exon 7 of SMN; (c) a fragment of the nucleic acid residues of
exon 8 of SMN; and (d) a reporter gene coding sequence lacking a
start codon, wherein: (i) in the mRNA transcript transcribed from
the nucleic acid construct, the regions of the mRNA transcripts
corresponding to the fragments of the nucleic acid residues of exon
6 and exon 7 of SMN do not contain a stop codon; (ii) the fragment
of the nucleic acid residues of exon 6 of SMN and the fragment of
the nucleic acid residues of exon 7 of SMN each comprise any number
of nucleotides of exon 6 of SMN and exon 7 of SMN, respectively, so
long as in the mRNA transcript transcribed from the nucleic acid
construct the open reading frame of the fragment of the nucleic
acid residues of exon 6 of SMN and the open reading frame of the
fragment of the nucleic acid residues of exon 8 of SMN are in frame
with one another; (iii) the open reading frame of the nucleic acid
residues of exon 6 of SMN, the open reading frame of the fragment
of the nucleic acid residues of exon 7 of SMN, and the open reading
frame of the nucleic acid residues of the nucleic acid residues of
exon 8 of SMN are in frame with one another in the mRNA transcript
from the nucleic acid construct; and (iv) the reporter gene coding
sequence is fused to the fragment of the nucleic acid residues of
exon 8 of SMN such that the open reading frames of the reporter
gene coding sequence and the fragment are out of frame with each
other in the mRNA transcript transcribed from the nucleic acid
construct and there is a stop codon in the region of the mRNA
transcript corresponding to the fragment of the nucleic acid
residues of exon 8 of SMN (i.e., upstream of the reporter coding
sequence). In one embodiment, the fragment of the nucleic acid
residues of exon 8 of SMN consists of the first 21 or 23
nucleotides from the 5' end of exon 8 of SMN. In certain
embodiments, an internal start codon (e.g., ATG) in the nucleic
acid residues of exon 6 of SMN or a fragment thereof is used as a
start codon for the nucleic acid construct. In other embodiments,
the nucleic acid construct comprises a start codon upstream (5') to
the nucleic acid residues of exon 6 of SMN or a fragment thereof.
In accordance with such embodiments, the first start codon and the
stop codon upstream of the reporter gene coding sequence in the
mRNA transcript are in the same contiguous open reading frame.
[0016] In another embodiment, a nucleic acid construct comprises,
in 5' to 3' order: (a) a start codon, (b) the nucleic acid residues
of exon 6 of SMN or a fragment thereof; (c) a fragment of the
nucleic acid residues of exon 7 of SMN; (d) a fragment of the
nucleic acid residues of exon 8 of SMN; and (e) a reporter gene
coding sequence lacking a start codon, wherein: (i) the reporter
gene coding sequence is fused to the fragment of the nucleic acid
residues of exon 8 of SMN such that the first codon of the reporter
gene coding sequence and the first codon of the fragment are out of
frame with each other in the mRNA transcript transcribed from the
nucleic acid construct and there is a stop codon in the region of
the mRNA transcript corresponding to the fragment of the nucleic
acid residues of exon 8 of SMN (i.e., upstream of the reporter
coding sequence); and (ii) the fragment of the nucleic acid
residues of exon 6 of SMN and the fragment of the nucleic acid
residues of exon 7 of SMN each comprise any number of nucleotides
of exon 6 of SMN and exon 7 of SMN, respectively, so long as in the
mRNA transcript transcribed from the nucleic acid construct the
first start codon and the stop codon upstream of the reporter gene
coding sequence are maintained in the same contiguous open reading
frame without any interruption by, e.g., a stop codon.
[0017] In another embodiment, a nucleic acid construct comprises,
in 5' to 3' order: (a) a fragment of the nucleic acid residues of
exon 7 of SMN; (b) the nucleic acid residues of intron 7 of SMN or
a fragment thereof, wherein the fragment of the nucleic acid
residues of intron 7 comprises any number of nucleotides of intron
7 of SMN required for a functional, minimum intron; (c) a fragment
of the nucleic acid residues of exon 8 of SMN; and (d) a reporter
gene coding sequence lacking a start codon, wherein: (i) in the
mRNA transcript transcribed from the nucleic acid construct, the
region of the mRNA transcript corresponding to the fragment of the
nucleic acid residues of exon 7 of SMN does not contain a stop
codon; (ii) the fragment of the nucleic acid residues of exon 7 of
SMN comprises a minimum of the nucleotides of exon 7 of SMN
required for splicing and in the mRNA transcript transcribed from
the nucleic acid construct the open reading frame of the fragment
of the nucleic acid residues of exon 7 of SMN and the open reading
frame of the fragment of the nucleic acid residues of exon 8 of SMN
are in frame with each other; and (iii) the reporter gene coding
sequence is fused to the fragment of the nucleic acid residues of
exon 8 of SMN such that the open reading frames of the reporter
gene coding sequence and the fragment are out of frame with each
other in the mRNA transcript transcribed from the nucleic acid
construct and there is a stop codon in the region of the mRNA
transcript corresponding to the fragment of the nucleic acid
residues of exon 8 of SMN (i.e., upstream of the reporter gene
coding sequence). In a specific embodiment, the fragment of the
nucleic acid residues of exon 8 of SMN consists of the first 21 or
23 nucleotides from the 5' end of exon 8 of SMN. In another
specific embodiment, the fragment of the nucleic acid residues of
exon 7 of SMN comprises the first two nucleotides from the 3' end
of exon 7 of SMN (i.e., nucleotide residues 53 and 54 from the 5'
end of exon 7 of SMN). In a specific embodiment, the fragment of
the nucleic acid residues of exon 7 of SMN comprises a minimum of
the first six nucleotides from the 3' end of exon 7 of SMN (i.e.,
nucleotide residues 49 to 54 from the 5' end of exon 7 of SMN). In
certain embodiments, the nucleic acid construct comprises a start
codon upstream (5') to the fragment of the nucleic acid residues of
exon 7 of SMN. In accordance with such embodiments, the first start
codon and the stop codon upstream of the reporter gene coding
sequence in the mRNA transcript are in the same contiguous open
reading frame.
[0018] In another embodiment, a nucleic acid construct comprises,
in 5' to 3' order: (a) a start codon; (b) a fragment of the nucleic
acid residues of exon 7 of SMN; (c) the nucleic acid residues of
intron 7 of SMN or a fragment thereof, wherein the fragment of the
nucleic acid residues of intron 7 comprises any number of
nucleotides of intron 7 of SMN required for a functional, minimum
intron; (d) a fragment of the nucleic acid residues of exon 8 of
SMN; and (e) a reporter gene coding sequence lacking a start codon,
wherein: (i) the reporter gene coding sequence is fused to the
fragment of the nucleic acid residues of exon 8 of SMN such that
the first codon of the reporter gene coding sequence and the first
codon of the fragment are out of frame with each other in the mRNA
transcript transcribed from the nucleic acid construct and there is
a stop codon in the region of the mRNA transcript corresponding to
the fragment of the nucleic acid residues of exon 8 of SMN (i.e.,
upstream of the reporter gene coding sequence); and (ii) the
fragment of the nucleic acid residues of exon 7 of SMN comprises a
minimum number of the nucleotides of exon 7 of SMN required for
splicing and that number of nucleotides maintains the start codon
and the stop codon upstream of the reporter gene coding sequence in
the same contiguous open reading frame without any interruption by,
e.g., stop codon.
[0019] In another embodiment, a nucleic acid construct comprises,
in 5' to 3' order: (a) the nucleic acid residues of exon 6 of SMN
or a fragment thereof; (b) a fragment of the nucleic acid residues
of exon 7 of SMN; (c) the nucleic acid residues of intron 7 of SMN
or a fragment thereof, wherein the fragment of the nucleic acid
residues of intron 7 comprises any number of nucleotides of intron
7 of SMN required for a functional, minimum intron; (d) a fragment
of the nucleic acid residues of exon 8 of SMN; and (e) a reporter
gene coding sequence lacking a start codon, wherein: (i) in the
mRNA transcript transcribed from the nucleic acid construct, the
regions of the mRNA transcript corresponding to the fragments of
the nucleic acid residues of exon 6 and exon 7 of SMN do not
contain a stop codon; (ii) the fragment of the nucleic acid
residues of exon 6 of SMN comprises any number of nucleotides of
exon 6 of SMN so long as in the mRNA transcript transcribed from
the nucleic acid construct the open reading frame of the fragment
of the nucleic acid residues of exon 6 of SMN and the open reading
frame of the fragment of the nucleic acid residues of exon 8 of SMN
are in frame with each other; (iii) the fragment of the nucleic
acid residues of exon 7 of SMN comprises a minimum of the
nucleotides of exon 7 of SMN required for splicing and in the mRNA
transcript transcribed from the nucleic acid construct the open
reading frame of the fragment of the nucleic acid residues of exon
7 of SMN, the open reading frame of the nucleic acid residues of
exon 6 of SMN or a fragment thereof, and the open reading frame of
the fragment of exon 8 of SMN are in frame with each other; and
(iv) the reporter gene coding sequence is fused to the fragment of
the nucleic acid residues of exon 8 of SMN such that the open
reading frames of the reporter gene coding sequence and the
fragment are out of frame with each other in the mRNA transcript
transcribed from the nucleic acid construct and there is a stop
codon in the region of the mRNA transcript corresponding to the
fragment of the nucleic acid residues of exon 8 of SMN (i.e.,
upstream of the reporter gene coding sequence). In a specific
embodiment, the fragment of the nucleic acid residues of exon 8 of
SMN consists of the first 21 or 23 nucleotides from the 5' end of
exon 8 of SMN. In another specific embodiment, the fragment of the
nucleic acid residues of exon 7 of SMN comprises the first two
nucleotides from the 3' end of exon 7 of SMN (i.e., nucleotide
residues 53 and 54 from the 5' end of exon 7 of SMN). In another
specific embodiment, the fragment of the nucleic acid residues of
exon 7 of SMN comprises a minimum of the first six nucleotides from
the 3' end of exon 7 of SMN (i.e., nucleotide residues 49 to 54
from the 5' end of exon 7 of SMN). In certain embodiments, an
internal start codon (e.g., ATG) of the nucleic acid residues of
exon 6 of SMN or a fragment thereof is used as a start codon for
the nucleic acid construct. In some embodiments, the nucleic acid
construct comprises a start codon upstream (5') to the nucleic acid
residues of exon 6 of SMN or a fragment thereof. In accordance with
such embodiments, the first start codon and the stop codon upstream
of the reporter gene coding sequence in the mRNA transcript are in
the same contiguous open reading frame.
[0020] In another embodiment, a nucleic acid construct comprises,
in 5' to 3' order: (a) a start codon; (b) the nucleic acid residues
of exon 6 of SMN or a fragment thereof; (c) a fragment of the
nucleic acid residues of exon 7 of SMN; (d) the nucleic acid
residues of intron 7 of SMN or a fragment thereof, wherein the
fragment of the nucleic acid residues of intron 7 comprises any
number of nucleotides of intron 7 of SMN required for a functional,
minimum intron; (e) a fragment of the nucleic acid residues of exon
8 of SMN; and (f) a reporter gene coding sequence lacking a start
codon, wherein: (i) the reporter gene coding sequence is fused to
the fragment of the nucleic acid residues of exon 8 of SMN such
that the first codon of the reporter gene coding sequence and the
first codon of the fragment are out of frame with each other in the
mRNA transcript transcribed from the nucleic acid construct and
there is a stop codon in the region of the mRNA transcript
corresponding to the fragment of the nucleic acid residues of exon
8 of SMN (i.e., upstream of the reporter gene coding sequence);
(ii) the fragment of the nucleic acid residues of exon 6 of SMN
comprises any number of nucleotides of exon 6 of SMN so long as in
the mRNA transcript transcribed from the nucleic acid construct the
first start codon and the stop codon upstream of the reporter gene
coding sequence are maintained in the same contiguous open reading
frame without any interruption by, e.g., stop codon; and (iii) the
fragment of the nucleic acid residues of exon 7 of SMN comprises a
minimum number of the nucleotides of exon 7 of SMN required for
splicing and that number of nucleotides maintains the first start
codon and the stop codon upstream of the reporter gene coding
sequence in the same contiguous open reading frame without any
interruption by, e.g., a stop codon.
[0021] In another embodiment, a nucleic acid construct comprises,
in 5' to 3' order: (a) the nucleic acid residues of exon 6 of SMN
or a fragment thereof; (b) the nucleic acid residues of intron 6 of
SMN or a fragment thereof, wherein the fragment of the nucleic acid
residues of intron 6 of SMN comprises any number of nucleotides of
intron 6 of SMN required for a functional, minimum intron; (c) a
fragment of the nucleic acid residues of exon 7 of SMN; (d) the
nucleic acid residues of intron 7 of SMN or a fragment thereof,
wherein the fragment of the nucleic acid residues of intron 7
comprises any number of nucleotides of intron 7 of SMN required for
a functional, minimum intron; (e) a fragment of the nucleic acid
residues of exon 8 of SMN; and (f) a reporter gene coding sequence
lacking a start codon, wherein: (i) in the mRNA transcript
transcribed from the nucleic acid construct, the regions of the
mRNA transcript corresponding to the fragments of the nucleic acid
residues of exon 6 and exon 7 of SMN do not contain a stop codon;
(ii) the fragment of the nucleic acid residues of exon 6 of SMN
comprises a minimum of the nucleotides of exon 6 of SMN required
for splicing and in the mRNA transcript transcribed from the
nucleic acid construct the open reading frame of the fragment of
the nucleic acid residues of exon 6 of SMN and the fragment of the
nucleic acid residues of exon 8 of SMN are in frame with each
other; (iii) the fragment of the nucleic acid residues of exon 7 of
SMN comprises a minimum of the nucleotides of exon 7 of SMN
required for splicing and in the mRNA transcript transcribed from
the nucleic acid construct the open reading frame of the fragment
of the nucleic acid residues of exon 7 of SMN, the open reading
frame of the nucleic acid residues of exon 6 of SMN or a fragment
thereof, and the open reading frame of the fragment of exon 8 of
SMN are in frame with each other; and (iv) the reporter gene coding
sequence is fused to the fragment of the nucleic acid residues of
exon 8 of SMN such that the open reading frames of the reporter
gene coding sequence and the fragment are out of frame with each
other in the mRNA transcript transcribed from the nucleic acid
construct and there is a stop codon in the region of the mRNA
transcript corresponding to the fragment of the nucleic acid
residues of exon 8 of SMN (i.e., upstream of the reporter gene
coding sequence). In a specific embodiment, the fragment of the
nucleic acid residues of exon 8 of SMN consists of the first 21 or
23 nucleotides from the 5' end of exon 8 of SMN. In another
specific embodiment, the fragment of the nucleic acid residues of
exon 7 of SMN comprises a minimum of the first nucleotide from the
5' end of exon 7 of SMN and the first two nucleotides from the 3'
end of exon 7 of SMN (i.e., nucleotide residues 53 and 54 from the
5' end of exon 7 of SMN). In another specific embodiment, the
fragment of the nucleic acid residues of exon 7 of SMN comprises a
minimum of the first six nucleotides from the 3' end of exon 7 of
SMN (i.e., nucleotide residues 49 to 54 from the 5' end of exon 7
of SMN). In certain specific embodiments, the fragment of the
nucleic acid residues of exon 6 of SMN comprises a minimum of the
first two nucleotides from the 3' end of exon 6 of SMN. In other
embodiments, the fragment of the nucleic acid residues of exon 6 of
SMN comprises a minimum of the first three nucleotides from the 3'
end of exon 6 of SMN. In certain embodiments, an internal start
codon (e.g., ATG) of the nucleic acid residues of exon 6 of SMN or
a fragment thereof is used as a start codon for the nucleic acid
construct. In some embodiments, the nucleic acid construct
comprises a start codon upstream (5') to the nucleic acid residues
of exon 6 of SMN or a fragment thereof. In accordance with such
embodiments, the first start codon and the stop codon upstream of
the reporter gene coding sequence in the mRNA transcript are in the
same contiguous open reading frame.
[0022] In another embodiment, a nucleic acid construct comprises,
in 5' to 3' order: (a) a start codon; (b) the nucleic acid residues
of exon 6 of SMN or a fragment thereof; (c) the nucleic acid
residues of intron 6 of SMN or a fragment thereof, wherein the
fragment of the nucleic acid residues of intron 6 of SMN comprises
any number of nucleotides of intron 6 of SMN required for a
functional, minimum intron; (d) a fragment of the nucleic acid
residues of exon 7 of SMN; (e) the nucleic acid residues of intron
7 of SMN or a fragment thereof, wherein the fragment of the nucleic
acid residues of intron 7 comprises any number of nucleotides of
intron 7 of SMN required for a functional, minimum intron; (f) a
fragment of the nucleic acid residues of exon 8 of SMN; and (g) a
reporter gene coding sequence lacking a start codon, wherein: (i)
the reporter gene coding sequence is fused to the fragment of the
nucleic acid residues of exon 8 of SMN such that the first codon of
the reporter gene coding sequence and the first codon of the
fragment are out of frame with each other in the mRNA transcript
transcribed from the nucleic acid construct and there is a stop
codon in the region of the mRNA transcript corresponding to the
fragment of the nucleic acid residues of exon 8 of SMN (i.e.,
upstream of the reporter gene coding sequence); and (ii) the
fragment of the nucleic acid residues of exon 6 of SMN and the
fragment of the nucleic acid residues of exon 7 of SMN each
comprise a minimum number of the nucleotides of exon 6 of SMN and
exon 7 of SMN, respectively, required for splicing and that number
of nucleotides maintains the first start codon and the stop codon
upstream of the reporter gene coding sequence in the same
contiguous open reading frame without any interruption by, e.g., a
stop codon.
[0023] In another embodiment, a nucleic acid construct comprises,
in 5' to 3' order: (a) the nucleic acid residues of exon 7 of SMN,
wherein any number of nucleotides are inserted after the 48th
nucleotide residue from the 5' end of exon 7 of SMN (i.e., before
the 6th nucleotide from the 3' end of exon 7 of SMN) as long as the
native stop codon of exon 7 of SMN is inactivated and any
additional stop codon is not generated; (b) a fragment of the
nucleic acid residues of exon 8 of SMN; and (c) a reporter gene
coding sequence lacking a start codon, wherein: (i) in the mRNA
transcript transcribed from the nucleic acid construct, the open
reading frame of the nucleic acid residues of exon 7 of SMN and the
open reading frame of the fragment of the nucleic acid residues of
exon 8 of SMN are in frame with each other; and (ii) the reporter
gene coding sequence is fused to the fragment of the nucleic acid
residues of exon 8 of SMN such that the open reading frames of the
reporter gene and the fragment are out of frame with each other in
the mRNA transcript transcribed from the nucleic acid construct and
there is a stop codon in the region of the mRNA transcript
corresponding to the fragment of the nucleic acid residues of exon
8 of SMN (i.e., upstream of the reporter gene coding sequence). In
a specific embodiment, the fragment of the nucleic acid residues of
exon 8 of SMN consists of the first 21 or 23 nucleotides from the
5' end of exon 8 of SMN. In another embodiment, the fragment of the
nucleic acid residues of SMN consists of the first 23 nucleotides
from the 5' end of exon 8 of SMN. In another specific embodiment, a
single nucleotide residue is inserted after the 48th nucleotide
residue from the 5' end of exon 7 of SMN (i.e., before the 6th
nucleotide from the 3' end of exon 7 of SMN). In certain
embodiments, the nucleic acid construct comprises a start codon
upstream (5') to the nucleic acid residues of exon 7 of SMN. In
accordance with such embodiments, the first start codon and the
stop codon upstream of the reporter gene coding sequence in the
mRNA transcript are in the same contiguous open reading frame
without any interruption by, e.g., a stop codon.
[0024] In another embodiment, a nucleic acid construct comprises,
in 5' to 3' order: (a) a start codon, (b) the nucleic acid residues
of exon 7 of SMN, wherein any number of nucleotides are inserted
after the 48th nucleotide residue from the 5' end of exon 7 of SMN
(i.e., before the 6th nucleotide from the 3' end of exon 7 of SMN)
as long as the native stop codon of exon 7 of SMN is inactivated
and any additional stop codon is not generated; (c) a fragment of
the nucleic acid residues of exon 8 of SMN; and (d) a reporter gene
coding sequence lacking a start codon, wherein: (i) the reporter
gene coding sequence is fused to the fragment of the nucleic acid
residues of exon 8 of SMN such that the first codon of the reporter
gene and the first codon of the fragment are out of frame with each
other in the mRNA transcript transcribed from the nucleic acid
construct and there is a stop codon in the region of the mRNA
transcript corresponding to the fragment of the nucleic acid
residues of exon 8 of SMN (i.e., upstream of the reporter gene
coding sequence); and (ii) in the mRNA transcript transcribed from
the nucleic acid construct, the first start codon and the stop
codon upstream from the reporter gene coding sequence are in the
same contiguous open reading frame without any interruption by,
e.g., a stop codon.
[0025] In another embodiment, a nucleic acid construct comprises,
in 5' to 3' order: (a) the nucleic acid residues of exon 6 of SMN
or a fragment thereof; (b) the nucleic acid residues of exon 7 of
SMN, wherein any number of nucleotides are inserted after the 48th
nucleotide residue from the 5' end of exon 7 of SMN (i.e., before
the 6th nucleotide from the 3' end of exon 7 of SMN) as long as the
native stop codon of exon 7 of SMN is inactivated and any
additional stop codon is not generated; (c) a fragment of the
nucleic acid residues of exon 8 of SMN; and (d) a reporter gene
coding sequence lacking a start codon, wherein: (i) the fragment of
the nucleic acid residues of exon 6 of SMN comprises any number of
nucleotides of exon 6 of SMN so long as in the mRNA transcript
transcribed from the nucleic acid construct the open reading frame
of the fragment of the nucleic acid residues of exon 6 of SMN and
the open reading frame of the fragment of the nucleic acid residues
of exon 8 of SMN are in frame with one another; (ii) the open
reading frame of the nucleic acid residues of exon 6 of SMN, the
open reading frame of the nucleic acid residues of exon 7 of SMN,
and the open reading frame of the fragment of exon 8 of SMN are in
frame with one another in the mRNA transcript transcribed from the
nucleic acid construct; (iii) the reporter gene coding sequence is
fused to the fragment of the nucleic acid residues of exon 8 of SMN
such that the open reading frames of the reporter gene coding
sequence and the fragment are out of frame with each other in the
mRNA transcript transcribed from the nucleic acid construct and
there is a stop codon in the region of the mRNA transcript
corresponding to the fragment of the nucleic acid residues of exon
8 of SMN. In a specific embodiment, the fragment of the nucleic
acid residues of exon 8 of SMN consists of the first 21 or 23
nucleotides from the 5' end of exon 8 of SMN. In another specific
embodiment, a single nucleotide residue is inserted after the 48th
nucleotide residue from the 5' end of exon 7 of SMN (i.e., before
the 6th nucleotide from the 3' end of exon 7 of SMN). In certain
embodiments, an internal start codon (e.g., ATG) in the nucleic
acid residues of exon 6 of SMN or a fragment thereof is used as a
start codon for the nucleic acid construct. In other embodiments,
the nucleic acid construct comprises a start codon upstream (5') to
the nucleic acid residues of exon 6 of SMN or a fragment thereof.
In accordance with such embodiments, the first start codon and the
stop codon upstream of the reporter gene coding sequence in the
mRNA transcript are in the same contiguous open reading frame
without any interruption by, e.g., a stop codon.
[0026] In another embodiment, a nucleic acid construct comprises,
in 5' to 3' order: (a) a start codon; (b) the nucleic acid residues
of exon 6 of SMN or a fragment thereof; (c) the nucleic acid
residues of exon 7 of SMN, wherein any number of nucleotides are
inserted after the 48th nucleotide residue from the 5' end of exon
7 of SMN (i.e., before the 6th nucleotide from the 3' end of exon 7
of SMN) as long as the native stop codon of exon 7 of SMN is
inactivated and any additional stop codon is not generated; (d) a
fragment of the nucleic acid residues of exon 8 of SMN; and (e) a
reporter gene coding sequence lacking a start codon, wherein: (i)
the reporter gene coding sequence is fused to the fragment of the
nucleic acid residues of exon 8 of SMN such that the first codon of
the reporter gene coding sequence and the first codon of the
fragment are out of frame with each other in the mRNA transcript
transcribed from the nucleic acid construct and there is a stop
codon in the region of the mRNA transcript corresponding to the
fragment of the nucleic acid residues of exon 8 of SMN (i.e.,
upstream of the reporter gene coding sequence); and (ii) in the
mRNA transcript transcribed from the nucleic acid construct, the
first start codon and the stop codon upstream from the reporter
gene coding sequence are in the same contiguous open reading frame
without any interruption by, e.g., a stop codon.
[0027] In another embodiment, a nucleic acid construct comprises,
in 5' to 3' order: (a) the nucleic acid residues of exon 7 of SMN,
wherein any number of nucleotides are inserted after the 48th
nucleotide residue from the 5' end of exon 7 of SMN (i.e., before
the 6th nucleotide from the 3' end of exon 7 of SMN) as long as the
native stop codon of exon 7 of SMN is inactivated and any
additional stop codon is not generated; (b) the nucleic acid
residues of intron 7 of SMN or a fragment thereof, wherein the
fragment of the nucleic acid residues of intron 7 comprises any
number of nucleotides of intron 7 of SMN required for a functional,
minimum intron; (c) a fragment of the nucleic acid residues of exon
8 of SMN; and (d) a reporter gene coding sequence lacking a start
codon, wherein: (i) the open reading frame of the nucleic acid
residues of exon 7 of SMN and the open reading frame of the nucleic
acid residues of exon 8 of SMN are in frame with each other in the
mRNA transcript transcribed from the nucleic acid construct; and
(ii) the reporter gene coding sequence is fused to the fragment of
the nucleic acid residues of exon 8 of SMN such that the open
reading frames of the reporter gene coding sequence and the
fragment are out of frame with each other in the mRNA transcript
transcribed from the nucleic acid construct and there is a stop
codon in the region of the mRNA transcript corresponding to the
fragment of the nucleic acid residues of exon 8 of SMN (i.e.,
upstream of the reporter gene coding sequence). In a specific
embodiment, the fragment of the nucleic acid residues of exon 8 of
SMN consists of the first 21 or 23 nucleotides from the 5' end of
exon 8 of SMN. In another specific embodiment, a single nucleotide
residue is inserted after the 48th nucleotide residue from the 5'
end of exon 7 of SMN (i.e., before the 6th nucleotide from the 3'
end of exon 7 of SMN). In certain embodiments, the nucleic acid
construct comprises a start codon upstream (5') to the nucleic acid
residues of exon 7 of SMN. In accordance with such embodiments, the
first start codon and the stop codon upstream of the reporter gene
coding sequence in the mRNA transcript are in the same contiguous
open reading frame without any interruption by, e.g., a stop
codon.
[0028] In another embodiment, a nucleic acid construct comprises,
in 5' to 3' order: (a) a start codon; (b) the nucleic acid residues
of exon 7 of SMN, wherein any number of nucleotides are inserted
after the 48th nucleotide residue from the 5' end of exon 7 of SMN
(i.e., before the 6th nucleotide from the 3' end of exon 7 of SMN)
as long as the native stop codon of exon 7 of SMN is inactivated
and any additional stop codon is not generated; (c) the nucleic
acid residues of intron 7 of SMN or a fragment thereof, wherein the
fragment of the nucleic acid residues of intron 7 comprises any
number of nucleotides of intron 7 of SMN required for a functional,
minimum intron; (d) a fragment of the nucleic acid residues of exon
8 of SMN; and (e) a reporter gene coding sequence lacking a start
codon, wherein: (i) the reporter gene coding sequence is fused to
the fragment of the nucleic acid residues of exon 8 of SMN such
that the first codon of the reporter gene coding sequence and the
first codon of the fragment are out of frame with each other in the
mRNA transcript transcribed from the nucleic acid construct and
there is a stop codon in the region of the mRNA transcript
corresponding to the fragment of the nucleic acid residues of exon
8 of SMN (i.e., upstream of the reporter gene coding sequence); and
(ii) in the mRNA transcript transcribed from the nucleic acid
construct, the first start codon and the stop codon upstream from
the reporter gene coding sequence are in the same contiguous open
reading frame without any interruption by, e.g., a stop codon.
[0029] In another embodiment, a nucleic acid construct comprises,
in 5' to 3' order: (a) the nucleic acid residues of exon 6 of SMN
or a fragment thereof; (b) the nucleic acid residues of exon 7 of
SMN, wherein any number of nucleotides are inserted after the 48th
nucleotide residue from the 5' end of exon 7 of SMN (i.e., before
the 6th nucleotide from the 3' end of exon 7 of SMN) as long as the
native stop codon of exon 7 of SMN is inactivated and any
additional stop codon is not generated; (c) the nucleic acid
residues of intron 7 of SMN or a fragment thereof, wherein the
fragment of the nucleic acid residues of intron 7 comprises any
number of nucleotides of intron 7 of SMN required for a functional,
minimum intron; (d) a fragment of the nucleic acid residues of exon
8 of SMN; and (e) a reporter gene coding sequence lacking a start
codon, wherein: (i) the fragment of exon 6 of SMN comprises any
number of nucleotides of exon 6 of SMN so long as in the mRNA
transcript transcribed from the nucleic acid construct the open
reading frame of the fragment of the nucleic acid residues of exon
6 of SMN and the fragment of the nucleic acid residues of exon 8 of
SMN are in frame with each other; (ii) the open reading frame of
the nucleic acid residues of exon 6 of SMN, the open reading frame
of the nucleic acid residues of exon 7 of SMN, and the open reading
frame of the fragment of exon 8 of SMN are in frame with one
another in the mRNA transcript transcribed from the nucleic acid
construct; and (iii) the reporter gene coding sequence is fused to
the fragment of the nucleic acid residues of exon 8 of SMN such
that the open reading frames of the reporter gene coding sequence
and the fragment are out of frame with each other in the mRNA
transcript transcribed from the nucleic acid construct and there is
a stop codon in the region of the mRNA transcript corresponding to
the fragment of the nucleic acid residues of exon 8 of SMN (i.e.,
upstream of the reporter gene coding sequence). In a specific
embodiment, the fragment of the nucleic acid residues of exon 8 of
SMN consists of the first 21 or 23 nucleotides from the 5' end of
exon 8 of SMN. In another specific embodiment, a single nucleotide
residue is inserted after the 48th nucleotide residue from the 5'
end of exon 7 of SMN (i.e., before the 6th nucleotide from the 3'
end of exon 7 of SMN). In certain embodiments, an internal start
codon (e.g., ATG) of the nucleic acid residues of exon 6 of SMN or
a fragment thereof is used as a start codon for the nucleic acid
construct. In some embodiments, the nucleic acid construct
comprises a start codon upstream (5') to the nucleic acid residues
of exon 6 of SMN or a fragment thereof. In accordance with such
embodiments, the first start codon and the stop codon upstream of
the reporter gene coding sequence in the mRNA transcript are in the
same contiguous open reading frame without any interruption by,
e.g., a stop codon.
[0030] In another embodiment, a nucleic acid construct comprises,
in 5' to 3' order: (a) a start codon; (b) the nucleic acid residues
of exon 6 of SMN or a fragment thereof; (c) the nucleic acid
residues of exon 7 of SMN, wherein any number of nucleotides are
inserted after the 48th nucleotide residue from the 5' end of exon
7 of SMN (i.e., before the 6th nucleotide from the 3' end of exon 7
of SMN) as long as the native stop codon of exon 7 of SMN is
inactivated and any additional stop codon is not generated; (d) the
nucleic acid residues of intron 7 of SMN or a fragment thereof,
wherein the fragment of the nucleic acid residues of intron 7
comprises any number of nucleotides of intron 7 of SMN required for
a functional, minimum intron; (e) a fragment of the nucleic acid
residues of exon 8 of SMN; and (f) a reporter gene coding sequence
lacking a start codon, wherein: (i) the reporter gene coding
sequence is fused to the fragment of the nucleic acid residues of
exon 8 of SMN such that the first codon of the reporter gene coding
sequence and the first codon of the fragment are out of frame with
each other in the mRNA transcript transcribed from the nucleic acid
construct and there is a stop codon in the region of the mRNA
transcript corresponding to the fragment of the nucleic acid
residues of exon 8 of SMN (i.e., upstream of the reporter gene
coding sequence); and (ii) in the mRNA transcript transcribed from
the nucleic acid construct, the first start codon and the stop
codon upstream from the reporter gene coding sequence are in the
same contiguous open reading frame without any interruption by,
e.g., a stop codon.
[0031] In another embodiment, a nucleic acid construct comprises,
in 5' to 3' order: (a) the nucleic acid residues of exon 6 of SMN
or a fragment thereof; (b) the nucleic acid residues of intron 6 of
SMN or a fragment thereof, wherein the fragment of the nucleic acid
residues of intron 6 of SMN comprises any number of nucleotides of
intron 6 of SMN required for a functional, minimum intron; (c) the
nucleic acid residues of exon 7 of SMN, wherein any number of
nucleotides are inserted after the 48th nucleotide residue from the
5' end of exon 7 of SMN (i.e., before the 6th nucleotide from the
3' end of exon 7 of SMN) as long as the native stop codon of exon 7
of SMN is inactivated and any additional stop codon is not
generated; (d) the nucleic acid residues of intron 7 of SMN or a
fragment thereof, wherein the fragment of the nucleic acid residues
of intron 7 comprises any number of nucleotides of intron 7 of SMN
required for a functional, minimum intron; (e) a fragment of the
nucleic acid residues of exon 8 of SMN; and (f) a reporter gene
coding sequence lacking a start codon, wherein: (i) the fragment of
the nucleic acid residues of exon 6 of SMN comprises a minimum of
the nucleotides of exon 6 of SMN required for splicing and in the
mRNA transcript transcribed from the nucleic acid construct the
open reading frame of the fragment of the nucleic acid residues of
exon 6 of SMN and the fragment of the nucleic acid residues of exon
8 of SMN are in frame with each other; (ii) the open reading frame
of the nucleic acid residues of exon 6 of SMN, the open reading
frame of the nucleic acid residues of exon 7 of SMN, and the open
reading frame of the fragment of the nucleic acid residues of exon
8 of SMN are in frame with one another in the mRNA transcript
transcribed from the nucleic acid construct; and (iii) the reporter
gene coding sequence is fused to the fragment of the nucleic acid
residues of exon 8 of SMN such that the open reading frames of the
reporter gene coding sequence and the fragment are out of frame
with each other in the mRNA transcript transcribed from the nucleic
acid construct and there is a stop codon in the region of the mRNA
transcript corresponding to the fragment of the nucleic acid
residues of exon 8 of SMN (i.e., upstream of the reporter gene
coding sequence). In a specific embodiment, the fragment of the
nucleic acid residues of exon 8 of SMN consists of the first 21 or
23 nucleotides from the 5' end of exon 8 of SMN. In another
specific embodiment, a single nucleotide residue is inserted after
the 48th nucleotide residue from the 5' end of exon 7 of SMN (i.e.,
before the 6th nucleotide from the 3' end of exon 7 of SMN). In
certain specific embodiments, the fragment of the nucleic acid
residues of exon 6 of SMN comprises a minimum of the first two
nucleotides from the 3' end of exon 6 of SMN. In other embodiments,
the fragment of the nucleic acid residues of exon 6 of SMN
comprises a minimum of the first three nucleotides from the 3' end
of exon 6 of SMN. In certain embodiments, an internal start codon
(e.g., ATG) of the nucleic acid residues of exon 6 of SMN or a
fragment thereof is used as a start codon for the nucleic acid
construct. In some embodiments, the nucleic acid construct
comprises a start codon upstream (5') to the nucleic acid residues
of exon 6 of SMN or a fragment thereof. In accordance with such
embodiments, the first start codon and the stop codon upstream of
the reporter gene coding sequence in the mRNA transcript are in the
same contiguous open reading frame without any interruption by,
e.g., a stop codon.
[0032] In another embodiment, a nucleic acid construct comprises,
in 5' to 3' order: (a) a start codon; (b) the nucleic acid residues
of exon 6 of SMN or a fragment thereof, wherein the fragment
comprises a minimum number of nucleotides required for splicing;
(c) the nucleic acid residues of intron 6 of SMN or a fragment
thereof, wherein the fragment of the nucleic acid residues of
intron 6 of SMN comprises any number of nucleotides of intron 6 of
SMN required for a functional, minimum intron; (d) the nucleic acid
residues of exon 7 of SMN, wherein any number of nucleotides are
inserted after the 48th nucleotide residue from the 5' end of exon
7 of SMN (i.e., before the 6th nucleotide from the 3' end of exon 7
of SMN) as long as the native stop codon of exon 7 of SMN is
inactivated and any additional stop codon is not generated; (e) the
nucleic acid residues of intron 7 of SMN or a fragment thereof,
wherein the fragment of the nucleic acid residues of intron 7
comprises any number of nucleotides of intron 7 of SMN required for
a functional, minimum intron; (f) a fragment of the nucleic acid
residues of exon 8 of SMN; and (g) a reporter gene coding sequence
lacking a start codon, wherein: (i) the reporter gene coding
sequence is fused to the fragment of the nucleic acid residues of
exon 8 of SMN such that the first codon of the reporter gene coding
sequence and the first codon of the fragment are out of frame with
each other in the mRNA transcript transcribed from the nucleic acid
construct and there is a stop codon in the region of the mRNA
transcript corresponding to the fragment of the nucleic acid
residues of exon 8 of SMN (i.e., upstream of the reporter gene
coding sequence); and (ii) in the mRNA transcript transcribed from
the nucleic acid construct, the first start codon and the stop
codon upstream from the reporter gene coding sequence are in the
same contiguous open reading frame without any interruption by,
e.g., a stop codon.
[0033] In another embodiment, a nucleic acid construct comprises,
in 5' to 3' order: (a) the nucleic acid residues of exon 7 of SMN,
wherein a single guanine residue is inserted after the 48th
nucleotide residue from the 5' end of exon 7 of SMN (i.e., before
the 6th nucleotide from the 3' end of exon 7 of SMN); (b) the
nucleic acid residues of intron 7 of SMN or a fragment thereof,
wherein the fragment of the nucleic acid residues of intron 7
comprises any number of nucleotides of intron 7 of SMN required for
a functional, minimum intron; (c) a fragment of the nucleic acid
residues of exon 8 of SMN; and (d) a reporter gene coding sequence
lacking a start codon, wherein: (i) the open reading frame of the
nucleic acid residues of exon 7 of SMN and the open reading frame
of the fragment of the nucleic acid residues of exon 8 of SMN are
in frame with each other in the mRNA transcript transcribed from
the nucleic acid construct; (ii) the reporter gene coding sequence
is fused to the fragment of the nucleic acid residues of exon 8 of
SMN such that the reporter gene coding sequence and the fragment
are out of frame with each other in the mRNA transcript transcribed
from the nucleic acid construct; and (iii) the production of the
mRNA transcript generates a stop codon in the region of the mRNA
transcript that corresponds to the fragment of the nucleic acid
residues of exon 8 of SMN. In a specific embodiment, the fragment
of the nucleic acid residues of exon 8 of SMN consists of the first
21 or 23 nucleotides from the 5' end of exon 8 of SMN. In another
embodiment, the fragment of the nucleic acid residues of SMN
consists of the first 23 nucleotides from the 5' end of exon 8 of
SMN. In certain embodiments, the nucleic acid construct comprises a
start codon upstream (5') to the nucleic acid residues of exon 7 of
SMN. In accordance with such embodiments, the first start codon and
the stop codon upstream of the reporter gene coding sequence in the
mRNA transcript are in the same contiguous open reading frame
without any interruption by, e.g., a stop codon.
[0034] In another embodiment, a nucleic acid construct comprises,
in 5' to 3' order: (a) a start codon; (b) the nucleic acid residues
of exon 7 of SMN, wherein a single guanine residue is inserted
after the 48th nucleotide residue from the 5' end of exon 7 of SMN
(i.e., before the 6th nucleotide from the 3' end of exon 7 of SMN);
(c) the nucleic acid residues of intron 7 of SMN or a fragment
thereof, wherein the fragment of the nucleic acid residues of
intron 7 comprises any number of nucleotides of intron 7 of SMN
required for a functional, minimum intron; (d) a fragment of the
nucleic acid residues of exon 8 of SMN; and (e) a reporter gene
coding sequence lacking a start codon, wherein: (i) the reporter
gene coding sequence is fused to the fragment of the nucleic acid
residues of exon 8 of SMN such that the first codon of the reporter
gene coding sequence and the first codon of the fragment are out of
frame with each other in the mRNA transcript transcribed from the
nucleic acid construct; (ii) the production of the mRNA transcript
generates a stop codon in the region of the mRNA transcript that
corresponds to the fragment of the nucleic acid residues of exon 8
of SMN (i.e., upstream of the reporter gene coding sequence); and
(iii) in the mRNA transcript transcribed from the nucleic acid
construct, the first start codon and the stop codon upstream from
the reporter gene coding sequence are in the same contiguous open
reading frame without any interruption by, e.g., a stop codon.
[0035] In another embodiment, a nucleic acid construct comprises,
in 5' to 3' order: (a) the nucleic acid residues of exon 6 of SMN
or a fragment thereof; (b) the nucleic acid residues of exon 7 of
SMN, wherein a single guanine residue is inserted after the 48th
nucleotide residue from the 5' end of exon 7 of SMN (i.e., before
the 6th nucleotide from the 3' end of exon 7 of SMN); (c) the
nucleic acid residues of intron 7 of SMN or a fragment thereof,
wherein the fragment of intron 7 of SMN comprises any number of
nucleotides of intron 7 of SMN required for a functional, minimum
intron; (d) a fragment of the nucleic acid residues of exon 8 of
SMN; and (e) a reporter gene coding sequence lacking a start codon,
wherein (i) the fragment of the nucleic acid residues of exon 6 of
SMN comprises any number of nucleotides of exon 6 of SMN so long as
in the mRNA transcript transcribed from the nucleic acid construct
the open reading frame of the fragment of the nucleic acid residues
of exon 6 of SMN and the open reading frame of the fragment of the
nucleic acid residues of exon 8 of SMN are in frame with each
other; (ii) the open reading frame of the nucleic acid residues of
exon 6 of SMN, the open reading frame of the nucleic acid residues
of exon 7 of SMN, and the open reading frame of the fragment of the
nucleic acid residues of exon 8 of SMN are in frame with each other
in the mRNA transcript transcribed from the nucleic acid construct;
(iii) the reporter gene coding sequence is fused to the fragment of
the nucleic acid residues of exon 8 of SMN such that the reporter
gene coding sequence and the fragment are out of frame with each
other in the mRNA transcript transcribed from the nucleic acid
construct; and (iii) the production of the mRNA transcript
generates a stop codon in the region of the mRNA transcript that
corresponds to the fragment of the nucleic acid residues of exon 8
of SMN. In a specific embodiment, the fragment of the nucleic acid
residues of exon 8 of SMN consists of the first 21 or 23
nucleotides from the 5' end of exon 8 of SMN. In certain
embodiments, an internal start codon (e.g., ATG) in the nucleic
acid residues of exon 6 of SMN or a fragment thereof is used as a
start codon for the nucleic acid construct. In other embodiments,
the nucleic acid construct comprises a start codon upstream (5') to
the nucleic acid residues of exon 6 of SMN or a fragment thereof.
In accordance with such embodiments, the first start codon and the
stop codon upstream of the reporter gene coding sequence in the
mRNA transcript are in the same contiguous open reading frame
without any interruption by, e.g., a stop codon.
[0036] In another embodiment, a nucleic acid construct comprises,
in 5' to 3' order: (a) a start codon; (b) the nucleic acid residues
of exon 6 of SMN or a fragment thereof; (c) the nucleic acid
residues of exon 7 of SMN, wherein a single guanine residue is
inserted after the 48th nucleotide residue from the 5' end of exon
7 of SMN (i.e., before the 6th nucleotide from the 3' end of exon 7
of SMN); (d) the nucleic acid residues of intron 7 of SMN or a
fragment thereof, wherein the fragment of intron 7 of SMN comprises
any number of nucleotides of intron 7 of SMN required for a
functional, minimum intron; (e) a fragment of the nucleic acid
residues of exon 8 of SMN; and (f) a reporter gene coding sequence
lacking a start codon, wherein (i) the reporter gene coding
sequence is fused to the fragment of the nucleic acid residues of
exon 8 of SMN such that the first codon of the reporter gene coding
sequence and the first codon of the fragment are out of frame with
each other in the mRNA transcript transcribed from the nucleic acid
construct; (ii) the production of the mRNA transcript generates a
stop codon in the region of the mRNA transcript that corresponds to
the fragment of the nucleic acid residues of exon 8 of SMN (i.e.,
upstream of the reporter gene coding sequence); and (iii) in the
mRNA transcript transcribed from the nucleic acid construct, the
first start codon and the stop codon upstream from the reporter
gene coding sequence are in the same contiguous open reading frame
without any interruption by, e.g., a stop codon.
[0037] In another embodiment, a nucleic acid construct comprises,
in 5' to 3' order: (a) the nucleic acid residues of exon 6 of SMN
or a fragment thereof; (b) the nucleic acid residues of intron 6 of
SMN or a fragment thereof, wherein the fragment of the nucleic acid
residues of intron 6 of SMN comprises any number of nucleotides of
intron 6 required for a functional, minimum intron; (c) the nucleic
acid residues of exon 7 of SMN, wherein a single guanine residue is
inserted after the 48th nucleotide residue from the 5' end of exon
7 of SMN (i.e., before the 6th nucleotide from the 3' end of exon 7
of SMN); (d) the nucleic acid residues of intron 7 of SMN or a
fragment thereof, wherein the fragment of intron 7 of SMN comprises
any number of nucleotides of intron 7 of SMN required for a
functional, minimum intron; (e) a fragment of the nucleic acid
residues of exon 8 of SMN; and (f) a reporter gene coding sequence
lacking a start codon, wherein (i) the fragment of the nucleic acid
residues of exon 6 of SMN comprises a minimum of the nucleotides of
exon 6 of SMN required for splicing and in the mRNA transcript
transcribed from the nucleic acid construct the open reading frame
of the fragment of the nucleic acid residues of exon 6 of SMN and
the open reading frame of the fragment of the nucleic acid residues
of exon 8 of SMN are in frame with each other; (ii) the open
reading frame of the nucleic acid residues of exon 6 of SMN, the
open reading frame of the nucleic acid residues of exon 7 of SMN,
and the open reading frame of the fragment of the nucleic acid
residues of exon 8 of SMN are in frame with each other in the mRNA
transcript transcribed from the nucleic acid construct; (iii) the
reporter gene coding sequence is fused to the fragment of the
nucleic acid residues of exon 8 of SMN such that the reporter gene
coding sequence and the fragment are out of frame with each other
in the mRNA transcript transcribed from the nucleic acid construct;
and (iv) the production of the mRNA transcript generates a stop
codon in the region of the mRNA transcript that corresponds to the
fragment of the nucleic acid residues of exon 8 of SMN (i.e.,
upstream of the reporter gene coding sequence). In a specific
embodiment, the fragment of the nucleic acid residues of exon 8 of
SMN consists of the first 21 or 23 nucleotides from the 5' end of
exon 8 of SMN. In certain specific embodiments, the fragment of the
nucleic acid residues of exon 6 of SMN comprises a minimum of the
first two nucleotides from the 3' end of exon 6 of SMN. In other
embodiments, the fragment of the nucleic acid residues of exon 6 of
SMN comprises a minimum of the first three nucleotides from the 3'
end of exon 6 of SMN. In certain embodiments, an internal ATG in
the nucleic acid residues of exon 6 of SMN or a fragment thereof is
used as a start codon for the nucleic acid construct. In accordance
with such embodiments, the first start codon and the stop codon
upstream of the reporter gene coding sequence in the mRNA
transcript are in the same contiguous open reading frame without
any interruption by, e.g., a stop codon.
[0038] In another embodiment, a nucleic acid construct comprises,
in 5' to 3' order: (a) a start codon; (b) the nucleic acid residues
of exon 6 of SMN or a fragment thereof; (c) the nucleic acid
residues of intron 6 of SMN or a fragment thereof, wherein the
fragment of the nucleic acid residues of intron 6 of SMN comprises
any number of nucleotides of intron 6 required for a functional,
minimum intron; (d) the nucleic acid residues of exon 7 of SMN,
wherein a single guanine residue is inserted after the 48th
nucleotide residue from the 5' end of exon 7 of SMN (i.e., before
the 6th nucleotide from the 3' end of exon 7 of SMN); (e) the
nucleic acid residues of intron 7 of SMN or a fragment thereof,
wherein the fragment of intron 7 of SMN comprises any number of
nucleotides of intron 7 of SMN required for a functional, minimum
intron; (f) a fragment of the nucleic acid residues of exon 8 of
SMN; and (g) a reporter gene coding sequence lacking a start codon,
wherein (i) the fragment of the nucleic acid residues of exon 6 of
SMN comprises a minimum of the nucleotides of exon 6 of SMN
required for splicing; (ii) the reporter gene coding sequence is
fused to the fragment of the nucleic acid residues of exon 8 of SMN
such that the first codon of the reporter gene coding sequence and
the first codon of the fragment are out of frame with each other in
the mRNA transcript transcribed from the nucleic acid construct;
(iii) the production of the mRNA transcript generates a stop codon
in the region of the mRNA transcript that corresponds to the
fragment of the nucleic acid residues of exon 8 of SMN (i.e.,
upstream of the reporter gene coding sequence); and (iv) in the
mRNA transcript transcribed from the nucleic acid construct, the
first start codon and the stop codon upstream from the reporter
gene coding sequence are in the same contiguous open reading frame
without any interruption by, e.g., a stop codon.
[0039] In another embodiment, a nucleic acid construct comprises,
in 5' to 3' order: (a) a minimum of one nucleotide; (b) a fragment
of the nucleic acid residues of exon 7 of SMN, wherein the fragment
of the nucleic acid residues of exon 7 of SMN comprises a minimum
of the first six nucleotides from the 3' end of exon 7 of SMN
(i.e., nucleotide residues 49 to 54 from the 5' end of exon 7 of
SMN) and wherein a single guanine residue is inserted into the
fragment of the nucleic acid residues of exon 7 of SMN at the
location that corresponds to the location in exon 7 of SMN that is
after the 48th nucleotide from the 5' end of exon 7 of SMN; (c) the
nucleic acid residues of intron 7 of SMN or a fragment thereof,
wherein the fragment of the nucleic acid residues of intron 7 of
SMN comprises any number of nucleotides of intron 7 required for a
functional, minimum intron; (d) a fragment of the nucleic acid
residues of exon 8 of SMN; and (e) a reporter gene coding sequence
lacking a start codon, wherein (i) in the mRNA transcript
transcribed from the nucleic acid construct, open reading of the
fragment of the nucleic acid residues of exon 7 of SMN and the open
reading frame of the fragment of the nucleic acid residues of exon
8 of SMN are in frame with each other; (ii) the reporter gene
coding sequence is fused to the fragment of the nucleic acid
residues of exon 8 of SMN such that the reporter gene coding
sequence and the fragment are out of frame with each other in the
mRNA transcript transcribed from the nucleic acid construct; and
(iii) the production of the mRNA transcript generates a stop codon
in the region of the mRNA transcript that corresponds to the
fragment of the nucleic acid residues of exon 8 of SMN. In a
specific embodiment, the fragment of the nucleic acid residues of
exon 8 of SMN consists of the first 21 or 23 nucleotides from the
5' end of exon 8 of SMN. In certain embodiments, the nucleic acid
construct comprises a start codon upstream (5') to the minimum of
one nucleotide. In accordance with such embodiments, the first
start codon and the stop codon upstream of the reporter gene coding
sequence in the mRNA transcript are in the same contiguous open
reading frame without any interruption by, e.g., a stop codon.
[0040] In another embodiment, a nucleic acid construct comprises,
in 5' to 3' order: (a) a start codon; (b) a minimum of one
nucleotide; (c) a fragment of the nucleic acid residues of exon 7
of SMN, wherein the fragment of the nucleic acid residues of exon 7
of SMN comprises a minimum of the first six nucleotides from the 3'
end of exon 7 of SMN (i.e., nucleotide residues 49 to 54 from the
5' end of exon 7 of SMN) and wherein a single guanine residue is
inserted into the fragment of the nucleic acid residues of exon 7
of SMN at the location that corresponds to the location in exon 7
of SMN that is after the 48th nucleotide from the 5' end of exon 7
of SMN; (d) the nucleic acid residues of intron 7 of SMN or a
fragment thereof, wherein the fragment of the nucleic acid residues
of intron 7 of SMN comprises any number of nucleotides of intron 7
required for a functional, minimum intron; (e) a fragment of the
nucleic acid residues of exon 8 of SMN; and (f) a reporter gene
coding sequence lacking a start codon, wherein (i) the reporter
gene coding sequence is fused to the fragment of the nucleic acid
residues of exon 8 of SMN such that the first codon of the reporter
gene coding sequence and the first codon of the fragment are out of
frame with each other in the mRNA transcript transcribed from the
nucleic acid construct; (ii) the production of the mRNA transcript
generates a stop codon in the region of the mRNA transcript that
corresponds to the fragment of the nucleic acid residues of exon 8
of SMN (i.e., upstream of the reporter gene coding sequence); and
(iii) in the mRNA transcript transcribed from the nucleic acid
construct, the first start codon and the stop codon upstream from
the reporter gene coding sequence are in the same contiguous open
reading frame without any interruption by, e.g., a stop codon.
[0041] In another embodiment, a nucleic acid construct comprises,
in 5' to 3' order: (a) a fragment of the nucleic acid residues of
exon 7 of SMN, wherein the fragment of the nucleic acid residues of
exon 7 of SMN comprises a minimum of the first six nucleotides from
the 3' end of exon 7 of SMN (i.e., nucleotide residues 49 to 54
from the 5' end of exon 7 of SMN), wherein a single guanine residue
is inserted into the fragment of the nucleic acid residues of exon
7 of SMN at the location that corresponds to the location in exon 7
of SMN that is after the 48th nucleotide from the 5' end of exon 7
of SMN, and wherein the fragment of the nucleic acid residues of
exon 7 of SMN comprises any number of nucleotides of exon 7
required for splicing; (b) the nucleic acid residues of intron 7 of
SMN or a fragment thereof, wherein the fragment of the nucleic acid
residues of intron 7 of SMN comprises any number of nucleotides of
intron 7 required for a functional, minimum intron; (c) a fragment
of the nucleic acid residues of exon 8 of SMN; and (d) a reporter
gene coding sequence lacking a start codon, wherein (i) in the mRNA
transcript transcribed from the nucleic acid construct, open
reading of the fragment of the nucleic acid residues of exon 7 of
SMN and the open reading frame of the fragment of the nucleic acid
residues of exon 8 of SMN are in frame with each other; (ii) the
reporter gene coding sequence is fused to the fragment of the
nucleic acid residues of exon 8 of SMN such that the reporter gene
coding sequence and the fragment are out of frame with each other
in the mRNA transcript transcribed from the nucleic acid construct;
and (iii) the production of the mRNA transcript generates a stop
codon in the region of the mRNA transcript that corresponds to the
fragment of the nucleic acid residues of exon 8 of SMN (i.e.,
upstream of the reporter gene coding sequence). In a specific
embodiment, the fragment of the nucleic acid residues of exon 8 of
SMN consists of the first 21 or 23 nucleotides from the 5' end of
exon 8 of SMN. In certain embodiments, the nucleic acid construct
comprises a start codon upstream (5') to the fragment of the
nucleic acid residues of exon 7 of SMN. In accordance with such
embodiments, the first start codon and the stop codon upstream of
the reporter gene coding sequence in the mRNA transcript are in the
same contiguous open reading frame without any interruption by,
e.g., a stop codon.
[0042] In another embodiment, a nucleic acid construct comprises,
in 5' to 3' order: (a) a start codon; (b) a fragment of the nucleic
acid residues of exon 7 of SMN, wherein the fragment of the nucleic
acid residues of exon 7 of SMN comprises a minimum of the first six
nucleotides from the 3' end of exon 7 of SMN (i.e., nucleotide
residues 49 to 54 from the 5' end of exon 7 of SMN), wherein a
single guanine residue is inserted into the fragment of the nucleic
acid residues of exon 7 of SMN at the location that corresponds to
the location in exon 7 of SMN that is after the 48th nucleotide
from the 5' end of exon 7 of SMN, and wherein the fragment of the
nucleic acid residues of exon 7 of SMN comprises any number of
nucleotides of exon 7 required for splicing; (c) the nucleic acid
residues of intron 7 of SMN or a fragment thereof, wherein the
fragment of the nucleic acid residues of intron 7 of SMN comprises
any number of nucleotides of intron 7 required for a functional,
minimum intron; (d) a fragment of the nucleic acid residues of exon
8 of SMN; and (e) a reporter gene coding sequence lacking a start
codon, wherein (i) the reporter gene coding sequence is fused to
the fragment of the nucleic acid residues of exon 8 of SMN such
that the first codon of the reporter gene coding sequence and the
first codon of the fragment are out of frame with each other in the
mRNA transcript transcribed from the nucleic acid construct; (ii)
the production of the mRNA transcript generates a stop codon in the
region of the mRNA transcript that corresponds to the fragment of
the nucleic acid residues of exon 8 of SMN (i.e., upstream of the
reporter gene coding sequence); and (iii) in the mRNA transcript
transcribed from the nucleic acid construct, the first start codon
and the stop codon upstream from the reporter gene coding sequence
are in the same contiguous open reading frame without any
interruption by, e.g., a stop codon.
[0043] In a specific embodiment, a nucleic acid construct
comprises, in 5' to 3' order: (a) a minimum of one nucleotide; (b)
a fragment of the nucleic acid residues of exon 7 of SMN, wherein
the fragment of the nucleic acid residues of exon 7 of SMN consists
of the first six nucleotides from the 3' end of exon 7 of SMN
(i.e., nucleotide residues 49 to 54 from the 5' end of exon 7 of
SMN) and wherein a single guanine residue is inserted upstream (5')
of the fragment of the nucleic acid residues of exon 7 of SMN; (c)
the nucleic acid residues of intron 7 of SMN or a fragment thereof,
wherein the fragment of the nucleic acid residues of intron 7 of
SMN comprises any number of nucleotides of intron 7 required for a
functional, minimum intron; (d) a fragment of the nucleic acid
residues of exon 8 of SMN; and (e) a reporter gene coding sequence
lacking a start codon, wherein (i) in the mRNA transcript
transcribed from the nucleic acid construct, open reading of the
fragment of the nucleic acid residues of exon 7 of SMN and the open
reading frame of the fragment of the nucleic acid residues of exon
8 of SMN are in frame with each other; (ii) the reporter gene
coding sequence is fused to the fragment of the nucleic acid
residues of exon 8 of SMN such that the reporter gene coding
sequence and the fragment are out of frame with each other in the
mRNA transcript transcribed from the nucleic acid construct; and
(iii) the production of the mRNA transcript generates a stop codon
in the region of the mRNA transcript that corresponds to the
fragment of the nucleic acid residues of exon 8 of SMN (i.e.,
upstream of the reporter gene coding sequence). In a specific
embodiment, the fragment of the nucleic acid residues of exon 8 of
SMN consists of the first 21 or 23 nucleotides from the 5' end of
exon 8 of SMN. In certain embodiments, the nucleic acid construct
comprises a start codon upstream (5') to the minimum one
nucleotide. In accordance with such embodiments, the first start
codon and the stop codon upstream of the reporter gene coding
sequence in the mRNA transcript are in the same contiguous open
reading frame without any interruption by, e.g., a stop codon.
[0044] In a specific embodiment, a nucleic acid construct
comprises, in 5' to 3' order: (a) a start codon; (b) a minimum of
one nucleotide; (c) a fragment of the nucleic acid residues of exon
7 of SMN, wherein the fragment of the nucleic acid residues of exon
7 of SMN consists of the first six nucleotides from the 3' end of
exon 7 of SMN (i.e., nucleotide residues 49 to 54 from the 5' end
of exon 7 of SMN) and wherein a single guanine residue is inserted
upstream (5') of the fragment of the nucleic acid residues of exon
7 of SMN; (d) the nucleic acid residues of intron 7 of SMN or a
fragment thereof, wherein the fragment of the nucleic acid residues
of intron 7 of SMN comprises any number of nucleotides of intron 7
required for a functional, minimum intron; (e) a fragment of the
nucleic acid residues of exon 8 of SMN; and (f) a reporter gene
coding sequence lacking a start codon, wherein (i) the reporter
gene coding sequence is fused to the fragment of the nucleic acid
residues of exon 8 of SMN such that the first codon of the reporter
gene coding sequence and the first codon of the fragment are out of
frame with each other in the mRNA transcript transcribed from the
nucleic acid construct; (ii) the production of the mRNA transcript
generates a stop codon in the region of the mRNA transcript that
corresponds to the fragment of the nucleic acid residues of exon 8
of SMN (i.e., upstream of the reporter gene coding sequence); and
(iii) in the mRNA transcript transcribed from the nucleic acid
construct, the first start codon and the stop codon upstream from
the reporter gene coding sequence are in the same contiguous open
reading frame without any interruption by, e.g., a stop codon.
[0045] In a specific embodiment, a nucleic acid construct
comprises, in 5' to 3' order: (a) a fragment of the nucleic acid
residues of exon 7 of SMN, wherein the fragment of the nucleic acid
residues of exon 7 of SMN consists of the first six nucleotides
from the 3' end of exon 7 of SMN (i.e., nucleotide residues 49 to
54 from the 5' end of exon 7 of SMN), wherein a single guanine
residue is inserted upstream (5') of the fragment of the nucleic
acid residues of exon 7 of SMN, and wherein the fragment of the
nucleic acid residues of exon 7 of SMN comprises any number of
nucleotides of exon 7 required for splicing; (b) the nucleic acid
residues of intron 7 of SMN or a fragment thereof, wherein the
fragment of the nucleic acid residues of intron 7 of SMN comprises
any number of nucleotides of intron 7 required for a functional,
minimum intron; (c) a fragment of the nucleic acid residues of exon
8 of SMN; and (d) a reporter gene coding sequence lacking a start
codon, wherein (i) in the mRNA transcript transcribed from the
nucleic acid construct, open reading of the fragment of the nucleic
acid residues of exon 7 of SMN and the open reading frame of the
fragment of the nucleic acid residues of exon 8 of SMN are in frame
with each other; (ii) the reporter gene coding sequence is fused to
the fragment of the nucleic acid residues of exon 8 of SMN such
that the reporter gene coding sequence and the fragment are out of
frame with each other in the mRNA transcript transcribed from the
nucleic acid construct; and (iii) the production of the mRNA
transcript generates a stop codon in the region of the mRNA
transcript that corresponds to the fragment of the nucleic acid
residues of exon 8 of SMN (i.e., upstream of the reporter gene
coding sequence). In a specific embodiment, the fragment of the
nucleic acid residues of exon 8 of SMN consists of the first 21 or
23 nucleotides from the 5' end of exon 8 of SMN. In certain
embodiments, the nucleic acid construct comprises a start codon
upstream (5') to the fragment of the nucleic acid residues of exon
7 of SMN. In accordance with such embodiments, the first start
codon and the stop codon upstream of the reporter gene coding
sequence in the mRNA transcript are in the same contiguous open
reading frame without any interruption by, e.g., a stop codon.
[0046] In a specific embodiment, a nucleic acid construct
comprises, in 5' to 3' order: (a) a start codon; (b) a fragment of
the nucleic acid residues of exon 7 of SMN, wherein the fragment of
the nucleic acid residues of exon 7 of SMN consists of the first
six nucleotides from the 3' end of exon 7 of SMN (i.e., nucleotide
residues 49 to 54 from the 5' end of exon 7 of SMN), wherein a
single guanine residue is inserted upstream (5') of the fragment of
the nucleic acid residues of exon 7 of SMN, and wherein the
fragment of the nucleic acid residues of exon 7 of SMN comprises
any number of nucleotides of exon 7 required for splicing; (c) the
nucleic acid residues of intron 7 of SMN or a fragment thereof,
wherein the fragment of the nucleic acid residues of intron 7 of
SMN comprises any number of nucleotides of intron 7 required for a
functional, minimum intron; (d) a fragment of the nucleic acid
residues of exon 8 of SMN; and (e) a reporter gene coding sequence
lacking a start codon, wherein (i) the reporter gene coding
sequence is fused to the fragment of the nucleic acid residues of
exon 8 of SMN such that the first codon of the reporter gene coding
sequence and the first codon of the fragment are out of frame with
each other in the mRNA transcript transcribed from the nucleic acid
construct; (ii) the production of the mRNA transcript generates a
stop codon in the region of the mRNA transcript that corresponds to
the fragment of the nucleic acid residues of exon 8 of SMN (i.e.,
upstream of the reporter gene coding sequence); and (iii) in the
mRNA transcript transcribed from the nucleic acid construct, the
first start codon and the stop codon upstream from the reporter
gene coding sequence are in the same contiguous open reading frame
without any interruption by, e.g., a stop codon.
[0047] In another embodiment, a nucleic acid construct comprises,
in 5' to 3' order: (a) the nucleic acid residues of exon 6 of SMN
or a fragment thereof; (b) a fragment of the nucleic acid residues
of exon 7 of SMN, wherein the fragment of the nucleic acid residues
of exon 7 of SMN comprises a minimum of the first six nucleotides
from the 3' end of exon 7 of SMN (i.e., nucleotide residues 49 to
54 from the 5' end of exon 7 of SMN) and wherein a single guanine
residue is inserted into the fragment of the nucleic acid residues
of exon 7 of SMN at the location that corresponds to the location
in exon 7 of SMN that is after the 48th nucleotide from the 5' end
of exon 7 of SMN; (c) the nucleic acid residues of intron 7 of SMN
or a fragment thereof, wherein the fragment of the nucleic acid
residues of intron 7 of SMN comprises any number of nucleotides of
intron 7 required for a functional, minimum intron; (d) a fragment
of the nucleic acid residues of exon 8 of SMN; and (e) a reporter
gene coding sequence lacking a start codon, wherein (i) the
fragment of the nucleic acid residues of exon 6 of SMN comprises
any number of nucleotides of exon 6 of SMN so long as in the mRNA
transcript transcribed from the nucleic acid construct the open
reading frame of the fragment of the nucleic acid resides of exon 6
of SMN, the open reading frame of the fragment of the nucleic acid
residues of exon 7 of SMN and the open reading frame of the
fragment of the nucleic acid residues of exon 8 of SMN are in frame
with each other; (ii) the open reading frame of the nucleic acid
residues of exon 6 of SMN, the open reading frame of the fragment
of the nucleic acid residues of exon 7 of SMN, and the open reading
frame of the fragment of the nucleic acid residues of exon 8 of SMN
are in frame with each other in the mRNA transcript transcribed
from the nucleic acid construct; (iii) the reporter gene coding
sequence is fused to the fragment of the nucleic acid residues of
exon 8 of SMN such that the reporter gene coding sequence and the
fragment are out of frame with each other in the mRNA transcript
transcribed from the nucleic acid construct; and (iv) the
production of the mRNA transcript generates a stop codon in the
region of the mRNA transcript that corresponds to the fragment of
the nucleic acid residues of exon 8 of SMN (i.e., upstream of the
reporter gene coding sequence). In a specific embodiment, the
fragment of the nucleic acid residues of exon 8 of SMN consists of
the first 21 or 23 nucleotides from the 5' end of exon 8 of SMN. In
certain embodiments, an internal start codon (e.g., ATG) in the
nucleic acid residues of exon 6 of SMN or a fragment thereof is
used as a start codon for the nucleic acid construct. In some
embodiments, the nucleic acid construct comprises a start codon
upstream (5') to the nucleic acid residues of exon 6 of SMN or a
fragment thereof. In accordance with such embodiments, the first
start codon and the stop codon upstream of the reporter gene coding
sequence in the mRNA transcript are in the same contiguous open
reading frame without any interruption by, e.g., a stop codon.
[0048] In another embodiment, a nucleic acid construct comprises,
in 5' to 3' order: (a) a start codon; (b) the nucleic acid residues
of exon 6 of SMN or a fragment thereof; (c) a fragment of the
nucleic acid residues of exon 7 of SMN, wherein the fragment of the
nucleic acid residues of exon 7 of SMN comprises a minimum of the
first six nucleotides from the 3' end of exon 7 of SMN (i.e.,
nucleotide residues 49 to 54 from the 5' end of exon 7 of SMN) and
wherein a single guanine residue is inserted into the fragment of
the nucleic acid residues of exon 7 of SMN at the location that
corresponds to the location in exon 7 of SMN that is after the 48th
nucleotide from the 5' end of exon 7 of SMN; (d) the nucleic acid
residues of intron 7 of SMN or a fragment thereof, wherein the
fragment of the nucleic acid residues of intron 7 of SMN comprises
any number of nucleotides of intron 7 required for a functional,
minimum intron; (e) a fragment of the nucleic acid residues of exon
8 of SMN; and (f) a reporter gene coding sequence lacking a start
codon, wherein (i) the reporter gene coding sequence is fused to
the fragment of the nucleic acid residues of exon 8 of SMN such
that the first codon of the reporter gene coding sequence and the
first codon fragment are out of frame with each other in the mRNA
transcript transcribed from the nucleic acid construct; (ii) the
production of the mRNA transcript generates a stop codon in the
region of the mRNA transcript that corresponds to the fragment of
the nucleic acid residues of exon 8 of SMN (i.e., upstream of the
reporter gene coding sequence); and (iii) in the mRNA transcript
transcribed from the nucleic acid construct, the first start codon
and the stop codon upstream from the reporter gene coding sequence
are in the same contiguous open reading frame without any
interruption by, e.g., a stop codon.
[0049] In another embodiment, a nucleic acid construct comprises,
in 5' to 3' order: (a) the nucleic acid residues of exon 6 of SMN
or a fragment thereof; (b) the nucleic acid residues of intron 6 of
SMN or a fragment thereof, wherein the fragment of the nucleic acid
residues of SMN comprises any number of nucleotides of intron 6 of
SMN for a functional, minimum intron; (c) a fragment of the nucleic
acid residues of exon 7 of SMN, wherein the fragment of the nucleic
acid residues of exon 7 of SMN comprises a minimum of the first six
nucleotides from the 3' end of exon 7 of SMN (i.e., nucleotide
residues 49 to 54 from the 5' end of exon 7 of SMN) and wherein a
single guanine residue is inserted into the fragment of the nucleic
acid residues of exon 7 of SMN at the location that corresponds to
the location in exon 7 of SMN that is after the 48th nucleotide
from the 5' end of exon 7 of SMN; (d) the nucleic acid residues of
intron 7 of SMN or a fragment thereof, wherein the fragment of the
nucleic acid residues of intron 7 of SMN comprises any number of
nucleotides of intron 7 required for a functional, minimum intron;
(e) a fragment of the nucleic acid residues of exon 8 of SMN; and
(f) a reporter gene coding sequence lacking a start codon, wherein
(i) the fragment of the nucleic acid residues of exon 6 of SMN
comprises a minimum of the nucleotides of exon 6 of SMN required
for splicing and in the mRNA transcript transcribed from the
nucleic acid construct the open reading frame of the fragment of
the nucleic acid resides of exon 6 of SMN, the open reading frame
of the fragment of the nucleic acid residues of exon 7 of SMN and
the open reading frame of the fragment of the nucleic acid residues
of exon 8 of SMN are in frame with each other; (ii) the open
reading frame of the nucleic acid residues of exon 6 of SMN, the
open reading frame of the fragment of the nucleic acid residues of
exon 7 of SMN, and the open reading frame of the fragment of the
nucleic acid residues of exon 8 of SMN are in frame with each other
in the mRNA transcript transcribed from the nucleic acid construct;
(iii) the reporter gene coding sequence is fused to the fragment of
the nucleic acid residues of exon 8 of SMN such that the reporter
gene coding sequence and the fragment are out of frame with each
other in the mRNA transcript transcribed from the nucleic acid
construct; and (iv) the production of the mRNA transcript generates
a stop codon in the region of the mRNA transcript that corresponds
to the fragment of the nucleic acid residues of exon 8 of SMN
(i.e., upstream of the reporter gene coding sequence). In a
specific embodiment, the fragment of the nucleic acid residues of
exon 8 of SMN consists of the first 21 or 23 nucleotides from the
5' end of exon 8 of SMN. In another specific embodiment, the
fragment of the nucleic acid residues of exon 7 of SMN comprises a
minimum of the first nucleotide from the 5' end of exon 7 of SMN
and the first two nucleotides from the 3' end of exon 7 of SMN
(i.e., nucleotide residues 53 and 54 from the 5' end of exon 7 of
SMN). In another specific embodiment, the fragment of the nucleic
acid residues of exon 7 of SMN comprises a minimum of the first six
nucleotides from the 3' end of exon 7 of SMN (i.e., nucleotide
residues 49 to 54 from the 5' end of exon 7 of SMN). In another
specific embodiment, the fragment of exon 6 of SMN comprises a
minimum of the first two nucleotides from the 3' end of exon 6 of
SMN. In certain embodiments, an internal start codon (e.g., ATG) in
the nucleic acid residues of exon 6 of SMN or a fragment thereof is
used as a start codon for the nucleic acid construct. In some
embodiments, the nucleic acid construct comprises a start codon
upstream (5') to the nucleic acid residues of exon 6 of SMN or a
fragment thereof. In accordance with such embodiments, the first
start codon and the stop codon upstream of the reporter gene coding
sequence in the mRNA transcript are in the same contiguous open
reading frame without any interruption by, e.g., a stop codon.
[0050] In another embodiment, a nucleic acid construct comprises,
in 5' to 3' order: (a) start codon; (b) the nucleic acid residues
of exon 6 of SMN or a fragment thereof; (c) the nucleic acid
residues of intron 6 of SMN or a fragment thereof, wherein the
fragment of the nucleic acid residues of SMN comprises any number
of nucleotides of intron 6 of SMN for a functional, minimum intron;
(d) a fragment of the nucleic acid residues of exon 7 of SMN,
wherein the fragment of the nucleic acid residues of exon 7 of SMN
comprises a minimum of the first six nucleotides from the 3' end of
exon 7 of SMN (i.e., nucleotide residues 49 to 54 from the 5' end
of exon 7 of SMN) and wherein a single guanine residue is inserted
into the fragment of the nucleic acid residues of exon 7 of SMN at
the location that corresponds to the location in exon 7 of SMN that
is after the 48th nucleotide from the 5' end of exon 7 of SMN; (e)
the nucleic acid residues of intron 7 of SMN or a fragment thereof,
wherein the fragment of the nucleic acid residues of intron 7 of
SMN comprises any number of nucleotides of intron 7 required for a
functional, minimum intron; (f) a fragment of the nucleic acid
residues of exon 8 of SMN; and (g) a reporter gene coding sequence
lacking a start codon, wherein (i) the fragment of the nucleic acid
residues of exon 6 of SMN comprises a minimum of the nucleotides of
exon 6 of SMN required for splicing; (ii) the reporter gene coding
sequence is fused to the fragment of the nucleic acid residues of
exon 8 of SMN such that the first codon of the reporter gene coding
sequence and the first codon fragment are out of frame with each
other in the mRNA transcript transcribed from the nucleic acid
construct; (iii) the production of the mRNA transcript generates a
stop codon in the region of the mRNA transcript that corresponds to
the fragment of the nucleic acid residues of exon 8 of SMN (i.e.,
upstream of the reporter gene coding sequence); and (iv) in the
mRNA transcript transcribed from the nucleic acid construct, the
first start codon and the stop codon upstream from the reporter
gene coding sequence are in the same contiguous open reading frame
without any interruption by, e.g., a stop codon.
[0051] In certain aspects of the invention, an RNA transcript
transcribed from a nucleic acid construct described above is
utilized in the cell-based and cell-free screening assays to
identify or validate compounds that modulate ribosomal
frameshifting (e.g., programmed ribosomal frameshifting). In a
specific embodiment, a mRNA transcript transcribed from a nucleic
acid construct described above is utilized in the cell-based and
cell-free screening assays to identify or validate compounds that
modulate ribosomal frameshifting (e.g., programmed ribosomal
frameshifting).
[0052] In one embodiment, the invention provides a method for the
identification or validation of a compound that modulates of
ribosomal frameshifting comprising: (a) contacting a compound with
a host cell containing a nucleic acid construct described herein;
and (b) detecting the activity or amount of a fusion protein
expressed from the nucleic acid construct, wherein an increase in
the activity or amount of the fusion protein expressed by the host
cell in the presence of a compound when compared to (i) a
previously determined reference range for a negative control, (ii)
the activity or amount of the fusion protein expressed by the host
cell in the absence of the compound, or (iii) the activity or
amount of the fusion protein expressed by the host cell in the
presence of a negative control indicates that the compound
modulates ribosomal frameshifting.
[0053] In another embodiment, the invention provides a method for
the identification or validation of a compound that modulates
ribosomal frameshifting comprising: (a) contacting a compound with
a host cell containing an RNA transcript transcribed from a nucleic
acid construct described herein; and (b) detecting the activity or
amount of a fusion protein translated from the RNA transcript,
wherein an increase in the activity or amount of the fusion protein
translated from the RNA transcript in the presence of a compound
when compared to (i) a previously determined reference range for a
negative control, (ii) the activity or amount of the fusion protein
translated from the RNA transcript in the absence of the compound,
or (iii) the activity or amount of the fusion protein translated
from the RNA transcript in the presence of a negative control
indicates that the compound modulates ribosomal frameshifting.
[0054] In one embodiment, the invention provides a method for the
identification or validation of a compound that modulates the
efficiency of programmed ribosomal frameshifting comprising: (a)
contacting a compound with a host cell containing a nucleic acid
construct described herein; and (b) detecting the activity or
amount of a fusion protein expressed from the nucleic acid
construct, wherein an increase in the activity or amount of the
fusion protein expressed by the host cell in the presence of a
compound when compared to (i) a previously determined reference
range for a negative control, (ii) the activity or amount of the
fusion protein expressed by the host cell in the absence of the
compound, or (iii) the activity or amount of the fusion protein
expressed by the host cell in the presence of a negative control
indicates that the compound modulates the efficiency of programmed
ribosomal frameshifting.
[0055] In another embodiment, the invention provides a method for
the identification or validation of a compound that modulates the
efficiency of programmed ribosomal frameshifting comprising: (a)
contacting a compound with a host cell containing an RNA transcript
transcribed from a nucleic acid construct described herein; and (b)
detecting the activity or amount of a fusion protein translated
from the RNA transcript, wherein an increase in the activity or
amount of the fusion protein translated from the RNA transcript in
the presence of a compound when compared to (i) a previously
determined reference range for a negative control, (ii) the
activity or amount of the fusion protein translated from the RNA
transcript in the absence of the compound, or (iii) the activity or
amount of the fusion protein translated from the RNA transcript in
the presence of a negative control indicates that the compound
modulates the efficiency of programmed ribosomal frameshifting.
[0056] In another embodiment, the invention provides a method for
identifying or validating a compound that modulates ribosomal
frameshifting comprising: (a) contacting a compound with a
cell-free extract and an RNA transcript transcribed from a nucleic
acid construct described herein; and (b) detecting the amount or
activity of the fusion protein translated from the RNA transcript,
wherein an increase in the activity or amount of the fusion protein
translated from the RNA transcript in the presence of a compound
when compared to (i) a previously determined reference range for a
negative control, (ii) the activity or amount of the fusion protein
translated from the RNA transcript in the absence of the compound,
or (iii) the activity or amount of the fusion protein translated
from the RNA transcript in the presence of a negative control
indicates that the compound modulates ribosomal frameshifting.
[0057] In another embodiment, the invention provides a method for
identifying or validating a compound that modulates the efficiency
of programmed ribosomal frameshifting comprising: (a) contacting a
compound with a cell-free extract and an RNA transcript transcribed
from a nucleic acid construct described herein; and (b) detecting
the amount or activity of the fusion protein translated from the
RNA transcript, wherein an increase in the activity or amount of
the fusion protein translated from the RNA transcript in the
presence of a compound when compared to (i) a previously determined
reference range for a negative control, (ii) the activity or amount
of the fusion protein translated from the RNA transcript in the
absence of the compound, or (iii) the activity or amount of the
fusion protein translated from the RNA transcript in the presence
of a negative control indicates that the compound modulates the
efficiency of programmed ribosomal frameshifting.
[0058] The compounds identified or validated in the cell-based or
cell-free assays may be used to modulate the efficiency of viral
programmed ribosomal frameshifting and may be used to inhibit or
reduce viral replication, inhibit or reduce viral infectivity,
treat a viral infection, prevent a viral disease, or treat a viral
disease. In one embodiment, the invention provides a method for
modulating the efficiency of viral programmed ribosomal
frameshifting, comprising contacting a compound with a mixture of a
cell and a virus that employs ribosomal frameshifting, wherein the
compound in vitro or in cells increases the amount or activity of a
fusion protein encoded by a nucleic acid construct or translated
from a RNA transcript (e.g., a mRNA transcript) transcribed from
the nucleic acid construct, and wherein the nucleic acid construct
comprises, in 5' to 3' order: (i) the nucleic acid residues of exon
6 of SMN; (ii) the nucleic acid residues of intron 6 of SMN; (iii)
the nucleic acid residues of exon 7 of SMN, wherein a single
guanine is inserted after the 48th nucleotide residue from the 5'
end of exon 7 of SMN (i.e., before the 6th nucleotide from the 3'
end of exon 7 of SMN); (iv) the nucleic acid residues of intron 7
of SMN; (v) a fragment of the nucleic acid residues of exon 8 of
SMN, wherein the fragment is composed of the first 23 nucleotides
from the 5' end of exon 8 of SMN; and (vi) a reporter gene lacking
a start codon, wherein the reporter gene is fused to the fragment
of the nucleic acid residues of exon 8 of SMN such that the
reporter gene and the fragment are out of frame with each other in
the mRNA transcript transcribed from the nucleic acid construct,
and wherein the production of the mRNA transcript generates a stop
codon in the region of the mRNA transcript that corresponds to the
fragment of the nucleic acid residues of exon 8 of SMN. In certain
embodiments, an internal start codon (e.g., an ATG) found in exon 6
is used as the start codon for the nucleic acid construct. In some
embodiments, the nucleic acid construct comprises a start codon 5'
to the nucleic acid residues of exon 6 of SMN. In one embodiment,
the compound is a compound of Formula (I) or a form thereof. In
another embodiment, the compound is a compound of Formula (II) or a
form thereof. In another embodiment, the compound is a compound of
Formula (Ia) or a form thereof. In another embodiment, the compound
is a compound of Formula (IIa) or a form thereof. In a specific
embodiment, the compound is Compound 1.
[0059] In another embodiment, the invention provides a method for
modulating the efficiency of viral programmed ribosomal
frameshifting, comprising contacting a compound with a cell
containing a virus or provirus that employs programmed ribosomal
frameshifting, wherein the compound in vitro or in cells increases
the amount or activity of a fusion protein encoded by a nucleic
acid construct or translated from a RNA transcript (e.g., a mRNA
transcript) transcribed from the nucleic acid construct, and
wherein the nucleic acid construct comprises, in 5' to 3' order:
(i) the nucleic acid residues of exon 6 of SMN; (ii) the nucleic
acid residues of intron 6 of SMN; (iii) the nucleic acid residues
of exon 7 of SMN, wherein a single guanine is inserted after the
48th nucleotide residue from the 5' end of exon 7 of SMN (i.e.,
before the 6th nucleotide from the 3' end of exon 7 of SMN); (iv)
the nucleic acid residues of intron 7 of SMN; (v) a fragment of the
nucleic acid residues of exon 8 of SMN, wherein the fragment is
composed of the first 23 nucleotides from the 5' end of exon 8 of
SMN; and (vi) a reporter gene lacking a start codon, wherein the
reporter gene is fused to the fragment of the nucleic acid residues
of exon 8 of SMN such that the reporter gene and the fragment are
out of frame with each other in the mRNA transcript transcribed
from the nucleic acid construct, and wherein the production of the
mRNA transcript generates a stop codon in the region of the mRNA
transcript that corresponds to the fragment of the nucleic acid
residues of exon 8 of SMN. In certain embodiments, an internal
start codon (e.g., an ATG) found in exon 6 is used as the start
codon for the nucleic acid construct. In some embodiments, the
nucleic acid construct comprises a start codon 5' to the nucleic
acid residues of exon 6 of SMN. In one embodiment, the compound is
a compound of Formula (I) or a form thereof. In another embodiment,
the compound is a compound of Formula (II) or a form thereof. In
another embodiment, the compound is a compound of Formula (Ia) or a
form thereof. In another embodiment, the compound is a compound of
Formula (IIa) or a form thereof. In a specific embodiment, the
compound is Compound 1.
[0060] In another embodiment, the invention provides a method for
altering the ratio of two viral genes expressed from overlapping
reading frames in which programmed ribosomal frameshifting
regulates the expression of the two genes, comprising contacting a
compound with a mixture of a cell and a virus that employs
programmed ribosomal frameshifting, wherein the compound in vitro
or in cells increases the amount or activity of a fusion protein
encoded by a nucleic acid construct or translated from a RNA
transcript (e.g., a mRNA transcript) transcribed from the nucleic
acid construct, and wherein the nucleic acid construct comprises,
in 5' to 3' order: (i) the nucleic acid residues of exon 6 of SMN;
(ii) the nucleic acid residues of intron 6 of SMN; (iii) the
nucleic acid residues of exon 7 of SMN, wherein a single guanine is
inserted after the 48th nucleotide residue from the 5' end of exon
7 of SMN (i.e., before the 6th nucleotide from the 3' end of exon 7
of SMN); (iv) the nucleic acid residues of intron 7 of SMN; (v) a
fragment of the nucleic acid residues of exon 8 of SMN, wherein the
fragment is composed of the first 23 nucleotides from the 5' end of
exon 8 of SMN; and (vi) a reporter gene lacking a start codon,
wherein the reporter gene is fused to the fragment of the nucleic
acid residues of exon 8 of SMN such that the reporter gene and the
fragment are out of frame with each other in the mRNA transcript
transcribed from the nucleic acid construct, and wherein the
production of the mRNA transcript generates a stop codon in the
region of the mRNA transcript that corresponds to the fragment of
the nucleic acid residues of exon 8 of SMN. In certain embodiments,
an internal start codon (e.g., an ATG) found in exon 6 is used as
the start codon for the nucleic acid construct. In some
embodiments, the nucleic acid construct comprises a start codon 5'
to the nucleic acid residues of exon 6 of SMN. In one embodiment,
the compound is a compound of Formula (I) or a form thereof. In
another embodiment, the compound is a compound of Formula (II) or a
form thereof. In another embodiment, the compound is a compound of
Formula (Ia) or a form thereof. In another embodiment, the compound
is a compound of Formula (IIa) or a form thereof. In a specific
embodiment, the compound is Compound 1.
[0061] In another embodiment, the invention provides a method for
altering the ratio of two viral genes expressed from overlapping
reading frames in which programmed ribosomal frameshifting
regulates the expression of the two genes, comprising contacting a
compound with a cell containing a virus or provirus that employs
programmed ribosomal frameshifting, wherein the compound in vitro
or in cells increases the amount or activity of a fusion protein
encoded by a nucleic acid construct or translated from a RNA
transcript (e.g., a mRNA transcript) transcribed from the nucleic
acid construct, and wherein the nucleic acid construct comprises,
in 5' to 3' order: (i) the nucleic acid residues of exon 6 of SMN;
(ii) the nucleic acid residues of intron 6 of SMN; (iii) the
nucleic acid residues of exon 7 of SMN, wherein a single guanine is
inserted after the 48th nucleotide residue from the 5' end of exon
7 of SMN (i.e., before the 6th nucleotide from the 3' end of exon 7
of SMN); (iv) the nucleic acid residues of intron 7 of SMN; (v) a
fragment of the nucleic acid residues of exon 8 of SMN, wherein the
fragment is composed of the first 23 nucleotides from the 5' end of
exon 8 of SMN; and (vi) a reporter gene lacking a start codon,
wherein the reporter gene is fused to the fragment of the nucleic
acid residues of exon 8 of SMN such that the reporter gene and the
fragment are out of frame with each other in the mRNA transcript
transcribed from the nucleic acid construct, and wherein the
production of the mRNA transcript generates a stop codon in the
region of the mRNA transcript that corresponds to the fragment of
the nucleic acid residues of exon 8 of SMN. In certain embodiments,
an internal start codon (e.g., an ATG) found in exon 6 is used as
the start codon for the nucleic acid construct. In some
embodiments, the nucleic acid construct comprises a start codon 5'
to the nucleic acid residues of exon 6 of SMN. In one embodiment,
the compound is a compound of Formula (I) or a form thereof. In
another embodiment, the compound is a compound of Formula (II) or a
form thereof. In another embodiment, the compound is a compound of
Formula (Ia) or a form thereof. In another embodiment, the compound
is a compound of Formula (IIa) or a form thereof. In a specific
embodiment, the compound is Compound 1.
[0062] In certain embodiments, the cells used in the cell-based
assays described herein are susceptible to infection with a
virus.
[0063] A compound identified or validated in the cell-based or
cell-free assays that modulate the efficiency of viral programmed
ribosomal frameshifting may be used as an antiviral agent.
Compounds that modulate the efficiency of programmed ribosomal
frameshifting have several advantages over other antiviral agents
currently in use. For example, a compound that modulates the
efficiency of programmed ribosomal frameshifting is expected to
have limited adverse effects on cellular gene expression because it
is likely that mammalian ribosomal frameshifting may be the result
of cis-acting sequences and transacting factors that are different
from those of viral (e.g., HIV-1) programmed -1 ribosomal
frameshifting. There are only two known mammalian genes (Edr and
Ma3) that are expressed by -1 ribosomal frameshifting (Shigemoto,
et al., 2001, Nucleic Acids Res. 29:4079-4088; Wills, et al., 2006,
J. Biol. Chem. 281:7082-7088). Edr is expressed during
embryogenesis. Ma3 is a member of family of mammalian genes whose
protein products are the target of immunity associated with
paraneoplastic disorders. However, downstream of both Edr and Ma3
ribosomal frameshifting signals there is a pseudoknot structure
(see, Shigemoto, et al., 2001, Nucleic Acids Res. 29:4079-4088;
Wills, et al., 2006, J. Biol. Chem. 281:7082-7088), which is
fundamentally different from that of the HIV ribosomal
frameshifting stem-loop (Gaudin, et al., 2005, J. Mol. Biol.
349:1024-1035).
[0064] In one embodiment, the invention provides a method for
reducing or inhibiting viral replication, comprising contacting a
cell or a population of cells containing a virus or provirus that
employs programmed ribosomal frameshifting with a compound, wherein
the compound in vitro or in cells increases the amount or activity
of a fusion protein encoded by a nucleic acid construct or
translated from a RNA transcript (e.g., a mRNA transcript)
transcribed from the nucleic acid construct, and wherein the
nucleic acid construct comprises, in 5' to 3' order: (i) the
nucleic acid residues of exon 6 of SMN; (ii) the nucleic acid
residues of intron 6 of SMN; (iii) the nucleic acid residues of
exon 7 of SMN, wherein a single guanine is inserted after the 48th
nucleotide residue from the 5' end of exon 7 of SMN (i.e., before
the 6th nucleotide from the 3' end of exon 7 of SMN); (iv) the
nucleic acid residues of intron 7 of SMN; (v) a fragment of the
nucleic acid residues of exon 8 of SMN, wherein the fragment is
composed of the first 23 nucleotides from the 5' end of exon 8 of
SMN; and (vi) a reporter gene lacking a start codon, wherein the
reporter gene is fused to the fragment of the nucleic acid residues
of exon 8 of SMN such that the reporter gene and the fragment are
out of frame with each other in the mRNA transcript transcribed
from the nucleic acid construct, and wherein the production of the
mRNA transcript generates a stop codon in the region of the mRNA
transcript that corresponds to the fragment of the nucleic acid
residues of exon 8 of SMN. In certain embodiments, an internal
start codon (e.g., an ATG) found in exon 6 is used as the start
codon for the nucleic acid construct. In some embodiments, the
nucleic acid construct comprises a start codon 5' to the nucleic
acid residues of exon 6 of SMN. In one embodiment, the compound is
a compound of Formula (I) or a form thereof. In another embodiment,
the compound is a compound of Formula (II) or a form thereof. In
another embodiment, the compound is a compound of Formula (Ia) or a
form thereof. In another embodiment, the compound is a compound of
Formula (IIa) or a form thereof. In a specific embodiment, the
compound is Compound 1.
[0065] In another embodiment, the invention provides a method for
reducing or inhibiting viral replication, comprising contacting a
compound with a mixture of a cell or a population of cells and a
virus that employs programmed ribosomal frameshifting, wherein the
compound in vitro or in cells increases the amount or activity of a
fusion protein encoded by a nucleic acid construct or translated
from a RNA transcript (e.g., a mRNA transcript) transcribed from
the nucleic acid construct, and wherein the nucleic acid construct
comprises, in 5' to 3' order: (i) the nucleic acid residues of exon
6 of SMN; (ii) the nucleic acid residues of intron 6 of SMN; (iii)
the nucleic acid residues of exon 7 of SMN, wherein a single
guanine is inserted after the 48th nucleotide residue from the 5'
end of exon 7 of SMN (i.e., before the 6th nucleotide from the 3'
end of exon 7 of SMN); (iv) the nucleic acid residues of intron 7
of SMN; (v) a fragment of the nucleic acid residues of exon 8 of
SMN, wherein the fragment is composed of the first 23 nucleotides
from the 5' end of exon 8 of SMN; and (vi) a reporter gene lacking
a start codon, wherein the reporter gene is fused to the fragment
of the nucleic acid residues of exon 8 of SMN such that the
reporter gene and the fragment are out of frame with each other in
the mRNA transcript transcribed from the nucleic acid construct,
and wherein the production of the mRNA transcript generates a stop
codon in the region of the mRNA transcript that corresponds to the
fragment of the nucleic acid residues of exon 8 of SMN. In certain
embodiments, an internal start codon (e.g., an ATG) found in exon 6
is used as the start codon for the nucleic acid construct. In some
embodiments, the nucleic acid construct comprises a start codon 5'
to the nucleic acid residues of exon 6 of SMN. In one embodiment,
the compound is a compound of Formula (I) or a form thereof. In
another embodiment, the compound is a compound of Formula (II) or a
form thereof. In another embodiment, the compound is a compound of
Formula (Ia) or a form thereof. In another embodiment, the compound
is a compound of Formula (IIa) or a form thereof. In a specific
embodiment, the compound is Compound 1.
[0066] In another embodiment, the invention provides a method for
reducing or inhibiting a viral infection, comprising contacting a
cell or a population of cells containing a virus or provirus that
employs programmed ribosomal frameshifting with a compound, wherein
the compound in vitro or in cells increases the amount or activity
of a fusion protein encoded by a nucleic acid construct or
translated from a RNA transcript (e.g., a mRNA transcript)
transcribed from the nucleic acid construct, and wherein the
nucleic acid construct comprises, in 5' to 3' order: (i) the
nucleic acid residues of exon 6 of SMN; (ii) the nucleic acid
residues of intron 6 of SMN; (iii) the nucleic acid residues of
exon 7 of SMN, wherein a single guanine is inserted after the 48th
nucleotide residue from the 5' end of exon 7 of SMN (i.e., before
the 6th nucleotide from the 3' end of exon 7 of SMN); (iv) the
nucleic acid residues of intron 7 of SMN; (v) a fragment of the
nucleic acid residues of exon 8 of SMN, wherein the fragment is
composed of the first 23 nucleotides from the 5' end of exon 8 of
SMN; and (vi) a reporter gene lacking a start codon, wherein the
reporter gene is fused to the fragment of the nucleic acid residues
of exon 8 of SMN such that the reporter gene and the fragment are
out of frame with each other in the mRNA transcript transcribed
from the nucleic acid construct, and wherein the production of the
mRNA transcript generates a stop codon in the region of the mRNA
transcript that corresponds to the fragment of the nucleic acid
residues of exon 8 of SMN. In certain embodiments, an internal
start codon (e.g., an ATG) found in exon 6 is used as the start
codon for the nucleic acid construct. In some embodiments, the
nucleic acid construct comprises a start codon 5' to the nucleic
acid residues of exon 6 of SMN. In one embodiment, the compound is
a compound of Formula (I) or a form thereof. In another embodiment,
the compound is a compound of Formula (II) or a form thereof. In
another embodiment, the compound is a compound of Formula (Ia) or a
form thereof. In another embodiment, the compound is a compound of
Formula (IIa) or a form thereof. In a specific embodiment, the
compound is Compound 1.
[0067] In another embodiment, the invention provides a method for
reducing or inhibiting a viral infection, comprising contacting a
compound with a mixture of a cell or a population of cells and a
virus that employs programmed ribosomal frameshifting, wherein the
compound in vitro or in cells increases the amount or activity of a
fusion protein encoded by a nucleic acid construct or translated
from a RNA transcript (e.g., a mRNA transcript) transcribed from
the nucleic acid construct, and wherein the nucleic acid construct
comprises, in 5' to 3' order: (i) the nucleic acid residues of exon
6 of SMN; (ii) the nucleic acid residues of intron 6 of SMN; (iii)
the nucleic acid residues of exon 7 of SMN, wherein a single
guanine is inserted after the 48th nucleotide residue from the 5'
end of exon 7 of SMN (i.e., before the 6th nucleotide from the 3'
end of exon 7 of SMN); (iv) the nucleic acid residues of intron 7
of SMN; (v) a fragment of the nucleic acid residues of exon 8 of
SMN, wherein the fragment is composed of the first 23 nucleotides
from the 5' end of exon 8 of SMN; and (vi) a reporter gene lacking
a start codon, wherein the reporter gene is fused to the fragment
of the nucleic acid residues of exon 8 of SMN such that the
reporter gene and the fragment are out of frame with each other in
the mRNA transcript transcribed from the nucleic acid construct,
and wherein the production of the mRNA transcript generates a stop
codon in the region of the mRNA transcript that corresponds to the
fragment of the nucleic acid residues of exon 8 of SMN. In certain
embodiments, an internal start codon (e.g., an ATG) found in exon 6
is used as the start codon for the nucleic acid construct. In some
embodiments, the nucleic acid construct comprises a start codon 5'
to the nucleic acid residues of exon 6 of SMN. In one embodiment,
the compound is a compound of Formula (I) or a form thereof. In
another embodiment, the compound is a compound of Formula (II) or a
form thereof. In another embodiment, the compound is a compound of
Formula (Ia) or a form thereof. In another embodiment, the compound
is a compound of Formula (IIa) or a form thereof. In a specific
embodiment, the compound is Compound 1.
[0068] In another embodiment, the invention provides a method for
inhibiting or reducing viral replication in a subject, comprising
administering to a subject in need thereof an effective amount of a
compound or a pharmaceutical composition thereof, wherein the
compound in vitro or in cells increases the amount or activity of a
fusion protein encoded by a nucleic acid construct or translated
from a RNA transcript (e.g., a mRNA transcript) transcribed from
the nucleic acid construct, and wherein the nucleic acid construct
comprises, in 5' to 3' order: (i) the nucleic acid residues of exon
6 of SMN; (ii) the nucleic acid residues of intron 6 of SMN; (iii)
the nucleic acid residues of exon 7 of SMN, wherein a single
guanine is inserted after the 48th nucleotide residue from the 5'
end of exon 7 of SMN (i.e., before the 6th nucleotide from the 3'
end of exon 7 of SMN); (iv) the nucleic acid residues of intron 7
of SMN; (v) a fragment of the nucleic acid residues of exon 8 of
SMN, wherein the fragment is composed of the first 23 nucleotides
from the 5' end of exon 8 of SMN; and (vi) a reporter gene lacking
a start codon, wherein the reporter gene is fused to the fragment
of the nucleic acid residues of exon 8 of SMN such that the
reporter gene and the fragment are out of frame with each other in
the mRNA transcript transcribed from the nucleic acid construct,
and wherein the production of the mRNA transcript generates a stop
codon in the region of the mRNA transcript that corresponds to the
fragment of the nucleic acid residues of exon 8 of SMN. In certain
embodiments, an internal start codon (e.g., an ATG) found in exon 6
is used as the start codon for the nucleic acid construct. In some
embodiments, the nucleic acid construct comprises a start codon 5'
to the nucleic acid residues of exon 6 of SMN. In one embodiment,
the compound is a compound of Formula (I) or a form thereof. In
another embodiment, the compound is a compound of Formula (II) or a
form thereof. In another embodiment, the compound is a compound of
Formula (Ia) or a form thereof. In another embodiment, the compound
is a compound of Formula (IIa) or a form thereof. In a specific
embodiment, the compound is Compound 1.
[0069] In another embodiment, the invention provides a method for
reducing viral titers in a subject, comprising administering to a
subject in need thereof an effective amount of a compound or
pharmaceutical composition thereof, wherein the compound in vitro
or in cells increases the amount or activity of a fusion protein
encoded by a nucleic acid construct or translated from a RNA
transcript (e.g., a mRNA transcript) transcribed from the nucleic
acid construct, and wherein the nucleic acid construct comprises,
in 5' to 3' order: (i) the nucleic acid residues of exon 6 of SMN;
(ii) the nucleic acid residues of intron 6 of SMN; (iii) the
nucleic acid residues of exon 7 of SMN, wherein a single guanine is
inserted after the 48th nucleotide residue from the 5' end of exon
7 of SMN (i.e., before the 6th nucleotide from the 3' end of exon 7
of SMN); (iv) the nucleic acid residues of intron 7 of SMN; (v) a
fragment of the nucleic acid residues of exon 8 of SMN, wherein the
fragment is composed of the first 23 nucleotides from the 5' end of
exon 8 of SMN; and (vi) a reporter gene lacking a start codon,
wherein the reporter gene is fused to the fragment of the nucleic
acid residues of exon 8 of SMN such that the reporter gene and the
fragment are out of frame with each other in the mRNA transcript
transcribed from the nucleic acid construct, and wherein the
production of the mRNA transcript generates a stop codon in the
region of the mRNA transcript that corresponds to the fragment of
the nucleic acid residues of exon 8 of SMN. In certain embodiments,
an internal start codon (e.g., an ATG) found in exon 6 is used as
the start codon for the nucleic acid construct. In some
embodiments, the nucleic acid construct comprises a start codon 5'
to the nucleic acid residues of exon 6 of SMN. In one embodiment,
the compound is a compound of Formula (I) or a form thereof. In
another embodiment, the compound is a compound of Formula (II) or a
form thereof. In another embodiment, the compound is a compound of
Formula (Ia) or a form thereof. In another embodiment, the compound
is a compound of Formula (IIa) or a form thereof. In a specific
embodiment, the compound is Compound 1.
[0070] In another embodiment, the invention provides a method for
treating a viral infection in a subject, comprising administering
to a subject in need thereof an effective amount of a compound or
pharmaceutical composition thereof, wherein the compound in vitro
or in cells increases the amount or activity of a fusion protein
encoded by a nucleic acid construct or translated from a RNA
transcript (e.g., a mRNA transcript) transcribed from the nucleic
acid construct, and wherein the nucleic acid construct comprises,
in 5' to 3' order: (i) the nucleic acid residues of exon 6 of SMN;
(ii) the nucleic acid residues of intron 6 of SMN; (iii) the
nucleic acid residues of exon 7 of SMN, wherein a single guanine is
inserted after the 48th nucleotide residue from the 5' end of exon
7 of SMN (i.e., before the 6th nucleotide from the 3' end of exon 7
of SMN); (iv) the nucleic acid residues of intron 7 of SMN; (v) a
fragment of the nucleic acid residues of exon 8 of SMN, wherein the
fragment is composed of the first 23 nucleotides from the 5' end of
exon 8 of SMN; and (vi) a reporter gene lacking a start codon,
wherein the reporter gene is fused to the fragment of the nucleic
acid residues of exon 8 of SMN such that the reporter gene and the
fragment are out of frame with each other in the mRNA transcript
transcribed from the nucleic acid construct, and wherein the
production of the mRNA transcript generates a stop codon in the
region of the mRNA transcript that corresponds to the fragment of
the nucleic acid residues of exon 8 of SMN. In certain embodiments,
an internal start codon (e.g., an ATG) found in exon 6 is used as
the start codon for the nucleic acid construct. In some
embodiments, the nucleic acid construct comprises a start codon 5'
to the nucleic acid residues of exon 6 of SMN. In one embodiment,
the compound is a compound of Formula (I) or a form thereof. In
another embodiment, the compound is a compound of Formula (II) or a
form thereof. In another embodiment, the compound is a compound of
Formula (Ia) or a form thereof. In another embodiment, the compound
is a compound of Formula (IIa) or a form thereof. In a specific
embodiment, the compound is Compound 1.
[0071] In another embodiment, the invention provides a method for
preventing a viral disease in a subject, comprising administering
to a subject in need thereof an effective amount of a compound or
pharmaceutical composition thereof, wherein the compound in vitro
or in cells increases the amount or activity of a fusion protein
encoded by a nucleic acid construct or translated from a RNA
transcript (e.g., a mRNA transcript) transcribed from the nucleic
acid construct, and wherein the nucleic acid construct comprises,
in 5' to 3' order: (i) the nucleic acid residues of exon 6 of SMN;
(ii) the nucleic acid residues of intron 6 of SMN; (iii) the
nucleic acid residues of exon 7 of SMN, wherein a single guanine is
inserted after the 48th nucleotide residue from the 5' end of exon
7 of SMN (i.e., before the 6th nucleotide from the 3' end of exon 7
of SMN); (iv) the nucleic acid residues of intron 7 of SMN; (v) a
fragment of the nucleic acid residues of exon 8 of SMN, wherein the
fragment is composed of the first 23 nucleotides from the 5' end of
exon 8 of SMN; and (vi) a reporter gene lacking a start codon,
wherein the reporter gene is fused to the fragment of the nucleic
acid residues of exon 8 of SMN such that the reporter gene and the
fragment are out of frame with each other in the mRNA transcript
transcribed from the nucleic acid construct, and wherein the
production of the mRNA transcript generates a stop codon in the
region of the mRNA transcript that corresponds to the fragment of
the nucleic acid residues of exon 8 of SMN. In certain embodiments,
an internal start codon (e.g., an ATG) found in exon 6 is used as
the start codon for the nucleic acid construct. In some
embodiments, the nucleic acid construct comprises a start codon 5'
to the nucleic acid residues of exon 6 of SMN. In one embodiment,
the compound is a compound of Formula (I) or a form thereof. In
another embodiment, the compound is a compound of Formula (II) or a
form thereof. In another embodiment, the compound is a compound of
Formula (Ia) or a form thereof. In another embodiment, the compound
is a compound of Formula (IIa) or a form thereof. In a specific
embodiment, the compound is Compound 1.
[0072] In another embodiment, the invention provides a method for
treating a viral disease in a subject, comprising administering to
a subject in need thereof an effective amount of a compound or
pharmaceutical composition thereof, wherein the compound in vitro
or in cells increases the amount or activity of a fusion protein
encoded by a nucleic acid construct or translated from a RNA
transcript (e.g., a mRNA transcript) transcribed from the nucleic
acid construct, and wherein the nucleic acid construct comprises,
in 5' to 3' order: (i) the nucleic acid residues of exon 6 of SMN;
(ii) the nucleic acid residues of intron 6 of SMN; (iii) the
nucleic acid residues of exon 7 of SMN, wherein a single guanine is
inserted after the 48th nucleotide residue from the 5' end of exon
7 of SMN (i.e., before the 6th nucleotide from the 3' end of exon 7
of SMN); (iv) the nucleic acid residues of intron 7 of SMN; (v) a
fragment of the nucleic acid residues of exon 8 of SMN, wherein the
fragment is composed of the first 23 nucleotides from the 5' end of
exon 8 of SMN; and (vi) a reporter gene lacking a start codon,
wherein the reporter gene is fused to the fragment of the nucleic
acid residues of exon 8 of SMN such that the reporter gene and the
fragment are out of frame with each other in the mRNA transcript
transcribed from the nucleic acid construct, and wherein the
production of the mRNA transcript generates a stop codon in the
region of the mRNA transcript that corresponds to the fragment of
the nucleic acid residues of exon 8 of SMN. In certain embodiments,
an internal start codon (e.g., an ATG) found in exon 6 is used as
the start codon for the nucleic acid construct. In some
embodiments, the nucleic acid construct comprises a start codon 5'
to the nucleic acid residues of exon 6 of SMN. In one embodiment,
the compound is a compound of Formula (I) or a form thereof. In
another embodiment, the compound is a compound of Formula (II) or a
form thereof. In another embodiment, the compound is a compound of
Formula (Ia) or a form thereof. In another embodiment, the compound
is a compound of Formula (IIa) or a form thereof. In a specific
embodiment, the compound is Compound 1.
[0073] In another embodiment, the invention provides a method for
inhibiting or reducing viral replication comprising contacting a
cell or a population of cells containing a virus or provirus with
an effective amount of a compound or a pharmaceutical composition
thereof, wherein the virus employs programmed ribosomal
frameshifting, and wherein the effective amount is an amount
sufficient to cause a statistically significant change in viral
programmed ribosomal frameshifting efficiency as measured by a
technique known to one of skill in the art or as described herein,
e.g., a dual luciferase construct assay. In another embodiment, the
invention provides a method for inhibiting viral replication
comprising contacting a cell or a population of cells containing a
virus or provirus with an effective amount of a compound or a
pharmaceutical composition thereof, wherein the virus employs
programmed ribosomal frameshifting, and wherein the effective
amount is an amount effective to alter the efficiency of programmed
ribosomal frameshifting by at least 20% (and in some embodiments,
at least 25%, at least 30%, at least 40%, at least 50%, or at least
75%) as measured by a technique known to one of skill in the art or
described herein, e.g., a dual luciferase construct assay.
[0074] In another embodiment, the invention provides a method for
inhibiting or reducing viral replication comprising contacting a
mixture of a cell or a population of cells and a virus with an
effective amount of a compound or a pharmaceutical composition
thereof, wherein the virus employs programmed ribosomal
frameshifting, and wherein the effective amount is an amount
sufficient to cause a statistically significant change in viral
programmed ribosomal frameshifting efficiency as measured by a
technique known to one of skill in the art or as described herein,
e.g., a dual luciferase construct assay. In another embodiment, the
invention provides a method for inhibiting or reducing viral
replication comprising contacting a mixture of a cell or a
population of cells and a virus with an effective amount of a
compound or a pharmaceutical composition thereof, wherein the virus
employs programmed ribosomal frameshifting, and wherein the
effective amount is an amount effective to alter the efficiency of
programmed ribosomal frameshifting by at least 20% (and in some
embodiments, at least 25%, at least 30%, at least 40%, at least 50%
or at least 75%) as measured by a technique known to one of skill
in the art or as described herein, e.g., a dual luciferase
construct assay.
[0075] In another embodiment, the invention provides a method for
inhibiting or reducing viral infectivity, comprising contacting a
cell or a population of cells containing a virus or provirus with
an effective amount of a compound or a pharmaceutical composition
thereof, wherein the virus employs programmed ribosomal
frameshifting, and wherein the effective amount is an amount
sufficient to cause a statistically significant change in viral
programmed ribosomal frameshifting efficiency as measured by a
technique known to one of skill in the art or as described herein,
e.g., a dual luciferase construct assay. In another embodiment, the
invention provides a method for inhibiting or reducing viral
infectivity, comprising contacting a cell or a population of cells
containing a virus or provirus with an effective amount of a
compound or a pharmaceutical composition thereof, wherein the virus
employs programmed ribosomal frameshifting, and wherein the
effective amount is an amount effective to alter the efficiency of
programmed ribosomal frameshifting by at least 20% (and in some
embodiments, at least 25%, at least 30%, at least 40%, at least 50%
or at least 75%) as measured by a technique known to one of skill
in the art or as described herein, e.g., a dual luciferase
construct assay.
[0076] In another embodiment, the invention provides a method for
inhibiting or reducing viral infectivity comprising contacting a
mixture of a cell or a population of cells and a virus with an
effective amount of a compound or a pharmaceutical composition
thereof, wherein the virus employs programmed ribosomal
frameshifting, and wherein the effective amount is an amount
sufficient to cause a statistically significant change in viral
programmed ribosomal frameshifting efficiency as measured by a
technique known to one of skill in the art or as described herein,
e.g., a dual luciferase construct assay. In another embodiment, the
invention provides a method for inhibiting or reducing viral
infectivity comprising contacting a mixture of a cell or a
population of cells and a virus with an effective amount of a
compound or a pharmaceutical composition thereof, wherein the virus
employs programmed ribosomal frameshifting, and wherein the
effective amount is an amount effective to alter the efficiency of
programmed ribosomal frameshifting by at least 20% (and in some
embodiments, at least 25%, at least 30%, at least 40%, at least 50%
or at least 75%) as measured by a technique known to one of skill
in the art or as described herein, e.g., a dual luciferase
construct assay.
[0077] In another embodiment, the invention provides a method for
preventing or treating a viral disease in a subject, comprising
administering to a subject in need thereof an effective amount of a
compound or a pharmaceutical composition thereof, wherein the viral
disease is a pathological condition resulting from the presence of
a virus, and wherein the effective amount is an amount effective to
sufficient to cause a statistically significant change in viral
programmed ribosomal frameshifting as measured by a technique known
to one of skill in the art or as described herein, e.g., a dual
luciferase construct assay. In another embodiment, the invention
provides a method for preventing or treating a viral disease in a
subject, comprising administering to a subject in need thereof an
effective amount of a compound or a pharmaceutical composition
thereof, wherein the viral disease is a pathological condition
resulting from the presence of a virus, and wherein the effective
amount is an amount effective to alter the efficiency of programmed
ribosomal frameshifting by at least 20% (and in some embodiments,
at least 25%, at least 30%, at least 40%, at least 50%, or at least
75%) as measured by a technique known to one of skill in the art or
as described herein, e.g., a dual luciferase construct assay.
[0078] In certain embodiments, a compound identified or validated
by a method described herein modulates the efficiency of programmed
-1 ribosomal frameshifting. In certain embodiments, a compound
identified or validated by a method described herein modulates the
efficiency of programmed+2 ribosomal frameshifting.
[0079] Non-limiting examples of viruses that employ programmed
ribosomal frameshifting include the viruses listed in Table 1,
infra, which is a modified version of Table 1 in Brierley, 1995, J.
Gen. Virol. 76:1885-1892. In a specific embodiment, the virus is
not a human immunodeficiency virus, hepatitis virus, or human
papillomavirus.
TABLE-US-00001 TABLE 1 Occurrence of established ribosomal
frameshift signals in viral RNAs Gene or Gene Family/Group Genus
Virus overlap Retroviridae Lentivirus Human immunodeficiency virus
type 1 (HIV-1) gag-pol Feline immunodeficiency virus (HIV) gag-pol
ALSV Rous sarcoma virus (RSV) gag-pol B-type Mouse mammary tumour
virus (MMTV) gag-pro D-type Simian retrovirus type 1 (SRV-1)
gag-pro HTLV/BLV Human T cell leukaemia virus type I (HTLV-I)
gag-pro HTLV-I pro-pol HTLV-II gag-pro-pol Coronaviridae
Coronavirus Infectious bronchitis virus (IBV) orf1a-orf1b Mouse
hepatitis virus (MHV) orf1a-orf1b Human coronavirus (HCoV)
orf1a-orf1b severe acute respiratory syndrome coronavirus orf3a
(SARS-CoV) Transmissible gastroenteritis virus (TGEV) orf1a-orf1b
Torovirus Berne virus (BEV) orf1a-orf1b Arterivirus Equine
arteritis virus (EAV) orf1a-orf1b Astroviridae Astrovirus Human
astrovirus serotype-1 (HAst-1) orf1a-orf1b Totiviridae Totivirus
Giardia lamblia virus (GLV) orf1-orf2 Saccharomyces cerevisiae
dsRNA virus L-A gag-pol (ScV/L-A) S. cerevisiae dsRNA virus L1
(ScV/L1) cap-pol Podoviridae T7 phage Bacteriophage T7 10A-10B
Siphoviridae .lamda. phage group Bacteriophage .lamda. gpG-T
Luteoviridae Luteovirus Barley yellow dwarf virus (BYDV) 39K-60K
Beet western yellows virus (BWYV) orf2-orf3 Potato leaf roll virus
(PLRV) orf2a-orf2b Dianthoviridae Dianthovirus Red clover necrotic
mosaic virus (RCNMV) p27-p57 Herpesviridae Simplexvirus Herpes
Simplex Virus (HSV) thymidine kinase gene
[0080] While not being limited by theory, within the scope of the
invention is the identification or validation of compounds that
modulate viral programmed ribosomal frameshifting such that an
attenuated virus or viral particle is produced. In a specific
embodiment, the attenuated virus or viral particle may be used as a
vaccine.
Terminology
[0081] As used herein, the term "about" or "approximately," when
used in conjunction with a number, refers to any number within 1, 5
or 10% of the referenced number.
[0082] As used herein, the terms "increase," "increases," and
"increased," in the context of the amount or activity of a fusion
protein refer, in some embodiments, to: (i) an increase of 0.5%,
1%. 1.5%, 2%, 5%, 10%, 20%, 30%, 40%, 50% or more; (ii) an increase
of 1.5, 2, 3, 4, or 5 fold or more; or (iii) a statistically
significant increase in the amount or activity of the fusion
protein relative to a negative control.
[0083] As used herein, the terms "change" or "changed," in the
context of the efficiency of programmed ribosomal frameshifting
refer, in some embodiments, to: (i) a change of 0.5%, 1%. 1.5%, 2%,
5%, 10%, 20%, 30%, 40%, 50% or more; (ii) a change of 1.5, 2, 3, 4,
or 5 fold or more; or (iii) a statistically significant change in
the efficiency of programmed ribosomal frameshifting.
[0084] As used herein, the term "not changed" refers to no
detectable change or no statistically significant change (i.e., a
change that has a p value of greater than 0.1).
[0085] As used herein, the term "statistically significant
increase" refers to an increase that has a p value of less than
0.1, 0.05, 0.01, 0.05, or 0.01.
[0086] As used herein, the term "not statistically significant
increase" refers to an increase that has a p value of more than
0.001, 0.01, 0.05, or 0.1.
[0087] As used herein, the term "statistically significantly
change" refers to a change that has a p value of less than 0.1,
0.05, 0.01, 0.05, or 0.01.
[0088] As used herein, unless otherwise specified, the term
"programmed ribosomal frameshifting" refers to a process in which
either a structural or sequence element directs the ribosomal
machinery to shift translational reading frame.
[0089] As used herein, the term "compound," unless otherwise
specified or clear from the context of the specification, refers to
any agent being tested for its ability to modulate the efficiency
of programmed ribosomal frameshifting, or is identified or
validated as modulating the efficiency of programmed ribosomal
frameshifting using a nucleic acid construct described herein. In
one embodiment, the term "compound" refers to a small molecule. In
a specific embodiment, the term "compound" refers to a compound of
Formula (I) or Formula (II) or a form thereof. In one embodiment,
the term "compound" refers to a compound selected from Compound
1.
[0090] As used herein, the term "small molecule" and analogous
terms include, but are not limited to, peptides, peptidomimetics,
amino acids, amino acid analogs, polynucleotides, polynucleotide
analogs, nucleotides, nucleotide analogs, other organic and
inorganic compounds (i.e., including heteroorganic and
organometallic compounds) and forms thereof having a molecular
weight of less than about 10,000 grams per mole, or less than about
5,000 grams per mole, or less than about 1,000 grams per mole, or
less than about 500 grams per mole, or less than about 100 grams
per mole.
[0091] As used herein, the term "viral disease" refers to a
pathological condition resulting from the presence of a virus in a
subject.
[0092] As used herein, the term "effective amount" in the context
of administering a compound to a subject refers to the amount of a
compound which is sufficient to achieve a prophylactic and/or
therapeutic effect. In specific embodiments, the tem "effective
amount" in the context of administering a compound to a subject
refers to the amount of a compound which is sufficient to achieve
at least one of the following effects: (i) reduce or ameliorate the
severity of a viral infection or disease or a symptom associated
therewith; (ii) reduce the duration of a viral infection or disease
or a symptom associated therewith; (iii) prevent the progression of
a viral infection or a disease or a symptom associated therewith;
(iv) cause regression of a viral infection or a symptom associated
therewith; (v) prevent the development or onset of a viral
infection or disease or a symptom associated therewith; (vi)
prevent the recurrence of a viral infection or disease or a symptom
associated therewith; (vii) reduce or prevent the spread of a virus
from one cell to another cell, or from one tissue to another
tissue; (viii) reduce or inhibit viral infectivity; (ix) prevent or
reduce the spread of a virus from one subject to another subject;
(x) reduce organ damage or failure associated with a viral
infection or a disease associated therewith; (xi) reduce
hospitalization of a subject; (xii) reduce hospitalization length;
(xiii) increase the survival of a subject with a viral infection or
a disease associated therewith; (xiv) reduce the viral titer in a
subject having a viral infection or a disease associated therewith;
(xv) eliminate a viral infection or a symptom associated therewith;
(xvi) inhibit or reduce viral replication; (xvii) eliminate viral
infectivity; (xviii) inhibit or reduce viral pathogenicity; (xix)
cure a viral infection or disease associated therewith; and/or (xx)
enhance or improve the prophylactic or therapeutic effect(s) of
another agent.
[0093] As used herein, the term "effective amount" in the context
of a compound for use in cell culture-related products refers to an
amount of a compound which is sufficient to reduce the viral titer
in cells, reduce the replication of a virus in cells or reduce the
infectivity of a virus in cells.
[0094] As used herein, the term "in combination," in the context of
the administration of a compound, refers to the administration of
two or more compounds of the present invention that modulate the
efficiency of programmed ribosomal frameshifting, or the
administration of one or more compounds of the present invention
that modulate the efficiency of programmed ribosomal frameshifting
and one or more additional agents. The use of the term "in
combination" does not restrict the order in which two or more of
the instant compounds or one or more of said compounds and another
agent are administered to a subject in need thereof. For example, a
compound can be administered prior to (e.g., 5 minutes, 15 minutes,
30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12
hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3
weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before),
concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes,
30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12
hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3
weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the
administration of another agent to a subject with a viral
infection.
[0095] As used herein, the term "elderly human" refers to a human
65 years or older.
[0096] As used herein, the term "human adult" refers to a human
that is 18 years or older.
[0097] As used herein, the term "human child" refers to a human
that is 1 year to 18 years old.
[0098] As used herein, the term "human toddler" refers to a human
that is 1 year to 3 years old.
[0099] As used herein, the term "human infant" refers to a newborn
to 1 year old year human.
[0100] As used herein, the term "premature human infant" refers to
a human infant born at less than 37 weeks of gestational age.
[0101] As used herein, the term "combination product" refers to a
product comprising: (i) two or more of the instant compounds that
modulate programmed ribosomal frameshifting; or (ii) one or more of
the instant compounds that modulate programmed ribosomal
frameshifting and one or more additional agents.
[0102] As used herein, the term "form" in the context of a compound
refers to a compound isolated for use as a pharmaceutically
acceptable salt, ester, hydrate, solvate, clathrate, polymorph,
geometric isomer, racemate, enantiomer, diastereomer or
tautomer.
[0103] As used herein, the italicized term "SMN," unless otherwise
specified herein, refers to human SMN1 or human SMN2. Nucleic acid
sequences for the human SMN1 and SMN2 are known in the art. See,
e.g., GENBANK.RTM. Accession Nos. DQ894095, NM.sub.--000344,
NM.sub.--022874, and BC062723 for nucleic acid sequences of human
SMN1. For nucleic acid sequences of human SMN2, see, e.g.,
NM.sub.--022875, NM.sub.--022876, NM.sub.--022877, NM.sub.--017411,
DQ894734 (Invitrogen, Carlsbad, Calif.), BC000908.2, and
BC015308.1.
[0104] The SMN1 gene can be found on human chromosome 5 from
approximately nucleotide 70,256,524 to approximately nucleotide
70,284,595 using Vega Gene ID: OTTHUMG00000099361 (see website for
ensembl.org/Homo_sapiens/geneview?gene=OTTHUMG00000099361;db=vega)
at cytogenetics location 5 of 13. See also GENBANK.RTM. Accession
No. NC.sub.--000005, Build 36.3 for the sequence of human
chromosome 5.
[0105] The approximate locations of exons 6, 7 and 8 and introns 6
and 7 of SMN1 on human chromosome 5 using Vega gene ID:
OTTHUMG00000099361 (see website for
ensembl.org/Homo_sapiens/geneview?gene=OTTHUMG00000099361;db=vega)
are as follows:
[0106] 70,277,649-70,277,759 exon 6
[0107] 70,277,760-70,283,523 intron 6
[0108] 70,283,524-70,283,577 exon 7
[0109] 70,283,578-70,284,021 intron 7
[0110] 70,284,022-70,284,595 exon 8
In specific embodiments, the nucleotide sequences delineated above
for exons 6, 7 and 8 and introns 6 and 7 of SMN1 are used in the
nucleic acid constructs described herein. In other specific
embodiments, the nucleotide sequences described in the example
below for exons 6, 7 and 8 and introns 6 and 7 are used in the
nucleic acid constructs described herein.
[0111] The SMN2 gene can be found on human chromosome 5 from
approximately nucleotide 69,381,106 to approximately nucleotide
69,409,175 using Vega gene ID: OTTHUMG00000099389 (see website for
ensembl.org/Homo_sapiens/geneview?gene=OTTHUMG00000099389;db=vega).
See also, GENBANK.RTM. Accession No. NC.sub.--000005, Build 36.3
for the sequence of human chromosome 5.
[0112] The approximate locations of exons 6, 7 and 8 and introns 6
and 7 of SMN2 on human chromosome 5 using Vega gene ID:
OTTHUMG00000099389 (see website for
ensembl.org/Homo_sapiens/geneview?gene=OTTHUMG00000099389;db=vega)
are as follows:
[0113] 69,402,224-69,402,334 exon 6
[0114] 69,402,335-69,408,103 intron 6
[0115] 69,408,104-69,408,157 exon 7
[0116] 69,408,158-69,408,601 intron 7
[0117] 69,408,602-69,409,175 exon 8.
In specific embodiments, the nucleotide sequences delineated above
for exons 6, 7 and 8 and introns 6 and 7 of SMN2 are used in the
nucleic acid constructs described herein. In other specific
embodiments, the nucleotide sequences of exons 6, 7 and 8 and
introns 6 and 7 of SMN2 are used in the nucleic acid constructs
described herein.
[0118] As used herein, the terms "virus," "viral," and analogous
terms refer to a virus that employs programmed ribosomal
frameshifting. In certain embodiments, the compounds identified and
validated using the assays of the present invention are expected to
be useful in modulating viral programmed ribosomal frameshifting.
In a specific embodiment the viral programmed ribosomal
frameshifting is -1 programmed ribosomal frameshifting. In other
embodiments, the programmed ribosomal frameshifting is selected
from +2 programmed ribosomal frameshifting, +1 programmed ribosomal
frameshifting and -2 programmed ribosomal frameshifting.
[0119] As used herein, the term "provirus" refers to a provirus
that encodes or codes for a virus that employs programmed ribosomal
frameshifting.
[0120] As used herein, the term "host cell" includes a particular
subject cell transformed or transfected with an instant nucleic
acid construct and the progeny or potential progeny of such a cell.
Progeny of such a cell may not be identical to the parent cell
transfected with the nucleic acid construct due to mutations or
environmental influences that may occur in succeeding generations
or integration of the nucleic acid construct into the host cell
genome.
[0121] As used herein, the term "isolated," as it refers to a
compound, means the physical state of a compound after being
separated and/or purified from precursors and other substances
found in a synthetic process (e.g., from a reaction mixture) or
natural source or combination thereof according to a process or
processes described herein or which are well known to the skilled
artisan (e.g., chromatography, recrystallization and the like) in
sufficient purity to be capable of characterization by standard
analytical techniques described herein or well known to the skilled
artisan. In a specific embodiment, the compound is at least 60%
pure, at least 65% pure, at least 70% pure, at least 75% pure, at
least 80% pure, at least 85% pure, at least 90% pure or at least
99% pure as assessed by techniques known to one of skill in the
art.
[0122] As used herein, the term "isolated," as it refers to a
nucleic acid, means the physical state of a nucleic acid after
being separated and/or purified from precursors and other
substances found in a synthetic process (e.g., from a reaction
mixture) or natural source or combination thereof according to a
process or processes described herein or which are well known to
the skilled artisan in sufficient purity to be capable of
characterization by standard analytical techniques described herein
or well known to the skilled artisan.
[0123] In some embodiments, the term "fragment" refers to a
nucleotide sequence comprising 2, 3, 6, 9, 12, 15, 21, 24, 27, 30
or more nucleotides from a longer nucleotide sequence. In certain
embodiments, the nucleotide sequences comprises 2, 3, 6, 9, 12, 15,
21, 24, 27, 30 or more contiguous nucleotides from a longer
nucleotide sequence.
[0124] In some embodiments, the terms "nucleic acid" and
"nucleotides" refer to deoxyribonucleotides, deoxyribonucleic
acids, ribonucleotides, and ribonucleic acids, and polymeric forms
thereof, and includes either single- or double-stranded forms. In
certain embodiments, such terms include known analogues of natural
nucleotides, for example, peptide nucleic acids ("PNA"s), that have
similar binding properties as the reference nucleic acid. In some
embodiments, nucleic acid refers to deoxyribonucleic acids (e.g.,
cDNA or DNA). In other embodiments, nucleic acid refers to
ribonucleic acids (e.g., mRNA or pre-mRNA).
[0125] As used herein, the term "nucleic acid residues of exon 6 of
SMN," unless otherwise specified herein, refers to a complete,
intact, non-truncated nucleic acid sequence exon 6 of SMN1 or SMN2.
In certain embodiments, a nucleic acid construct described herein
comprises, in part, a complete, intact, non-truncated nucleic acid
sequence of exon 6 of SMN1 or SMN2.
[0126] As used herein, the term "nucleic acid residues of intron 6
of SMN," unless otherwise specified herein, refers to a complete,
intact, non-truncated nucleic acid sequence of intron 6 of SMN1 or
SMN2. In certain embodiments, a nucleic acid construct described
herein comprises, in part, a complete, intact, non-truncated
nucleic acid sequence of intron 6 of SMN1 or SMN2.
[0127] As used herein, the term "nucleic acid residues of exon 7 of
SMN," unless otherwise specified herein, refers to a complete,
intact, non-truncated nucleic acid sequence of exon 7 of SMN1 or
SMN2. In certain embodiments, a nucleic acid construct described
herein comprises, in part, a complete, intact, non-truncated
nucleic acid sequence of exon 7 of SMN1 or SMN2.
[0128] As used herein, the term "nucleic acid residues of intron 7
of SMN," unless otherwise specified herein, refers to a complete,
intact, non-truncated nucleic acid sequence of intron 7 of SMN1 or
SMN2. In certain embodiments, a nucleic acid construct described
herein comprises, in part, a complete, intact, non-truncated
nucleic acid sequence of intron 7 of SMN1 or SMN2.
[0129] As used herein, the term "nucleic acid residues of exon 8 of
SMN," unless otherwise specified herein, refers to a complete,
intact, non-truncated nucleic acid sequence of exon 8 of SMN1 or
SMN2.
[0130] As used herein, the term "ORF" refers to a mRNA open reading
frame, i.e., the region of the mRNA that can translated into
protein.
[0131] Reference to the term "open reading frame" in the context of
two or more open reading frames being in frame with each other
refers to two nucleic acid sequences (e.g., nucleic acid residues
of an exon(s) of SMN or a fragment thereof and/or a nucleotide
sequence encoding an amino acid sequence), wherein each of the two
or more nucleic acid sequences are in the same contiguous open
reading frame which is defined by the first start codon and an
aberrant stop codon, i.e., the stop codon upstream (5') of the
reporter gene coding sequence.
[0132] Reference to the term "open reading frame" in the context of
two or more open reading frames being out of frame with each other
refers to two nucleic acid sequences (e.g., nucleic acid residues
of an exon(s) of SMN or a fragment thereof and/or a reporter gene
coding sequence), wherein each of the two or more nucleic acid
sequences are not in the same contiguous open reading frame which
is defined by the first start codon and an aberrant stop codon,
i.e., the stop codon upstream (5') of the reporter gene coding
sequence.
[0133] As used herein, the term "previously determined reference
range" in the context of detecting the amount or activity of a
protein refers to a reference range for the amount or the activity
of a fusion protein encoded by a nucleic acid construct or
transcribed from a mRNA transcript. Ideally, testing laboratories
will establish a reference range for each cell type and each
cell-free extract in the practice of such assays. In a specific
embodiment, at least one positive control or at least one negative
control is included for each compound analyzed. In a specific
embodiment, the previously determined reference range is the amount
or activity of a fusion protein detected in the presence of a
negative control, such as phosphate-buffered saline ("PBS") or
dimethyl sulfoxide ("DMSO").
[0134] As used herein, the terms "subject" and "patient" are used
interchangeably, and refer to an animal (e.g., birds, reptiles, and
mammals), such as a mammal including a non-primate (e.g., a camel,
donkey, zebra, cow, pig, horse, goat, sheep, cat, dog, rat, and
mouse) and a primate (e.g., a monkey, chimpanzee, and a human). In
a specific embodiment, the subject is a human.
[0135] As used herein, the term "infection" means the invasion by a
virus, the multiplication of a virus and/or presence of a virus or
provirus in a cell or a subject. In one embodiment, an infection is
an "active" infection, i.e., one in which the virus is replicating
in a cell or a subject. Such an infection is characterized by the
spread of the virus to other cells, tissues, and/or organs, from
the cells, tissues, and/or organs initially infected by the virus.
In another embodiment, an infection is a latent infection, i.e.,
one in which the virus is not actively replicating in a cell or a
subject but has the potential to replicate or one in which viral
particles are present in the cell or subject without the ability to
replicate. In another embodiment, an infection is a "persistent"
infection, i.e., one in which the virus is not cleared but remains
in cells or a subject. A persistent infection may involve stages of
both silent and productive infection without rapidly killing or
even producing excessive damage of cells. In a specific embodiment,
an infection refers to the pathological state resulting from the
presence of the virus in a cell or a subject, or by the invasion of
a cell or subject by the virus.
[0136] As used herein, the term "infectivity" means the ability of
a virus or viral particles to establish an infection.
[0137] As used herein, the terms "prevent," "preventing," and
"prevention," in the context of the administration of a compound of
the present invention either alone or in combination with another
agent to a subject in need thereof to prevent a viral disease
(i.e., a pathological condition resulting from the presence of a
virus or provirus in a subject), refer to the inhibition of the
development or onset of a viral disease or the inhibition of the
recurrence of a viral disease.
[0138] As used herein, the terms "treat," "treatment," and
"treating" refer, in the context of the administration of a
compound alone or in combination with another agent to a subject to
treat a viral infection or a viral disease, to a therapeutic
benefit achieved. In a specific embodiment, such terms refer to at
least one or more of the following effects resulting from the
administration of a compound or other agent to a subject: (i) the
reduction or amelioration of the severity of a viral infection or
disease or a symptom associated therewith; (ii) the reduction in
the duration of a viral infection or disease or a symptom
associated therewith; (iii) the regression of a viral infection or
a symptom associated therewith; (iv) the prevention of the
development, onset or recurrence of a symptom associated with a
viral infection or disease associated therewith; (v) the reduction
of the titer of a virus; (vi) the reduction in organ damage or
failure associated with a viral infection or disease associated
therewith; (vii) the reduction in hospitalization of a subject;
(viii) the reduction in hospitalization length; (ix) the increase
in the survival of a subject with a viral infection or disease
associated therewith; (x) the elimination of viral infectivity;
(xi) the elimination of a viral infection or a symptom associated
therewith; (xii) the inhibition of the progression of a viral
infection or disease or a symptom associated therewith; (xiii) the
prevention or reduction in the spread of a virus from a cell,
tissue or subject to another cell, tissue or subject; (xiv) the
inhibition or reduction in viral pathogenicity; (xv) the cure of a
viral infection or disease associated therewith; and/or (xvi) the
enhancement or improvement the therapeutic effect of another agent.
In some embodiments, treatment does not refer to a cure for a viral
infection or disease associated therewith, but the inhibition of
the progression or worsening of the viral infection or disease
associated therewith.
BRIEF DESCRIPTION OF THE DRAWINGS
[0139] FIG. 1: Fold inhibition of luciferase activity in the
presence of Compound 1.
[0140] FIG. 2A-2C: DNA sequence of the minigene from the SMN2-G
minigene construct (SEQ ID NO:11). Within the sequence shown in
FIG. 2A-2C, the following subsequences can be found:
[0141] 1-70: 5'UTR (deg)
[0142] 71-79: start codon and BamHI site (atgggatcc)
[0143] 80-190: exon 6
[0144] 191-5959: intron 6
[0145] 5960-6014: exon 7 with G insert (position 6008)
[0146] 6015-6458: intron 7
[0147] 6459-6481: part of exon 8
[0148] 6482-8146: BamHI site, luciferase coding sequence starting
with codon 2, NotI site, TAA stop codon
[0149] 8147-8266: 3'UTR (deg). FIG. 2A nucleic acids 1-4009 of the
DNA sequence; FIG. 2B nucleic acids 4010-7885 of the DNA sequence;
FIG. 2C nucleic acids 7886-8266 of the DNA sequence.
DETAILED DESCRIPTION OF THE INVENTION
[0150] The present invention relates to compounds that modulate
ribosomal frameshifting and nucleic acid constructs for use in
methods for identifying or validation such compounds. In
particular, the present invention provides nucleic acid constructs
and screening assays for the identification and validation of
compounds that are capable of modulating the efficiency of
programmed ribosomal frameshifting. Compounds identified or
validated according to the methods of the invention are expected to
be useful in the treatment of viral infections, as well as the
treatment and possibly the prevention of viral disease.
[0151] In one aspect, the invention provides a method for
identifying or validating a compound that modulates the efficiency
of ribosomal frameshifting. In another aspect, the invention
provides a method for identifying or validating a compound that
modulates the efficiency of programmed ribosomal frameshifting. In
another aspect, the invention provides compounds that modulate the
efficiency of programmed ribosomal frameshifting, and which may be
used to treat a viral infection, or prevent or treat a viral
disease. In one embodiment, a compound of Formula (I) or a form
thereof is used to treat a viral infection, or prevent or treat a
viral disease. In another embodiment, a compound of Formula (II) or
a form thereof is used to treat a viral infection, or prevent or
treat a viral disease. In another embodiment, a compound of Formula
(Ia) or a form thereof is used to treat a viral infection, or
prevent or treat a viral disease. In another embodiment, a compound
of Formula (IIa) or a form thereof is used to treat a viral
infection, or prevent or treat a viral disease. In a specific
embodiment, Compound I is used to treat a viral infection, or
prevent or treat a viral disease.
Compounds
[0152] The compounds described in International Publication
WO2007/109211 (which is incorporated by reference in its entirety)
may be used in accordance with the methods described herein.
[0153] Embodiments of the present invention include uses of
compounds of Formula (I) or a form thereof or Formula (II) or a
form thereof, wherein Formula (I) and Formula (II) have the
following structures:
##STR00001##
[0154] wherein,
[0155] W is selected from the group consisting of C(O), C(S), and
CH.sub.2;
[0156] B is CH.sub.2 or CH(C.sub.nH.sub.2+1), wherein n is an
integer from 1 to 8;
[0157] Ring C is selected from the group consisting of a fused
thienyl ring, a fused pyridinyl ring, and a fused cyclohexyl ring,
any of which can be saturated or contain, one or two non-conjugated
double bonds;
[0158] R.sub.1 and R.sub.2 are independently selected from the
group consisting of H and C.sub.1-C.sub.3 alkyl, or R.sub.1 and
R.sub.2 may be taken together with the carbon atom to which they
are attached to form a C.sub.3-C.sub.6 cycloalkyl ring or a
carbonyl group;
[0159] R.sub.3 is selected from the group consisting of H, halogen,
C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 alkoxy, C.sub.1-C.sub.4
haloalkyl, CN, NO.sub.2, heteroaryl, and phenyl optionally
substituted with any combination of one to five halogen, NO.sub.2,
CN, C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 haloalkyl, or
C.sub.1-C.sub.4 alkoxy substituents;
[0160] R.sub.4, R.sub.5, R.sub.6 and R.sub.7 are independently
selected from the group consisting of H, hydroxyl, halogen, CN,
NO.sub.2, sulfonamide, C.sub.1-C.sub.8 alkyl, C.sub.3-C.sub.6
cycloalkyl, cycloalkyloxy, C.sub.1-C.sub.6 alkoxy, C.sub.1-C.sub.6
haloalkoxy, C.sub.1-C.sub.4 haloalkyl, C.sub.2-C.sub.8 alkenyl,
amino, C.sub.1-C.sub.4 alkylamino, C.sub.1-C.sub.4 dialkylamino,
C.sub.3-C.sub.6 cycloalkylamino, morpholinyl, heteroaryl,
arylamino, arylalkylamino, phenyl, C(O)R', NR'(COR''),
NR'SO.sub.2R'' and NR'(CONR''R'''), wherein R', R'' and R''' are
independently H, C.sub.1-C.sub.6 alkyl, phenyl, or substituted
phenyl, and wherein C.sub.1-C.sub.8 alkyl is optionally substituted
with one or more substituents selected from the group consisting of
C.sub.1-C.sub.4 alkoxy, C.sub.1-C.sub.4 haloalkyl, C.sub.1-C.sub.6
dialkylamino, C.sub.1-C.sub.6 alkylamino, cycloalkylamino, and
morpholinyl, and the phenyl is optionally substituted with one or
more substituents selected from the group consisting of halogen,
NO.sub.2, CN, C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 haloalkyl, and
C.sub.1-C.sub.4 alkoxy, or R.sub.4 and R.sub.5, R.sub.5 and
R.sub.6, or R.sub.6 and R.sub.7, taken together with the carbon to
which they are attached, form a ring;
[0161] X is selected from the group consisting of H; CN;
C(O)OR.sub.8, wherein R.sub.8 is H or C.sub.1-C.sub.8 alkyl, and
C.sub.1-C.sub.8 alkyl optionally is substituted with one or more
substituents selected from the group consisting of C.sub.1-C.sub.4
alkoxy, C.sub.1-C.sub.4 haloalkyl, C.sub.1-C.sub.6 dialkylamino,
C.sub.1-C.sub.6 alkylamino, cycloalkylamino, phenyl, and
morpholinyl; C(O)NR.sub.9R.sub.10 or CH.sub.2NR.sub.9R.sub.10,
wherein R.sub.9 and R.sub.10 are independently selected from the
group consisting of H and C.sub.1-C.sub.6 alkyl, or R.sub.9 and
R.sub.10 together with the nitrogen to which they are attached form
a heterocyclyl ring; CH.sub.2OR.sub.11, wherein R.sub.11 is H,
C.sub.1-C.sub.8 alkyl, or C.sub.3-C.sub.6 cycloalkyl, wherein
C.sub.1-C.sub.8 alkyl is optionally substituted with one or more
substituents selected from the group consisting of C.sub.1-C.sub.4
alkoxy, C.sub.1-C.sub.4 haloalkyl, C.sub.1-C.sub.6 dialkylamino,
C.sub.1-C.sub.6 alkylamino, cycloalkylamino, and morpholinyl;
CH.sub.2Z, wherein Z is halogen; C(O)NHOH; C(O)NHCN;
C(O)N(R.sub.1)SO.sub.2R.sub.13, wherein R.sub.13 is C.sub.1-C.sub.4
alkyl, phenyl, or substituted phenyl; C.sub.1-C.sub.8 alkyl,
optionally substituted with one or more substituents selected from
the group consisting of C.sub.1-C.sub.4 alkoxy, C.sub.1-C.sub.4
haloalkyl, C.sub.1-C.sub.6 dialkylamino, and C.sub.1-C.sub.6
alkylamino; and C.sub.2-C.sub.8 alkenyl, optionally substituted
with one or more substituents selected from the group consisting of
C.sub.1-C.sub.4 alkoxy, C.sub.1-C.sub.4 haloalkyl, C.sub.1-C.sub.6
dialkylamino, and C.sub.1-C.sub.6 alkylamino.
[0162] In certain embodiments, the present invention includes the
use of compounds of Formula (Ia) or a form thereof or Formula (IIa)
or a form thereof, wherein Formula (Ia) and Formula (IIa) have the
following structures:
##STR00002##
[0163] wherein,
[0164] W.sub.1 is selected from the group consisting of C(O), C(S),
and CH.sub.2;
[0165] B.sub.1 is CH.sub.2 or CH(C.sub.mH.sub.2m+1), wherein m is
an integer from 1 to 8;
[0166] Ring C.sub.1 is selected from the group consisting of a
thienyl ring, a pyridinyl ring, a cyclohexyl ring, a
benzo[d][1,3]dioxolyl ring and a 2,3-dihydrobenzo[b][1,4]dioxinyl
ring, each of said rings fused to the moiety of Formula (IIa),
wherein benzo[d][1,3]dioxolyl and 2,3-dihydrobenzo[b][1,4]dioxinyl,
each having a benzo ring portion, are fused via said benzoportion,
and wherein any of the foregoing rings may optionally be fully or
partially saturated;
[0167] R.sub.20 and R.sub.21 are independently selected from the
group consisting of H and C.sub.1-C.sub.3 alkyl, or R.sub.20 and
R.sub.21 may be taken together with the carbon atom to which they
are attached to form a C.sub.3-C.sub.6 cycloalkyl ring or a
carbonyl group;
[0168] R.sub.22 is selected from the group consisting of H,
halogen, C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 alkoxy,
C.sub.1-C.sub.4 haloalkyl, cyano, nitro, heteroaryl, and phenyl
optionally substituted with any combination of one to five halogen,
nitro, cyano, C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 haloalkyl or
C.sub.1-C.sub.4 alkoxy substituents;
[0169] R.sub.23, R.sub.24, R.sub.25 and R.sub.26 are independently
selected from the group consisting of H, hydroxyl, halogen, cyano,
nitro, sulfonamide, C.sub.1-C.sub.8 alkyl, C.sub.1-C.sub.6 alkoxy,
C.sub.1-C.sub.6 alkoxyalkoxy, C.sub.1-C.sub.6 alkoxyalkyl,
C.sub.1-C.sub.6 haloalkoxy, C.sub.1-C.sub.4 haloalkyl,
C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.4 haloalkenyl, formyl,
C.sub.1-C.sub.6 alkylcarbonyl, amino, C.sub.1-C.sub.4 alkylamino,
C.sub.1-C.sub.4 dialkylamino, C.sub.1-C.sub.4 aminoalkyl,
C.sub.1-C.sub.4 alkylaminoalkyl, C.sub.1-C.sub.4 dialkylaminoalkyl,
phenyl, C.sub.3-C.sub.6 cycloalkyl, C.sub.3-C.sub.6
cycloalkylalkyl, C.sub.3-C.sub.6 cycloalkylalkoxy, cycloalkyloxy,
heterocyclyl, heterocyclylalkyl, heteroaryl, and
phenylcarbonyl,
[0170] wherein amino is optionally disubstituted with one
substituent selected from hydrogen, C.sub.1-C.sub.6 alkyl or phenyl
and the other is selected from formyl, phenyl, C.sub.3-C.sub.6
cycloalkyl, C.sub.1-C.sub.6 alkylcarbonyl, aminocarbonyl,
C.sub.1-C.sub.6 alkylaminocarbonyl, C.sub.1-C.sub.6
dialkylaminocarbonyl, phenylcarbonyl, phenylaminocarbonyl,
N-phenyl-N--C.sub.1-C.sub.6 alkyl-aminocarbonyl, C.sub.1-C.sub.6
alkylsulfonyl, aminosulfonyl, C.sub.1-C.sub.6 alkylaminosulfonyl,
C.sub.1-C.sub.6 dialkylaminosulfonyl or phenylsulfonyl,
[0171] wherein each instance of C.sub.1-C.sub.6 alkylcarbonyl is
optionally substituted on the alkyl portion with one or more
substituents selected from the group consisting of halogen,
C.sub.1-C.sub.4 alkoxy, C.sub.1-C.sub.6 alkylamino, C.sub.1-C.sub.6
dialkylamino, cycloalkylamino and heterocyclyl,
[0172] wherein each instance of phenyl is optionally substituted
with one or more substituents selected from the group consisting of
halogen, nitro, cyano, C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4
haloalkyl and C.sub.1-C.sub.4 alkoxy, and
[0173] alternatively, R.sub.23 and R.sub.24, R.sub.24 and R.sub.25
or R.sub.25 and R.sub.26 may be taken together with the carbon to
which they are attached to form a C.sub.3-C.sub.6 cycloalkyl ring
fused to the moiety of Formula (Ia);
[0174] X.sub.1 is absent or is selected from the group consisting
of H, cyano, C.sub.1-C.sub.8 alkyl, C.sub.1-C.sub.4 alkoxy, amino,
C.sub.1-C.sub.4 alkylamino, C.sub.1-C.sub.4 dialkylamino, carboxy,
C.sub.1-C.sub.8 alkoxycarbonyl, aminocarbonyl, C.sub.1-C.sub.8
alkylaminocarbonyl, C.sub.1-C.sub.8 dialkylaminocarbonyl,
hydroxylaminocarbonyl, cyanoaminocarbonyl, phenylaminocarbonyl,
aminosulfonylaminocarbonyl, C.sub.1-C.sub.8
alkylaminosulfonylaminocarbonyl, C.sub.1-C.sub.8
dialkylaminosulfonylaminocarbonyl, C.sub.1-C.sub.8
alkylsulfonylaminocarbonyl, phenylsulfonylaminocarbonyl and
heterocyclylcarbonyl,
[0175] wherein C.sub.1-C.sub.4 alkoxy and the C.sub.1-C.sub.8
alkoxy portion of C.sub.1-C.sub.8 alkoxycarbonyl is optionally
substituted with one or more substituents selected from the group
consisting of halogen, C.sub.1-C.sub.4 alkoxy, C.sub.1-C.sub.4
haloalkyl, amino, C.sub.1-C.sub.6 alkylamino, C.sub.1-C.sub.6
dialkylamino, cycloalkylamino, phenyl and heterocyclyl,
[0176] wherein C.sub.1-C.sub.8 alkyl is optionally substituted with
one or more substituents selected from the group consisting of
halogen, hydroxyl, C.sub.1-C.sub.4 haloalkyl, C.sub.2-C.sub.8
alkenyl, C.sub.1-C.sub.6 alkoxy, C.sub.1-C.sub.4 alkoxyalkoxy,
C.sub.3-C.sub.6 cycloalkyloxy, amino, C.sub.1-C.sub.6 alkylamino,
C.sub.1-C.sub.6 dialkylamino, cycloalkylamino, aminocarbonyl,
C.sub.1-C.sub.6 alkylaminocarbonyl, C.sub.1-C.sub.6
dialkylaminocarbonyl, hydroxylaminocarbonyl, cyanoaminocarbonyl,
C.sub.1-C.sub.6 alkylsulfonylaminocarbonyl,
phenylsulfonylaminocarbonyl and heterocyclyl, wherein
C.sub.1-C.sub.4 alkoxy or C.sub.2-C.sub.8 alkenyl are each further
optionally substituted with one or more substituents selected from
the group consisting of C.sub.1-C.sub.4 alkoxy, C.sub.1-C.sub.4
haloalkyl, amino, C.sub.1-C.sub.6 alkylamino and C.sub.1-C.sub.6
dialkylamino.
[0177] As used herein, unless otherwise specified, the term "alkyl"
means a saturated straight chain or branched non-cyclic hydrocarbon
chain radical having an indicated number of carbon atoms (e.g.,
C.sub.1-C.sub.20, C.sub.1-C.sub.10, C.sub.1-C.sub.8,
C.sub.1-C.sub.6, C.sub.1-C.sub.4, C.sub.1-C.sub.3, etc.).
Representative saturated straight chain alkyl radicals include
methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl,
n-octyl, n-nonyl and n-decyl; while representative saturated
branched chain alkyl radicals include isopropyl, sec-butyl,
isobutyl, tert-butyl, isopentyl, 2-methylbutyl, 3-methylbutyl,
2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2-methylhexyl,
3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2,3-dimethylbutyl,
2,3-dimethylpentyl, 2,4-dimethylpentyl, 2,3-dimethylhexyl,
2,4-dimethylhexyl, 2,5-dimethylhexyl, 2,2-dimethylpentyl,
2,2-dimethylhexyl, 3,3-dimtheylpentyl, 3,3-dimethylhexyl,
4,4-dimethylhexyl, 2-ethylpentyl, 3-ethylpentyl, 2-ethylhexyl,
3-ethylhexyl, 4-ethylhexyl, 2-methyl-2-ethylpentyl,
2-methyl-3-ethylpentyl, 2-methyl-4-ethylpentyl,
2-methyl-2-ethylhexyl, 2-methyl-3-ethylhexyl,
2-methyl-4-ethylhexyl, 2,2-diethylpentyl, 3,3-diethylhexyl,
2,2-diethylhexyl, 3,3-diethylhexyl and the like. An alkyl radical
can be unsubstituted or substituted.
[0178] As used herein, unless otherwise specified, the term
"cycloalkyl" means a monocyclic or polycyclic saturated ring
comprising carbon and hydrogen atoms and having no carbon-carbon
multiple bonds. Examples of cycloalkyl radicals include, but are
not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,
and cycloheptyl, and saturated cyclic and bicyclic terpenes. A
cycloalkyl radical can include C.sub.3-C.sub.14 cycloalkyl,
C.sub.5-C.sub.8 cycloalkyl, C.sub.3-C.sub.8 cycloalkyl,
C.sub.5-C.sub.6 cycloalkyl, C.sub.3-C.sub.6 cycloalkyl,
C.sub.3-C.sub.5 cycloalkyl, and the like, each of which may be
unsubstituted or substituted. Preferably, the cycloalkyl radical is
a monocyclic ring or bicyclic ring.
[0179] As used herein, unless otherwise specified, the term
"alkenyl" means a straight chain or branched non-cyclic hydrocarbon
chain radical having an indicated number of carbon atoms (e.g.,
C.sub.2-C.sub.20, C.sub.2-C.sub.10, C.sub.2-C.sub.8,
C.sub.2-C.sub.6, C.sub.2-C.sub.4, etc.) and including at least one
carbon-carbon double bond. Representative straight chain and
branched chain alkenyl radicals include vinyl, allyl, 1-butenyl,
2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl,
3-methyl-1-butenyl, 2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl,
1-hexenyl, 2-hexenyl, 3-hexenyl, 1-heptenyl, 2-heptenyl,
3-heptenyl, 1-octenyl, 2-octenyl, 3-octenyl, 1-nonenyl, 2-nonenyl,
3-nonenyl, 1-decenyl, 2-decenyl, 3-decenyl and the like. The double
bond of an alkenyl radical can be unconjugated or conjugated to
another unsaturated radical. An alkenyl radical can be
unsubstituted or substituted.
[0180] As used herein, unless otherwise specified the term
"alkynyl" means a straight chain or branched non-cyclic hydrocarbon
chain radical having an indicated number of carbon atoms (e.g.,
C.sub.2-C.sub.20, C.sub.2-C.sub.10, C.sub.2-C.sub.8,
C.sub.2-C.sub.6, C.sub.2-C.sub.4, etc.), and including at least one
carbon-carbon triple bond. Representative straight chain and
branched chain alkynyl radicals include acetylenyl, propynyl,
1-butyryl, 2-butyryl, 1-pentynyl, 2-pentynyl, 3-methyl-1-butyryl,
4-pentynyl, 1-hexynyl, 2-hexynyl, 5-hexynyl, 1-heptynyl,
2-heptynyl, 6-heptynyl, 1-octynyl, 2-octynyl, 7-octynyl, 1-nonynyl,
2-nonynyl, 8-nonynyl, 1-decynyl, 2-decynyl, 9-decynyl and the like.
The triple bond of an alkynyl radical can be unconjugated or
conjugated to another unsaturated group. An alkynyl radical can be
unsubstituted or substituted.
[0181] As used herein, unless otherwise specified, the term
"halogen" or "halo" means fluorine, chlorine, bromine, or iodine.
Furthermore, unless otherwise specified, the term "haloalkyl" means
alkyl substituted with one or more halogens, wherein alkyl and
halogen are defined as above.
[0182] As used herein, unless otherwise specified, the term
"alkoxy" means --O-(alkyl), wherein alkyl is defined above.
[0183] Furthermore, as used herein, the term "haloalkoxy" means
alkoxy substituted with one or more halogens, wherein alkoxy and
halogen are defined as above.
[0184] As used herein, unless otherwise specified, the term
"heteroaryl" means an carbocyclic aromatic ring containing from 5
to 14 ring atoms comprising at least one heteroatom, preferably 1
to 3 heteroatoms, independently selected from a nitrogen, oxygen,
and sulfur atom. Heteroaryl ring structures include compounds
having one or more ring structures, such as mono-, bi-, or
tricyclic compounds, as well as fused heterocyclic moities.
Representative heteroaryls are triazolyl, tetrazolyl, thiadiazolyl,
oxadiazolyl, pyridyl, furanyl, benzofuranyl, thiophenyl (also
referred to as thienyl), thiazolyl, benzothiophenyl,
benzoisoxazolyl, benzoisothiazolyl, quinolinyl, isoquinolinyl,
pyrrolyl, indolyl, indazolyl, isoindolyl, azaindolyl, oxazolyl,
benzoxazolyl, imidazolyl, benzimidazolyl, thiazolyl,
benzothiazolyl, isoxazolyl, pyrazolyl, isothiazolyl, pyridazinyl,
pyrimidinyl, pyrazinyl, triazinyl, cinnolinyl, phthalazinyl,
quinazolinyl, benzoquinazolinyl, acridinyl and the like. A
heteroaryl ring can be substituted or unsubstituted on a carbon or
nitrogen atom, wherein substitution on a nitrogen atom may
optionally form a quaternary salt moiety.
[0185] As used herein, unless otherwise specified, the term
"heterocyclyl" means a saturated or partially saturated monocyclic,
bicyclic or polycyclic carbocyclic ring containing from 5 to 14
ring atoms comprising at least one heteroatom, preferably 1 to 3
heteroatoms, independently selected from a nitrogen, oxygen, and
sulfur atom. Representative heterocyclyls are oxiranyl, oxetanyl,
azetidinyl, dihydrofuranyl, tetrahydrofuranyl, dihydrothienyl,
tetrahydrothienyl, pyrrolinyl, pyrrolidinyl, dihydropyrazolyl,
pyrazolinyl, pyrazolidinyl, dihydroimidazolyl, imidazolinyl,
imidazolidinyl, isoxazolinyl, isoxazolidinyl, isothiazolinyl,
isothiazolidinyl, oxazolinyl, oxazolidinyl, thiazolinyl,
thiazolidinyl, triazolinyl, triazolidinyl, oxadiazolinyl,
oxadiazolidinyl, thiadiazolinyl, thiadiazolidinyl, tetrazolinyl,
tetrazolidinyl, dihydro-2H-pyranyl, tetrahydro-2H-pyranyl,
tetrahydro-thiopyranyl, dihydro-pyridinyl, tetrahydro-pyridinyl,
hexahydro-pyridinyl, dihydro-pyrimidinyl, tetrahydro-pyrimidinyl,
dihydro-pyrazinyl, tetrahydro-pyrazinyl, dihydro-pyridazinyl,
tetrahydro-pyridazinyl, piperazinyl, piperidinyl, morpholinyl,
thiomorpholinyl, dihydro-triazinyl, tetrahydro-triazinyl,
hexahydro-triazinyl, dihydro-indole, tetrahydro-indole,
dihydro-indazolyl, tetrahydro-indazolyl, dihydro-isoindolyl,
tetrahydro-isoindolyl, dihydro-benzofuranyl,
tetrahydro-benzofuranyl, dihydro-benzothienyl,
tetrahydro-benzothienyl, dihydro-benzimidazolyl,
tetrahydro-benzimidazolyl, dihydro-benzoxazolyl,
tetrahydro-benzoxazolyl, benzo[1,3]dioxolyl, benzo[1,4]dioxanyl,
dihydro-purinyl, tetrahydro-purinyl, dihydro-quinolinyl,
tetrahydro-quinolinyl, dihydro-isoquinolinyl,
tetrahydro-isoquinolinyl, dihydro-quinazolinyl,
tetrahydro-quinazolinyl, dihydro-quinoxalinyl,
tetrahydro-quinoxalinyl and the like. A heterocyclyl radical can be
unsubstituted or substituted on a carbon or nitrogen atom, wherein
substitution on a nitrogen atom may form a quaternary salt
moiety.
[0186] As used herein, unless otherwise specified, the term
"CH(C.sub.nH.sub.2n+1)," wherein n is an integer from 1 to 8,
refers to an alkyl chain radical of the formula:
--(CH.sub.2).sub.1-7--CH.sub.3 substituted on the B variable of
either Formula (I) or Formula (II), wherein B is --CH--.
[0187] As used herein, unless otherwise specified, the term
"CH(C.sub.mH.sub.2m+1)," wherein m is an integer from 1 to 8,
refers to an alkyl chain radical of the formula:
--(CH.sub.2).sub.1-7--CH.sub.3 substituted on the B.sub.1 variable
of either Formula (Ia) or Formula (IIa), wherein B.sub.1 is
--CH--.
[0188] As used herein, unless otherwise specified, the term
"alkanoyl" refers to a radical of the formula: --C(O)-alkyl,
wherein alkyl is defined above.
[0189] As used herein, unless otherwise specified, the term
"alkanoylamino" refers to a radical of the formula:
--NH--C(O)-alkyl, wherein alkyl is defined above.
[0190] As used herein, unless otherwise specified, the term
"alkanoyloxy" refers to a radical of the formula: --O--C(O)-alkyl,
wherein alkyl is defined above.
[0191] As used herein, the term "alkoxyalkoxy" refers to a radical
of the formula: --O-alkyl-O-alkyl, wherein alkyl is defined above
(e.g., C.sub.1-C.sub.6 alkoxyalkoxy and the like).
[0192] As used herein, the term "alkoxyalkyl" refers to a radical
of the formula: -alkyl-O-alkyl, wherein alkyl is defined above
(e.g., C.sub.1-C.sub.6 alkoxyalkyl and the like).
[0193] As used herein, the term "alkoxycarbonyl" refers to a
radical of the formula: --C(O)--O-alkyl, wherein alkyl is defined
above.
[0194] As used herein, unless otherwise specified, the term
"alkylamino" refers to a radical of the formula: --NH-alkyl,
wherein alkyl is defined above.
[0195] As used herein, unless otherwise specified, the term
"alkylaminoalkyl" refers to a radical of the formula:
-alkyl-NH-alkyl, wherein alkyl is defined above.
[0196] As used herein, unless otherwise specified, the term
"alkylaminocarbonyl" refers to a radical of the formula:
--C(O)--NH-alkyl, wherein alkyl is defined above.
[0197] As used herein, unless otherwise specified, the term
"alkylaminosulfonyl" refers to a radical of the formula:
--SO.sub.2--NH-alkyl, wherein alkyl is defined above.
[0198] As used herein, unless otherwise specified, the term
"alkylaminosulfonylaminocarbonyl" refers to a radical of the
formula: --C(O)--NH--SO.sub.2--NH-alkyl, wherein alkyl is defined
above.
[0199] As used herein, the term "alkylcarbonyl" refers to a radical
of the formula: --C(O)-alkyl, wherein alkyl is defined above.
[0200] As used herein, unless otherwise specified, the term
"alkylsulfonyl" refers to a radical of the formula:
--SO.sub.2-alkyl, wherein alkyl is defined above.
[0201] As used herein, unless otherwise specified, the term
"alkylsulfonylaminocarbonyl" refers to a radical of the formula:
--C(O)--NH--SO.sub.2-alkyl, wherein alkyl is defined above.
[0202] As used herein, unless otherwise specified, the terms
"alkylthio" and "alkylthioether" refer to a radical of the formula:
--S-alkyl, wherein alkyl is defined above.
[0203] As used herein, unless otherwise specified, the term
"alkylthiono" refers to a radical of the formula: --C(S)-alkyl,
wherein alkyl is defined above.
[0204] As used herein, unless otherwise specified, the term "amino"
refers to a radical of the formula: --NH.sub.2.
[0205] As used herein, unless otherwise specified, the term
"aminoalkyl" refers to a radical of the formula: -alkyl-NH.sub.2,
wherein alkyl is defined above.
[0206] As used herein, unless otherwise specified, the term
"aminocarbonyl" refers to a radical of the formula:
--C(O)--NH.sub.2, wherein alkyl is defined above.
[0207] As used herein, unless otherwise specified, the terms
"aminosulfonyl", "sulfonamide" and "sulfonamido" refer to a radical
of the formula: --SO.sub.2--NH.sub.2.
[0208] As used herein, unless otherwise specified, the term
"aminosulfonylaminocarbonyl" refers to a radical of the formula:
--C(O)--NH--SO.sub.2--NH.sub.2.
[0209] As used herein, unless otherwise specified, the term
"aralkanoylamino" refers to a radical of the formula:
--NH--C(O)-alkyl-aryl, wherein alkyl and aryl are defined
above.
[0210] As used herein, unless otherwise specified, the terms
"aroyl" and "arylcarbonyl" refer to a radical of the formula:
--C(O)-aryl, wherein aryl is defined above (e.g., phenylcarbonyl
and the like).
[0211] As used herein, unless otherwise specified, the term
"aroylamino" refers to a radical of the formula: --NH--C(O)-aryl,
wherein aryl is defined above.
[0212] As used herein, unless otherwise specified, the term
"arylalkoxycarbonyl" refers to a radical of the formula:
--C(O)--O-alkyl-aryl, wherein alkyl and aryl are defined above
(e.g., benzyloxycarbonyl, and the like).
[0213] As used herein, unless otherwise specified, the term
"arylalkyl" refers to a radical of the formula: -alkyl-aryl,
wherein alkyl and aryl are defined above.
[0214] As used herein, unless otherwise specified, the term
"arylalkylamino" refers to a radical of the formula:
--NH-alkyl-aryl, wherein alkyl and aryl are defined above.
[0215] As used herein, unless otherwise specified, the term
"N-aryl-N-alkyl-aminocarbonyl" refers to a radical, wherein amino
is disubstituted, of the formula: --C(O)--N(aryl-alkyl), wherein
alkyl and aryl are defined above (e.g.,
N-phenyl-N-alkyl-aminocarbonyl and the like).
[0216] As used herein, unless otherwise specified, the term
"arylalkylsulfonyl" refers to a radical of the formula:
--SO.sub.2-alkyl-aryl, wherein alkyl and aryl are defined
above.
[0217] As used herein, unless otherwise specified, the term
"arylalkylthio" refers to a radical of the formula: --S-alkyl-aryl,
wherein alkyl and aryl are defined above.
[0218] As used herein, unless otherwise specified, the term
"arylalkylthiono" refers to a radical of the formula:
--C(S)-alkyl-aryl, wherein alkyl and aryl are defined above.
[0219] As used herein, unless otherwise specified, the term
"arylamino" refers to a radical of the formula: --NH-aryl, wherein
aryl is defined above.
[0220] As used herein, unless otherwise specified, the term
"arylaminocarbonyl" refers to a radical of the formula:
--C(O)--NH-aryl, wherein aryl is defined above (e.g.,
phenylaminocarbonyl and the like).
[0221] As used herein, unless otherwise specified, the term
"aryloxy" refers to a radical of the formula: --O-aryl, wherein
aryl is defined above.
[0222] As used herein, unless otherwise specified, the term
"arylsulfonyl" refers to a radical of the formula: --SO.sub.2-aryl,
wherein aryl is defined above (e.g., phenylsulfonyl and the
like).
[0223] As used herein, unless otherwise specified, the term
"arylsulfonylaminocarbonyl" refers to a radical of the formula:
--C(O)--NH--SO.sub.2-aryl, wherein aryl is defined above (e.g.,
phenylsulfonylaminocarbonyl, and the like).
[0224] As used herein, unless otherwise specified, the term
"arylthio" refers to a radical of the formula: --S-aryl, wherein
aryl is defined above.
[0225] As used herein, unless otherwise specified, the term
"arylthiono" refers to a radical of the formula: --C(S)-aryl,
wherein aryl is defined above.
[0226] As used herein, the term "carbamyl" refers to a radical of
the formula: --C(O)--NH.sub.2.
[0227] As used herein, the term "carbonyl" refers to a radical of
the formula: --C(O)--.
[0228] As used herein, the term "carboxy" refers to a radical of
the formula: --COOH or --CO.sub.2H.
[0229] As used herein, unless otherwise specified, the term
"cyanoaminocarbonyl" refers to a radical of the formula:
--C(O)--NH--C.ident.N or --C(O)--NH--CN.
[0230] As used herein, unless otherwise specified, the term
"cycloalkylalkoxy" refers to a radical of the formula:
--O-alkyl-cycloalkyl, wherein cycloalkyl is defined above (e.g.,
cyclopentyl-alkoxy, cyclobutyl-alkoxy and the like).
[0231] As used herein, unless otherwise specified, the term
"cycloalkylalkyl" refers to a radical of the formula:
-alkyl-cycloalkyl, wherein cycloalkyl is defined above (e.g.,
C.sub.3-C.sub.6 cycloalkylalkyl and the like).
[0232] As used herein, unless otherwise specified, the term
"cycloalkylamino" refers to a radical of the formula:
--NH-cycloalkyl, wherein cycloalkyl is defined above.
[0233] As used herein, unless otherwise specified, the term
"cycloalkyloxy" refers to a radical of the formula: --O-cycloalkyl,
wherein cycloalkyl is defined above (e.g., C.sub.3-C.sub.6
cycloalkyloxy and the like).
[0234] As used herein, unless otherwise specified, the term
"cycloalkylthio" refers to a radical of the formula:
--S-cycloalkyl, wherein cycloalkyl is defined above.
[0235] As used herein, unless otherwise specified, the term
"dialkylamino" refers to a radical of the formula:
--N(alkyl).sub.2, wherein alkyl is defined above.
[0236] As used herein, unless otherwise specified, the term
"dialkylaminoalkyl" refers to a radical of the formula:
-alkyl-N(alkyl).sub.2, wherein alkyl is defined above.
[0237] As used herein, unless otherwise specified, the term
"dialkylaminocarbonyl" refers to a radical of the formula:
--C(O)--N(alkyl).sub.2, wherein alkyl is defined above (e.g.,
C.sub.1-C.sub.6 dialkylaminocarbonyl and the like).
[0238] As used herein, unless otherwise specified, the term
"dialkylaminosulfonyl" refers to a radical of the formula:
--SO.sub.2--N(alkyl).sub.2, wherein alkyl is defined above (e.g.,
C.sub.1-C.sub.6 dialkylaminosulfonyl and the like).
[0239] As used herein, unless otherwise specified, the term
"dialkylaminosulfonylaminocarbonyl" refers to a radical of the
formula: --C(O)--NH--SO.sub.2--N(alkyl).sub.2, wherein alkyl is
defined above.
[0240] As used herein, the term "formyl" refers to a radical of the
formula: --C(O)H.
[0241] As used herein, the term "guanidino" refers to a radical of
the formula: --NH--C(NH)--NH.sub.2.
[0242] As used herein, the term "halo" or "halogen" generally
refers to a halogen atom radical, such as fluoro, chloro, bromo and
iodo.
[0243] As used herein, the term "haloalkenyl" refers to a radical
of the formula: -alkenyl-halo, wherein alkenyl and halo are defined
above and may be partially or completely substituted where allowed
by available valences with one or more halogen atoms (e.g.,
trifluoroalkenyl, and the like).
[0244] As used herein, the term "haloalkoxy" refers to a radical of
the formula: --O-alkyl-halo, wherein alkyl and halo are defined
above and may be partially or completely substituted where allowed
by available valences with one or more halogen atoms (e.g.,
trifluoroalkoxy, difluoroalkoxy, and the like).
[0245] As used herein, the term "haloalkyl" refers to a radical of
the formula: -alkyl-halo, wherein alkyl and halo are defined above
and may be partially or completely substituted where allowed by
available valences with one or more halogen atoms (e.g.,
trifluoroalkyl and the like).
[0246] As used herein, unless otherwise specified, the term
"heteroarylalkyl" refers to a radical of the formula:
-alkyl-heteroaryl, wherein alkyl and heteroaryl are defined
above.
[0247] As used herein, unless otherwise specified, the terms
"heterocyclylalkyl" and "alkylheterocyclyl" refer to a radical of
the formula: -alkyl-heterocyclyl, wherein alkyl and heterocyclyl
are defined above (e.g., C.sub.1-C.sub.4 morpholinylalkyl and the
like)).
[0248] As used herein, unless otherwise specified, the term
"heterocyclylamino" refers to a radical of the formula:
--NH-heterocyclyl, wherein heterocyclyl is defined above.
[0249] As used herein, unless otherwise specified, the term
"heterocyclylcarbonyl" refers to a radical of the formula:
--C(O)-heterocyclyl, wherein alkyl and aryl are defined above
(e.g., morpholinylcarbonyl, piperidinylcarbonyl, and the like).
[0250] As used herein, unless otherwise specified, the term
"heterocyclyloxy" refers to a radical of the formula:
--O-heterocyclyl, wherein heterocyclyl is defined above.
[0251] As used herein, unless otherwise specified, the term
"heterocyclylthio" refers to a radical of the formula:
--S-heterocyclyl, wherein heterocyclyl is defined above.
[0252] As used herein, the term "hydroxylalkyl" refers to a radical
of the formula: -alkyl-OH, wherein alkyl is defined above and may
be partially or completely substituted where allowed by available
valences with one or more hydroxyl substituents.
[0253] As used herein, unless otherwise specified, the term
"hydroxylaminocarbonyl" refers to a radical of the formula:
--C(O)--NH--OH.
[0254] As used herein, the term "thiol" refers to a radical of the
formula: --SH.
[0255] For the purposes of this invention, where one or more
functionalities encompassing substituent variables for a compound
of Formula (I) are incorporated into a compound of Formula (I),
each functionality appearing at any location within the disclosed
compound may be independently selected, and as appropriate,
independently and/or optionally substituted.
[0256] As used herein, the term "substituent" means positional
variables on the atoms of a core molecule that are known to those
skilled in the art to be chemical moieties that are appropriate for
substitution at a designated atom position, replacing one or more
hydrogens on the designated atom with a selection from the
indicated group, provided that the designated atom's normal valency
under the existing circumstances is not exceeded, and that the
substitution results in a stable compound. Combinations of
substituents and/or variables are permissible only if such
combinations result in stable compounds. It should also be noted
that any carbon as well as heteroatom with unsatisfied valences as
described or shown herein is assumed to have a sufficient number of
hydrogen atom(s) to satisfy the valences described or shown.
[0257] As used herein, the terms "independently substituted," or
"each selected from", and variations thereof, mean that, when any
substituent occurs more than once in a substituent list or as a
portion of a substituent in the list for Formula (I) or another
structural formulae of the invention, the pattern of substitution
on any particular substituent at each occurrence is independent of
the pattern at any other occurrence. Further, the use of a generic
substituent variable on any formula or structure position for a
compound of the present invention is understood to include the
replacement of the generic substituent with species substituents
that are included within the particular genus, e.g., aryl may be
replaced with phenyl or naphthalenyl and the like, and that the
resulting compound is to be included within the scope of the
compounds representative of the present invention.
[0258] As used herein, the term "optionally substituted" means
optional substitution with the specified groups, radicals or
moieties.
[0259] As used herein, unless otherwise specified, the term
"substituted" means a group substituted by one to four or more
substituents, such as, alkyl, alkenyl, alkynyl, cycloalkyl, aroyl,
halo, haloalkyl (e.g., trifluoromethyl), haloalkoxy (e.g.,
trifluoromethoxy), hydroxyl, alkoxy, alkylthioether, cycloalkyloxy,
heterocyclyloxy, oxo, alkanoyl, aryl, arylalkyl, heteroaryl,
heteroarylalkyl, heterocyclyl, aryloxy, alkanoyloxy, amino,
arylamino, arylalkylamino, cycloalkylamino, heterocyclylamino,
mono- and di-substituted amino (in which the one or two
substituents on the amino group are selected from alkyl, aryl or
arylalkyl), alkanoylamino, aroylamino, aralkanoylamino, substituted
alkanoylamino, substituted arylamino, substituted aralkanoylamino,
thiol, alkylthio, arylthio, arylalkylthio, cycloalkylthio,
heterocyclylthio, alkylthiono, arylthiono, arylalkylthiono,
alkylsulfonyl, arylsulfonyl, arylalkylsulfonyl, sulfonamido (e.g.,
SO.sub.2NH.sub.2), substituted sulfonamido, nitro, cyano, carboxy,
carbamyl (e.g., CONH.sub.2), substituted carbamyl (e.g.,
CONH-alkyl, CONH-aryl, CONH-arylalkyl or instances where there are
two substituents on nitrogen selected from alkyl or arylalkyl),
alkoxycarbonyl, aryl, substituted aryl, guanidino, substituted or
unsubstituted heterocyclylalkyl, substituted or unsubstituted
heteroaryl.
[0260] Whenever a range of the number of atoms in a structure is
indicated (e.g., a C.sub.1-C.sub.8, C.sub.1-C.sub.6,
C.sub.1-C.sub.4, or C.sub.1-C.sub.3 alkyl, C.sub.1-C.sub.4
haloalkyl or C.sub.1-C.sub.4 alkylamino, C.sub.2-C.sub.8 alkenyl,
etc.), it is specifically contemplated that any sub-range or
individual number of carbon atoms falling within the indicated
range also can be used. Thus, for instance, the recitation of a
range of 1-8 carbon atoms (e.g., C.sub.1-C.sub.8), 1-6 carbon atoms
(e.g., C.sub.1-C.sub.6), 1-4 carbon atoms (e.g., C.sub.1-C.sub.4),
1-3 carbon atoms (e.g., C.sub.1-C.sub.3), 2-8 carbon atoms (e.g.,
C.sub.2-C.sub.8) as used with respect to any chemical group (e.g.,
alkyl, haloalkyl, alkylamino, alkenyl, etc.) referenced herein
encompasses and specifically describes 1, 2, 3, 4, 5, 6, 7, or 8
carbon atoms, as appropriate, as well as any sub-range thereof
(e.g., 1-2 carbon atoms, 1-3 carbon atoms, 1-4 carbon atoms, 1-5
carbon atoms, 1-6 carbon atoms, 1-7 carbon atoms, 1-8 carbon atoms,
2-3 carbon atoms, 2-4 carbon atoms, 2-5 carbon atoms, 2-6 carbon
atoms, 2-7 carbon atoms, 2-8 carbon atoms, 3-4 carbon atoms, 3-5
carbon atoms, 3-6 carbon atoms, 3-7 carbon atoms, 3-8 carbon atoms,
4-5 carbon atoms, 4-6 carbon atoms, 4-7 carbon atoms, 4-8 carbon
atoms, 5-6 carbon atoms, 5-7 carbon atoms, 5-8 carbon atoms, 6-7
carbon atoms, or 6-8 carbon atoms, as appropriate).
[0261] Compound names used herein were obtained using the Autonom
batch naming feature of ChemDraw Ultra Version 10.0.4, provided by
CambridgeSoft. When the compound name disclosed herein conflicts
with the structure depicted, the structure shown will supercede the
use of the name to define the compound intended.
[0262] The invention encompasses uses of all compounds described by
Formulas (I) and (II) without limitation. However, for the purposes
of further illustration, preferred aspects and elements of the
invention are discussed herein.
[0263] With respect to Formulas (I) and (II), W is selected from
the group consisting of C(O), C(S), and CH.sub.2. According to
certain aspects of the invention, W is C(O), especially with
respect to compounds of Formula (I) and B is CH.sub.2 or
CH(C.sub.nH.sub.2n+1), wherein n is an integer from 1 to 8.
[0264] R.sub.1 and R.sub.2 can be the same or different, and are
selected from the group consisting of H and C.sub.1-C.sub.3 alkyl,
or R.sub.1 and R.sub.2 may be taken together with the carbon atom
to which they are attached to form a C.sub.3-C.sub.5 or
C.sub.3-C.sub.6 cycloalkyl ring or a carbonyl group. Preferably,
R.sub.1 and R.sub.2 are H or C.sub.1-C.sub.3 alkyl. More
preferably, R.sub.1 is H and R.sub.2 is C.sub.1-C.sub.3 alkyl.
[0265] R.sub.3 is selected from the group consisting of H, halogen,
C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 alkoxy, C.sub.1-C.sub.4
haloalkyl, CN, NO.sub.2, heteroaryl, and phenyl optionally
substituted with any combination of one to five halogen, NO.sub.2,
CN, C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 haloalkyl, or
C.sub.1-C.sub.4 alkoxy substituents. However, R.sub.3 preferably is
H.
[0266] According to one aspect of the invention, at least one of
R.sub.4, R.sub.5, R.sub.6 and R.sub.7 is not H. Thus, according to
this aspect of the invention, at least one of R.sub.4, R.sub.5,
R.sub.6 and R.sub.7, and in one embodiment R.sub.5, R.sub.6, and/or
R.sub.7, is selected from the group consisting of hydroxyl,
halogen, CN, NO.sub.2, sulfonamide, C.sub.1-C.sub.8 alkyl,
C.sub.3-C.sub.6 cycloalkyl, cycloalkyloxy, C.sub.1-C.sub.6 alkoxy,
C.sub.1-C.sub.6 haloalkoxy, C.sub.1-C.sub.4 haloalkyl,
C.sub.2-C.sub.8 alkenyl, amino, C.sub.1-C.sub.4 dialkylamino,
C.sub.1-C.sub.4 alkylamino, morpholinyl, heteroaryl, arylamino,
arylalkylamino, phenyl, and C(O)R', NR'(COR''), NR'SO.sub.2R'' and
NR'(CONR''R'''), wherein R', R'' and R''' are independently H,
C.sub.1-C.sub.6 alkyl, phenyl or substituted phenyl, and wherein
C.sub.1-C.sub.8 alkyl is optionally substituted with one or more
substituents selected from the group consisting of C.sub.1-C.sub.4
alkoxy, C.sub.1-C.sub.4 haloalkyl, C.sub.1-C.sub.6 dialkylamino,
C.sub.1-C.sub.6 alkylamino, cycloalkylamino, and morpholinyl, and
the phenyl is optionally substituted with one or more substituents
selected from the group consisting of halogen, NO.sub.2, CN,
C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 haloalkyl, and
C.sub.1-C.sub.4 alkoxy, or any sub-group or sub-combination
thereof.
[0267] In a further embodiment, either R.sub.5 or R.sub.6, or both,
are not H. Thus, either R.sub.5 or R.sub.6, or both, are
independently selected as above or from the group consisting of
halogen, C.sub.1-C.sub.8 alkyl, C.sub.3-C.sub.6 cycloalkyl,
cycloalkyloxy, C.sub.1-C.sub.6 alkoxy, C.sub.1-C.sub.4 haloalkyl,
amino, C.sub.1-C.sub.4 dialkylamino, C.sub.1-C.sub.4 alkylamino,
and morpholinyl, wherein C.sub.1-C.sub.8 alkyl is optionally
substituted with one or more substituents selected from the group
consisting of C.sub.1-C.sub.4 alkoxy, C.sub.1-C.sub.4 haloalkyl,
C.sub.1-C.sub.6 dialkylamino, C.sub.1-C.sub.6 alkylamino,
cycloalkylamino, and morpholinyl.
[0268] In one embodiment, R.sub.5 is H and R.sub.6 is selected as
described above, or R.sub.6 is selected from the group consisting
of halogen, C.sub.1-C.sub.3 alkyl, C.sub.1-C.sub.6 alkoxy,
cycloalkyloxy, C.sub.1-C.sub.4 dialkylamino, and C.sub.1-C.sub.4
haloalkyl. More specific examples of suitable R.sub.6 groups
include chloro, bromo, methyl, ethyl, propyl, i-propyl, methoxy,
ethoxy, propoxy, i-propoxy, cyclohexyloxy, dimethylamino, and
CF.sub.3. When R.sub.6 is not H, it is suitable that each of
R.sub.4, R.sub.5, and R.sub.7 are H.
[0269] When R.sub.5 is not H, R.sub.5 advantageously can be
selected as described above, or from the group consisting of CN,
halogen, C.sub.1-C.sub.6 alkyl, C.sub.3-C.sub.6 cycloalkyl,
C.sub.1-C.sub.6 alkoxy, C.sub.1-C.sub.4 haloalkyl, amino,
C.sub.1-C.sub.4 dialkyl amino, C.sub.1-C.sub.4 alkylamino and
morpholinyl, wherein the C.sub.1-C.sub.8 alkyl is optionally
substituted with one or more substituents selected from the group
consisting of C.sub.1-C.sub.4 alkoxy, C.sub.1-C.sub.4 haloalkyl,
C.sub.1-C.sub.6 dialkylamino, C.sub.1-C.sub.6 alkylamino,
cycloalkylamino, and morpholinyl. Specific examples of suitable
R.sub.5 groups include methyl, ethyl, propyl, or CN. When R.sub.5
is not H, it is suitable that each of R.sub.4, R.sub.6, and R.sub.7
are H.
[0270] When R.sub.7 is not H, R.sub.7 can be selected as described
above, or from the group consisting of halogen, C.sub.1-C.sub.8
alkyl, C.sub.3-C.sub.6 cycloalkyl, C.sub.1-C.sub.6 alkoxy,
C.sub.1-C.sub.4 haloalkyl, amino, C.sub.1-C.sub.4 dialkylamino,
C.sub.1-C.sub.4 alkylamino, C.sub.3-C.sub.6 cycloalkylamino, and
morpholinyl, wherein the C.sub.1-C.sub.8 alkyl is optionally
substituted with one or more substituents selected from the group
consisting of C.sub.1-C.sub.4 alkoxy, C.sub.1-C.sub.4 haloalkyl,
C.sub.1-C.sub.6 dialkylamino, C.sub.1-C.sub.6 alkylamino,
cycloalkylamino, and morpholinyl. More specific examples of R.sub.7
include C.sub.1-C.sub.8 alkyl, amino, or C.sub.1-C.sub.4
alkylamino, such as methyl, ethyl, propyl, or amino. When R.sub.7
is not H, it is suitable that R.sub.4, R.sub.5, and R.sub.6 are
H.
[0271] Alternatively, or in addition, R.sub.4 and R.sub.5, R.sub.5
and R.sub.6, or R.sub.6 and R.sub.7, may be taken together with the
carbon atoms to which they are attached to form a ring, preferably
a 5 or 6 membered heterocyclyl ring, fused to the benzo portion of
the compound of Formula (I). Non-limiting examples of such fused
heterocyclyl rings include a fused [1,4]dioxanyl or fused
[1,3]dioxolanyl ring.
[0272] Additional compounds of Formula (I) are those in which
wherein at least one of R.sub.3, R.sub.4, R.sub.5, R.sub.6, or
R.sub.7 is hydroxyl, C.sub.1-C.sub.6 haloalkoxy, C.sub.2-C.sub.6
alkenyl, or C.sub.1-C.sub.8 alkyl substituted with an arylamino or
arylalkylamino. In a further embodiment, at least one of R.sub.3,
R.sub.4, R.sub.5, R.sub.6, or R.sub.7 is a C.sub.1-C.sub.6
haloalkoxy. Non-limiting examples of haloalkoxy groups include
--OCHF.sub.2.
[0273] Alternatively, or in addition, at least one of R.sub.3,
R.sub.4, R.sub.5, R.sub.6, or R.sub.7 is C(O)R', NR'(COR''),
NR'SO.sub.2R'' and NR'(CONR''R''), wherein R', R'' and R''' are
independently H, C.sub.1-C.sub.6 alkyl, phenyl or substituted
phenyl. Non-limiting examples of such NR'(CONR''R'') groups include
urea (e.g., NH(CO)NH.sub.2).
[0274] X is selected from the group consisting of H, CN,
C(O)OR.sub.8, wherein R.sub.8 is H or C.sub.1-C.sub.8 alkyl, and
C.sub.1-C.sub.8 alkyl optionally is substituted with one or more
substituents selected from the group consisting of C.sub.1-C.sub.4
alkoxy, C.sub.1-C.sub.4 haloalkyl, C.sub.1-C.sub.6 dialkylamino,
C.sub.1-C.sub.6 alkylamino, cycloalkylamino, phenyl, and
morpholinyl; C(O)NR.sub.9R.sub.10, wherein R.sub.9 and R.sub.10 are
independently selected from the group consisting of H and
C.sub.1-C.sub.6 alkyl, or R.sub.9 and R.sub.10 together with the
nitrogen to which they are attached form a heterocyclyl ring such
as morpholinyl; CH.sub.2OR.sub.11, wherein R.sub.11 is H,
C.sub.1-C.sub.8 alkyl, or C.sub.3-C.sub.6 cycloaklyl, wherein
C.sub.1-C.sub.8 alkyl optionally is substituted with one or
substituents selected from the group consisting of C.sub.1-C.sub.4
alkoxy, C.sub.1-C.sub.4 haloalkyl, C.sub.1-C.sub.6 dialkylamino,
C.sub.1-C.sub.6 alkylamino, cycloalkylamino, and morpholinyl;
CH.sub.2NR.sub.9R.sub.10, wherein R.sub.9 and R.sub.10 are as
defined above; CH.sub.2Z, wherein Z is halogen; C(O)NHOH; C(O)NHCN;
C(O)N(R.sub.1)SO.sub.2R.sub.13, wherein R.sub.13 is C.sub.1-C.sub.4
alkyl, phenyl, or substituted phenyl; C.sub.1-C.sub.8 alkyl,
optionally substituted with one or more substituents selected from
the group consisting of C.sub.1-C.sub.4 alkoxy, C.sub.1-C.sub.4
haloalkyl, C.sub.1-C.sub.6 dialkylamino, and C.sub.1-C.sub.6
alkylamino; and C.sub.2-C.sub.8 alkenyl, optionally substituted
with one or more substituents selected from the group consisting of
C.sub.1-C.sub.4 alkoxy, C.sub.1-C.sub.4 haloalkyl, C.sub.1-C.sub.6
dialkylamino, and C.sub.1-C.sub.6 alkylamino; provided that when
the compound is a compound of Formula (I), and each of R.sub.4,
R.sub.5, R.sub.6 and R.sub.7 are H, then X is not C(O)OH.
[0275] While X can be chosen as described above, X can also be
selected from the group consisting of CN; C(O)OR.sub.8, wherein
R.sub.8 is C.sub.1-C.sub.8 alkyl, optionally substituted with
phenyl; C(O)NR.sub.9R.sub.10, wherein R.sub.9 and R.sub.10 are
independently selected from the group consisting of H and
C.sub.1-C.sub.6 alkyl, or R.sub.9 and R.sub.10 together with the
nitrogen to which they are attached form a heterocyclyl ring such
as morpholinyl; CH.sub.2OR.sub.11, wherein R.sub.11 is H,
C.sub.1-C.sub.8 alkyl, or C.sub.3-C.sub.6 cycloaklyl, wherein
C.sub.1-C.sub.8 alkyl optionally is substituted with one or
substituents selected from the group consisting of C.sub.1-C.sub.4
alkoxy, C.sub.1-C.sub.4 haloalkyl, C.sub.1-C.sub.6 dialkylamino,
C.sub.1-C.sub.6 alkylamino, cycloalkylamino, and morpholinyl;
CH.sub.2NR.sub.9R.sub.10, wherein R.sub.9 and R.sub.10 are as
defined above; and CH.sub.2Z, wherein Z is halogen; C(O)NHOH;
C(O)NHCN; C(O)N(R.sub.1)SO.sub.2R.sub.13, wherein in R.sub.13 is
C.sub.1-C.sub.4 alkyl, phenyl, or substituted phenyl.
[0276] In another embodiment, X is selected from the group
consisting of CN; C(O)OR.sub.8, wherein R.sub.8 is C.sub.1-C.sub.8
alkyl, optionally substituted with a phenyl; CH.sub.2OR.sub.11,
wherein R.sub.11 is H, C.sub.1-C.sub.8 alkyl, or C.sub.3-C.sub.6
cycloalkyl, wherein C.sub.1-C.sub.8 alkyl is optionally substituted
with one or more substituents selected from the group consisting of
C.sub.1-C.sub.4 alkoxy, C.sub.1-C.sub.4 haloalkyl, C.sub.1-C.sub.6
dialkylamino, C.sub.1-C.sub.6 alkylamino, cycloalkylamino, and
morpholinyl; CH.sub.2NR.sub.9R.sub.10, wherein R.sub.9 and R.sub.10
are independently selected from the group consisting of H and
C.sub.1-C.sub.6 alkyl, or R.sub.9 and R.sub.10 together with the
nitrogen to which they are attached form a heterocyclyl ring such
as morpholinyl; and CH.sub.2Z, wherein Z is halogen. X can be
selected as C(O)OR.sub.8, wherein R.sub.8 is C.sub.1-C.sub.6 alkyl,
optionally substituted with a phenyl, or CH.sub.2Z, wherein Z is
halogen. More specific examples of suitable X groups include
C(O)OR.sub.8, wherein R.sub.8 is methyl, ethyl, propyl, butyl,
t-butyl, or benzyl.
[0277] According to another aspect of the invention, when X is
selected as described above, and is not C(O)OH, each of R.sub.4,
R.sub.5, R.sub.6 and R.sub.7 can be H. Also, when at least one of
R.sub.4, R.sub.5, R.sub.6 and R.sub.7 is not H, and is instead
selected as described above, X can be C(O)OH. This aspect of the
invention is especially applicable to Formula (I) compounds.
[0278] According to another aspect of the invention, compounds of
Formula (I) are selected such that W is C(S) or CH.sub.2, B is
CH.sub.2, and R.sub.1-R.sub.7 are selected as described above.
[0279] In other embodiments, W.sub.1 is selected from C(O) or
CH.sub.2. In certain embodiments, W.sub.1 is selected from
CH(C.sub.mH.sub.2m+1) and m is an integer selected from 1, 2 or 3.
In certain embodiments, Ring C.sub.1 is selected from the group
consisting of a thienyl ring, a pyridinyl ring, a cyclohexyl ring,
a cyclohexenyl ring, a cyclohexa-1,4-dienyl ring, a
benzo[d][1,3]dioxolyl ring and a 2,3-dihydrobenzo[b][1,4]dioxinyl
ring, each of said rings fused to the moiety of Formula (IIa),
wherein benzo[d][1,3]dioxolyl and 2,3-dihydrobenzo[b][1,4]dioxinyl,
each having a benzo ring portion, are fused via said
benzoportion.
[0280] In other embodiments, R.sub.20 and R.sub.21 are each H. In
certain embodiments, R.sub.20 and R.sub.21 are each C.sub.1-C.sub.3
alkyl. In certain embodiments, R.sub.20 and R.sub.21 are taken
together with the carbon atom to which they are attached to form
carbonyl. In certain embodiments, when X is absent, then R.sub.20
and R.sub.21 may be taken together with the carbon atom to which
they are attached to form a C.sub.3-C.sub.5 or C.sub.3-C.sub.6
cycloalkyl ring selected from cyclopropyl, cyclopentyl or
cyclohexyl.
[0281] In certain embodiments, R.sub.22 is selected from the group
consisting of H, halogen, C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4
alkoxy, C.sub.1-C.sub.4 haloalkyl, cyano, thienyl, furanyl,
pyridinyl, pyrimidinyl and phenyl, wherein phenyl is optionally
substituted with one or two halogen, C.sub.1-C.sub.4 alkyl or
C.sub.1-C.sub.4 alkoxy substituents.
[0282] In certain embodiments, when one, two or three of R.sub.23,
R.sub.24, R.sub.25 and R.sub.26 are each H, then three, two or one
of R.sub.23, R.sub.24, R.sub.25 and R.sub.26, respectively, are
each selected from hydroxyl, halogen, cyano, nitro, C.sub.1-C.sub.8
alkyl, C.sub.1-C.sub.6 alkoxy, C.sub.1-C.sub.6 alkoxyalkoxy,
C.sub.1-C.sub.6 alkoxyalkyl, C.sub.1-C.sub.6 difluoroalkoxy,
C.sub.1-C.sub.6 trifluoroalkoxy, C.sub.1-C.sub.4 trifluoroalkyl,
C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.4 trifluoroalkenyl, amino,
C.sub.1-C.sub.4 alkylamino, C.sub.1-C.sub.4 dialkylamino,
C.sub.1-C.sub.4 aminoalkyl, C.sub.1-C.sub.4 alkylaminoalkyl or
C.sub.1-C.sub.4 dialkylaminoalkyl.
[0283] In certain embodiments, when three of R.sub.23, R.sub.24,
R.sub.25 and R.sub.26 are each H, then one of R.sub.23, R.sub.24,
R.sub.25 and R.sub.26 is selected from phenyl, cyclopentyl,
cyclopropyl, benzyloxy, C.sub.1-C.sub.4 cyclopentylalkoxy,
C.sub.1-C.sub.4 cyclobutylalkoxy, cyclopentyloxy, pyrrolidinyl,
piperidinyl, morpholinyl, C.sub.1-C.sub.4 morpholinylalkyl,
thienyl, pyridinyl, pyrimidinyl, or amino, wherein amino is
optionally disubstituted with one substituent selected from
hydrogen or C.sub.1-C.sub.6 alkyl and the other is selected from
phenyl, C.sub.1-C.sub.4 alkylcarbonyl, aminocarbonyl,
C.sub.1-C.sub.4 alkylaminocarbonyl, C.sub.1-C.sub.4
dialkylaminocarbonyl, phenylcarbonyl, phenylaminocarbonyl,
N-phenyl-N--C.sub.1-C.sub.4 alkyl-aminocarbonyl, C.sub.1-C.sub.6
alkylsulfonyl or phenylsulfonyl, and wherein each instance of
phenyl is optionally substituted with one or two substituents
selected from halogen, C.sub.1-C.sub.4 alkyl or C.sub.1-C.sub.4
alkoxy.
[0284] In certain embodiments, X.sub.1 is absent or is selected
from the group consisting of H, cyano, C.sub.1-C.sub.4 alkyl,
C.sub.1-C.sub.4 alkoxy, C.sub.1-C.sub.4 haloalkyl, C.sub.1-C.sub.6
hydroxylalkyl, C.sub.1-C.sub.6 alkoxyalkyl, C.sub.1-C.sub.4
morpholinylalkyl, amino, C.sub.1-C.sub.4 alkylamino,
C.sub.1-C.sub.4 dialkylamino, C.sub.1-C.sub.4 aminoalkyl,
C.sub.1-C.sub.4 alkylaminoalkyl, C.sub.1-C.sub.4 dialkylaminoalkyl,
carboxy, C.sub.1-C.sub.6 alkoxycarbonyl, benzyloxycarbonyl,
aminocarbonyl, C.sub.1-C.sub.8 alkylaminocarbonyl, C.sub.1-C.sub.8
dialkylaminocarbonyl, hydroxylaminocarbonyl, cyanoaminocarbonyl,
phenylaminocarbonyl, aminosulfonylaminocarbonyl, C.sub.1-C.sub.8
alkylaminosulfonylaminocarbonyl C.sub.1-C.sub.8
dialkylaminosulfonylaminocarbonyl, C.sub.1-C.sub.8
alkylsulfonylaminocarbonyl, phenylsulfonylaminocarbonyl,
morpholinylcarbonyl and piperidinylcarbonyl.
[0285] In one embodiment, a compound useful for the methods
provided herein is Compound 1:
##STR00003##
[0286] In other embodiments, Compound 1 is also referred to as
2-(4-isopropylphenyl)-6-methoxyisoindolin-1-one.
[0287] As those of ordinary skill in the art will appreciate, many
of the molecules described herein may contain one or more chiral
centers, wherein more than one stereoisomer (e.g., diastereomer or
enantiomer) of the molecule may exist. If the stereochemistry of a
structure or a portion of a structure is not indicated, for
example, with bold or dashed lines, the structure or portion of the
structure is to be interpreted as encompassing all stereoisomers of
it. The invention specifically contemplates any individual
stereoisomers (e.g., diastereomers or enantiomers) of the compounds
described herein, as well as mixtures thereof (e.g., racemic
mixtures).
[0288] The compounds described herein can be prepared by any of
several techniques known by those skilled in the art. By way of a
non-limiting example, the compounds can be prepared as described in
International Publication No. WO2007/109211, pub. Sep. 27, 2007,
which is incorporated by reference herein in its entirety. In a
specific embodiment, the compounds for use in the methods described
herein include those described in International Publication No. WO
2007/109211, pub. Sep. 27, 2007, which is incorporated by reference
herein in its entirety.
Nucleic Acid Constructs
[0289] In one aspect, the present invention provides nucleic acid
constructs for use in cell-based and cell-free screening assays for
the identification or validation of compounds that modulate
ribosomal frameshifting. In another aspect, the present invention
provides nucleic acid constructs for use in cell-based and
cell-free screening assays for the identification or validation of
compounds that modulate the efficiency of programmed ribosomal
frameshifting.
[0290] Presented herein are nucleic acid constructs comprising
nucleic acid residues of an exon(s) of SMN or a fragment thereof, a
reporter gene coding sequence lacking a start codon, and in some
instances, nucleic acid residues of an intron(s) of SMN. In
specific aspects, a nucleic acid construct described herein
comprises a fragment of the nucleic residues of an exon 8 of SMN
fused to a reporter gene coding sequence lacking the start codon,
wherein that the first codon of the reporter gene coding sequence
and the first codon of the fragment are out of frame with each
other in the mRNA transcript transcribed from the nucleic acid
construct and the presence of a stop codon in the mRNA transcript
causes translation termination prior to translation of the reporter
gene coding sequence (i.e., an aberrant stop codon). In such mRNA
transcripts, the first start codon and the aberrant stop codon are
in the same contiguous open reading frame without any interruption
by, e.g., a stop codon. In the presence of certain compounds, the
open reading frame may shift so that the start codon and the
aberrant stop codon are no longer in the same open reading frame,
and instead the first start codon and the stop codon found at the
end of the reporter gene coding sequence are in the same contigous
open reading frame without any interruptions. As a result, an
increase in fusion protein with activity of the reporter gene
coding sequence can be detected.
[0291] In one aspect, the nucleic acid constructs described herein
comprise deoxyribonucleic acid (DNA) residues or analogs thereof.
In one embodiment, a nucleic acid construct comprises, in 5' to 3'
order: (i) a fragment of the nucleic acid residues of exon 8 of
SMN; and (ii) a reporter gene coding sequence lacking a start
codon, wherein the reporter gene coding sequence is fused to the
fragment of the nucleic acid residues of exon 8 of SMN such that
the first codon of the reporter gene coding sequence and the first
codon of the fragment are out of frame with each other in the mRNA
transcript transcribed from the nucleic acid construct and a stop
codon is upstream of the reporter gene in the mRNA transcript. In a
specific embodiment, the first codon of the fragment of the nucleic
acid residues of exon 8 of SMN and the first codon of the reporter
gene coding sequence are out of frame with each other by one
nucleotide in the mRNA transcript transcribed from the nucleic acid
construct. In certain embodiments, the fragment of the nucleic acid
residues of exon 8 of SMN consists of the first 3, 5, 7, or 9
nucleotides from the 5' end of exon 8 of SMN. In other embodiments,
the fragment of the nucleic acid residues of exon 8 of SMN consists
of the first 11, 13, 15, 17, or 19 nucleotides from the 5' end of
exon 8 of SMN. In a specific embodiment, the fragment of the
nucleic acid residues of exon 8 SMN consists of the first 21
nucleotides from the 5' end of exon 8 of SMN. In another specific
embodiment, the fragment of the nucleic acid residues of exon 8 of
SMN consists of the first 23 nucleotides from the 5' end of exon 8
of SMN. In certain embodiments, the nucleic acid construct
comprises a start codon upstream (5') to the fragment of the
nucleic acid residues of exon 8 of SMN. In some embodiments, the
nucleic acid construct comprises one or more nucleotide sequences
encoding one or more amino acid sequence (e.g., peptides or
polypeptides), wherein said one or more nucleotide sequences are
upstream (5') of the fragment of the nucleic acid residues of exon
8 of SMN, and wherein the first codon of each of the one or more
nucleotide sequences and the first codon of the fragment are in
frame with the each other in the mRNA transcript transcribed from
the nucleic acid construct. In a specific embodiment, the one or
more nucleotide sequences encoding one or more amino acid sequence
(e.g., peptides or polypeptides) upstream (5') of the fragment of
the nucleic acid residues of exon 8 of SMN contains a start codon.
In accordance with such embodiments, the first start codon and the
stop codon upstream of the reporter gene coding sequence are in the
same contiguous open reading frame.
[0292] In another embodiment, a nucleic acid construct comprises,
in 5' to 3' order: (a) a start codon; (b) a fragment of the nucleic
acid residues of exon 8 of SMN; and (c) a reporter gene coding
sequence lacking a start codon, wherein (i) the reporter gene
coding sequence is fused to the fragment of the nucleic acid
residues of exon 8 of SMN such that the first codon of the reporter
gene coding sequence and the first codon of the fragment are out of
frame with each other in the mRNA transcript transcribed from the
nucleic acid construct and a stop codon is upstream of the reporter
gene coding sequence in the mRNA transcript; and (ii) the first
start codon and the stop codon upstream of the reporter gene coding
sequence in the mRNA transcript are in the same contiguous open
reading frame without any interruption by, e.g., a stop codon.
[0293] In another embodiment, a nucleic acid construct comprises,
in 5' to 3' order: (a) a start codon, (b) a fragment of the nucleic
acid residues of exon 7 of SMN; (c) a fragment of the nucleic acid
residues of exon 8 of SMN; and (d) a reporter gene coding sequence
lacking a start codon, wherein: (i) the reporter gene coding
sequence is fused to the fragment of the nucleic acid residues of
exon 8 of SMN such that the first codon of the reporter gene coding
sequence and the first codon of the fragment are out of frame with
each other in the mRNA transcript transcribed from the nucleic acid
construct and there is a stop codon in the region of the mRNA
transcript corresponding to the fragment of the nucleic acid
residues of exon 8 of SMN (i.e., upstream of the reporter gene
coding sequence); (ii) in the mRNA transcript transcribed from the
nucleic acid construct, the first start codon and the stop codon
upstream of the reporter gene coding sequence are in the same
contiguous open reading frame without any interruption by, e.g., a
stop codon; and (iii) the fragment of the nucleic acid residues of
exon 7 of SMN comprises any number of nucleotides of exon 7 of SMN
so long as in the mRNA transcript transcribed from the nucleic acid
construct the first start codon and the stop codon upstream of the
reporter gene coding sequence are maintained in the same contiguous
open reading frame without any interruption by, e.g., stop codon.
In certain embodiments, the first codon of the fragment of the
nucleic acid residues of exon 7 of SMN and the first codon of the
fragment of the nucleic acid residues of exon 8 of SMN are in frame
with each other in the mRNA transcript transcribed from the nucleic
acid construct. In certain embodiments, the first codon of the
fragment of the nucleic acid residues of exon 8 of SMN and the
first codon of the reporter gene coding sequence are out of frame
with each other by one nucleotide in the mRNA transcript
transcribed from the nucleic acid construct. In one embodiment, the
fragment of the nucleic acid residues of exon 8 of SMN consists of
the first 21 nucleotides from the 5' end of exon 8 of SMN. In
another embodiment, the fragment of the nucleic acid residues of
exon 8 of SMN consists of the first 23 nucleotides from the 5' end
of exon 8 of SMN. In some embodiments, the nucleic acid construct
comprises one or more nucleotide sequences encoding one or more
amino acid sequences (e.g., peptides or polypeptides), wherein said
one or more nucleotide sequences are upstream (5') of the fragment
of the nucleic acid residues of exon 7 of SMN and downstream (3')
to the start codon. In certain embodiments, the first codon of each
of the one or more nucleotide sequences encoding the one or more
amino acid sequences, the first codon of the fragment of the
nucleic acid residues of exon 7 of SMN, and the first codon of the
fragment of the nucleic acid residues of exon 8 of SMN are in frame
with one another in the mRNA transcript transcribed from the
nucleic acid construct. In accordance with such embodiments, the
first start codon and the stop codon upstream of the reporter gene
coding sequence in the mRNA transcript are in the same contiguous
open reading frame without any interruption by, e.g., a stop
codon.
[0294] In another embodiment, a nucleic acid construct comprises,
in 5' to 3' order: (a) the nucleic acid residues of exon 6 of SMN
or a fragment thereof; (b) a fragment of the nucleic acid residues
of exon 7 of SMN; (c) a fragment of the nucleic acid residues of
exon 8 of SMN; and (d) a reporter gene coding sequence lacking a
start codon, wherein: (i) the reporter gene coding sequence is
fused to the fragment of the nucleic acid residues of exon 8 of SMN
such that the first codon of the reporter gene coding sequence and
the first codon of the fragment are out of frame with each other in
the mRNA transcript transcribed from the nucleic acid construct and
there is a stop codon in the region of the mRNA transcript
corresponding to the fragment of the nucleic acid residues of exon
8 of SMN (i.e., upstream of the reporter coding sequence); (ii) in
the mRNA transcript transcribed from the nucleic acid construct,
the first start codon and the stop codon upstream of the reporter
gene coding sequence are in the same contiguous open reading frame
without any interruption by, e.g., a stop codon; and (iii) the
fragment of the nucleic acid residues of exon 7 of SMN comprises
any number of nucleotides of exon 7 of SMN so long as in the mRNA
transcript transcribed from the nucleic acid construct the first
start codon and the stop codon upstream of the reporter gene coding
sequence are maintained in the same contiguous open reading frame
without any interruption by, e.g., stop codon. In one embodiment,
the fragment of the nucleic acid residues of exon 8 of SMN consists
of the first 21 nucleotides from the 5' end of exon 8 of SMN. In
another embodiment, the fragment of the nucleic acid residues of
exon 8 of SMN consists of the first 23 nucleotides from the 5' end
of exon 8 of SMN. In certain embodiments, an internal start codon
(e.g., ATG) in the nucleic acid residues of exon 6 of SMN or a
fragment thereof is used as a start codon for the nucleic acid
construct. In other embodiments, the nucleic acid construct
comprises a start codon upstream (5') to the nucleic acid residues
of exon 6 of SMN or a fragment thereof. In some embodiments, the
first codon of the nucleic acid residues of exon 6 of SMN or a
fragment thereof, the first codon of the fragment of the nucleic
acid residues of exon 7 of SMN, and the first codon of the fragment
of exon 8 of SMN are in frame with each other in the mRNA
transcript transcribed from the nucleic acid construct. In some
embodiments, the nucleic acid construct comprises one or more
nucleotide sequences encoding one or more amino acid sequences
(e.g., peptides or polypeptides), wherein said one or more
nucleotide sequences are upstream (5') of the nucleic acid residues
of exon 6 of SMN or a fragment thereof, and wherein the first codon
of each of the one or more nucleotide sequences, the first codon of
the nucleic acid residues of exon 6 of SMN or a fragment thereof,
and the first codon of the fragment of the nucleic acid residues of
exon 8 of SMN are in frame with one another in the mRNA transcript
transcribed from the nucleic acid construct. In certain
embodiments, the one or more nucleotide sequences encoding amino
acid sequences (e.g., peptides or polypeptides) upstream of the
nucleic acid residues of exon 6 of SMN or a fragment thereof
contains a start codon. In accordance with such embodiments, the
first start codon and the stop codon upstream of the reporter gene
coding sequence in the mRNA transcript are in the same contiguous
open reading frame.
[0295] In another embodiment, a nucleic acid construct comprises,
in 5' to 3' order: (a) a start codon; (b) a fragment of the nucleic
acid residues of exon 7 of SMN; (c) the nucleic acid residues of
intron 7 of SMN or a fragment thereof, wherein the fragment of the
nucleic acid residues of intron 7 comprises any number of
nucleotides of intron 7 of SMN required for a functional, minimum
intron; (d) a fragment of the nucleic acid residues of exon 8 of
SMN; and (e) a reporter gene coding sequence lacking a start codon,
wherein: (i) the reporter gene coding sequence is fused to the
fragment of the nucleic acid residues of exon 8 of SMN such that
the first codon of the reporter gene coding sequence and the first
codon of the fragment are out of frame with each other in the mRNA
transcript transcribed from the nucleic acid construct and there is
a stop codon in the region of the mRNA transcript corresponding to
the fragment of the nucleic acid residues of exon 8 of SMN (i.e.,
upstream of the reporter gene coding sequence); (ii) in the mRNA
transcript transcribed from the nucleic acid construct, the first
start codon and the stop codon upstream of the reporter gene coding
sequence are in the same contiguous open reading frame without any
interruption by, e.g., a stop codon; and (iii) the fragment of the
nucleic acid residues of exon 7 of SMN comprises any number of
nucleotides of exon 7 of SMN required for splicing and so long as
in the mRNA transcript transcribed from the nucleic acid construct
the start codon and the stop codon upstream of the reporter gene
coding sequence are maintained in the same contiguous open reading
frame without any interruption by, e.g., stop codon. In a specific
embodiment, the first codon of the fragment of the nucleic acid
residues of exon 8 of SMN and the first codon of the reporter gene
coding sequence are out of frame with each other by one nucleotide
in the mRNA transcript transcribed from the nucleic acid construct.
In one embodiment, the fragment of the nucleic acid residues of
exon 8 of SMN consists of the first 21 nucleotides from the 5' end
of exon 8 of SMN. In another embodiment, the fragment of the
nucleic acid residues of exon 8 of SMN consists of the first 23
nucleotides from the 5' end of exon 8 of SMN. In a specific
embodiment, the fragment of the nucleic acid residues of exon 7 of
SMN comprises the first two nucleotides from the 3' end of exon 7
of SMN (i.e., nucleotide residues 53 and 54 from the 5' end of exon
7 of SMN). In another specific embodiment, the fragment of the
nucleic acid residues of exon 7 of SMN comprises a minimum of the
first two or six nucleotides from the 3' end of exon 7 of SMN
(i.e., nucleotide residues 49 to 54 from the 5' end of exon 7 of
SMN). In some embodiments, the nucleic acid construct comprises one
or more nucleotide sequences encoding one or more amino acid
sequences (e.g., peptides or polypeptides), wherein said one or
more nucleotide sequences are upstream (5') of the fragment of the
nucleic acid residues of exon 7 of SMN and downstream (3') to the
start codon. In certain embodiments, the first codon of each of the
one or more nucleotide sequences encoding the one or more amino
acid sequences, the first codon of the fragment of the nucleic acid
residues of exon 7 of SMN, and the first codon of the fragment of
the nucleic acid residues of exon 8 of SMN are in frame with one
another in the mRNA transcript transcribed from the nucleic acid
construct. In accordance with such embodiments, the first start
codon and the stop codon upstream of the reporter gene coding
sequence in the mRNA transcript are in the same contiguous open
reading frame without any interruption by, e.g., a stop codon.
[0296] In another embodiment, a nucleic acid construct comprises,
in 5' to 3' order: (a) the nucleic acid residues of exon 6 of SMN
or a fragment thereof; (b) a fragment of the nucleic acid residues
of exon 7 of SMN; (c) the nucleic acid residues of intron 7 of SMN
or a fragment thereof, wherein the fragment of the nucleic acid
residues of intron 7 comprises any number of nucleotides of intron
7 of SMN required for a functional, minimum intron; (d) a fragment
of the nucleic acid residues of exon 8 of SMN; and (e) a reporter
gene coding sequence lacking a start codon, wherein: (i) the
reporter gene coding sequence is fused to the fragment of the
nucleic acid residues of exon 8 of SMN such that the first codon of
the reporter gene coding sequence and the first codon of the
fragment are out of frame with each other in the mRNA transcript
transcribed from the nucleic acid construct and there is a stop
codon in the region of the mRNA transcript corresponding to the
fragment of the nucleic acid residues of exon 8 of SMN (i.e.,
upstream of the reporter gene coding sequence); (ii) the fragment
of the nucleic acid residues of exon 6 of SMN comprises any number
of nucleotides of exon 6 of SMN so long as in the mRNA transcript
transcribed from the nucleic acid construct the first codon of the
fragment of the nucleic acid residues of exon 6 of SMN and the
first codon of the fragment of the nucleic acid residues of exon 8
of SMN are in frame with each other; and (iii) the fragment of the
nucleic acid residues of exon 7 of SMN comprises a minimum of the
nucleotides of exon 7 of SMN required for splicing and in the mRNA
transcript transcribed from the nucleic acid construct the first
start codon and the stop codon upstream of the reporter gene coding
sequence are in the same contiguous open reading frame without any
interruption by, e.g., a stop codon. In some embodiments, in the
mRNA transcript transcribed from the nucleic acid construct, the
regions of the mRNA transcript corresponding to the fragments of
the nucleic acid residues of exon 6 and exon 7 of SMN do not
contain a stop codon. In a specific embodiment, the first codon of
the fragment of the nucleic acid residues of exon 8 of SMN and the
first codon of the reporter gene coding sequence are out of frame
with each other by one nucleotide in the mRNA transcript
transcribed from the nucleic acid construct. In one embodiment, the
fragment of the nucleic acid residues of exon 8 of SMN consists of
the first 21 nucleotides from the 5' end of exon 8 of SMN. In
another embodiment, the fragment of the nucleic acid residues of
exon 8 of SMN consists of the first 23 nucleotides from the 5' end
of exon 8 of SMN. In a specific embodiment, the fragment of the
nucleic acid residues of exon 7 of SMN comprises a minimum of the
first nucleotide from the 5' end of exon 7 of SMN and the first two
nucleotides from the 3' end of exon 7 of SMN (i.e., nucleotide
residues 53 and 54 from the 5' end of exon 7 of SMN). In another
specific embodiment, the fragment of the nucleic acid residues of
exon 7 of SMN comprises a minimum of the first two or six
nucleotides from the 3' end of exon 7 of SMN (i.e., nucleotide
residues 49 to 54 from the 5' end of exon 7 of SMN). In certain
embodiments, an internal start codon (e.g., ATG) in the nucleic
acid residues of exon 6 of SMN or a fragment thereof is used as a
start codon for the nucleic acid construct. In some embodiments,
the nucleic acid construct comprises a start codon upstream (5') to
the nucleic acid residues of exon 6 of SMN or a fragment thereof.
In some embodiments, the nucleic acid construct comprises one or
more nucleotide sequences encoding one or more amino acid sequences
(e.g., peptides or polypeptides), wherein said one or more
nucleotide sequences are upstream (5') of the nucleic acid residues
of exon 6 of SMN or a fragment thereof, and wherein the first codon
of each of the one or more nucleotide sequences. In some
embodiments, the first codon of the nucleic acid residues of exon 6
of SMN or a fragment thereof, and the first codon of the fragment
of the nucleic acid residues of exon 8 of SMN are in frame with one
another in the mRNA transcript transcribed from the nucleic acid
construct. In certain embodiments, the one or more nucleotide
sequences encoding amino acid sequences (e.g., peptides or
polypeptides) upstream of the nucleic acid residues of exon 6 of
SMN or a fragment thereof contains a start codon. In accordance
with such embodiments, the first start codon and the stop codon
upstream of the reporter gene coding sequence in the mRNA
transcript are in the same contiguous open reading frame without
any interruption by, e.g., a stop codon.
[0297] In another embodiment, a nucleic acid construct comprises,
in 5' to 3' order: (a) the nucleic acid residues of exon 6 of SMN
or a fragment thereof; (b) the nucleic acid residues of intron 6 of
SMN or a fragment thereof, wherein the fragment of the nucleic acid
residues of intron 6 of SMN comprises any number of nucleotides of
intron 6 of SMN required for a functional, minimum intron; (c) a
fragment of the nucleic acid residues of exon 7 of SMN; (d) the
nucleic acid residues of intron 7 of SMN or a fragment thereof,
wherein the fragment of the nucleic acid residues of intron 7
comprises any number of nucleotides of intron 7 of SMN required for
a functional, minimum intron; (e) a fragment of the nucleic acid
residues of exon 8 of SMN; and (f) a reporter gene coding sequence
lacking a start codon, wherein: (i) the reporter gene coding
sequence is fused to the fragment of the nucleic acid residues of
exon 8 of SMN such that the first codon of the reporter gene coding
sequence and the first codon of the fragment are out of frame with
each other in the mRNA transcript transcribed from the nucleic acid
construct and there is a stop codon in the region of the mRNA
transcript corresponding to the fragment of the nucleic acid
residues of exon 8 of SMN (i.e., upstream of the reporter gene
coding sequence); (ii) the fragment of the nucleic acid residues of
exon 6 of SMN comprises a minimum of the nucleotides of exon 6 of
SMN required for splicing so long as in the mRNA transcript
transcribed from the nucleic acid construct the first start codon
and the stop codon upstream of the reporter gene coding sequence
are in the same contiguous open reading frame without any
interruption by, e.g., a stop codon; and (iii) the fragment of the
nucleic acid residues of exon 7 of SMN comprises a minimum of the
nucleotides of exon 7 of SMN required for splicing so long as in
the mRNA transcript transcribed from the nucleic acid construct the
first start codon and the stop codon upstream of the reporter gene
coding sequence are in the same contiguous open reading frame
without any interruption by, e.g., a stop codon. In certain
embodiments, the first codon of the nucleic acid residues of exon 6
of SMN or fragment thereof, the first codon of the fragment of the
nucleic acid residues of exon 7 of SMN and the first codon of the
fragment of the nucleic acid residues of exon 8 of SMN are in frame
with each other. In some embodiments, in the mRNA transcript
transcribed from the nucleic acid construct, the regions of the
mRNA transcript corresponding to the fragments of the nucleic acid
residues of exon 6 and exon 7 of SMN do not contain a stop codon.
In a specific embodiment, the first codon of the fragment of the
nucleic acid residues of exon 8 of SMN and the first codon of the
reporter gene coding sequence are out of frame with each other by
one nucleotide in the mRNA transcript transcribed from the nucleic
acid construct. In one embodiment, the fragment of the nucleic acid
residues of exon 8 of SMN consists of the first 21 nucleotides from
the 5' end of exon 8 of SMN. In another embodiment, the fragment of
the nucleic acid residues of exon 8 of SMN consists of the first 23
nucleotides from the 5' end of exon 8 of SMN. In a specific
embodiment, the fragment of the nucleic acid residues of exon 7 of
SMN comprises a minimum of the first nucleotide from the 5' end of
exon 7 of SMN and the first two nucleotides from the 3' end of exon
7 of SMN (i.e., nucleotide residues 53 and 54 from the 5' end of
exon 7 of SMN). In another specific embodiment, the fragment of the
nucleic acid residues of exon 7 of SMN comprises a minimum of the
first two or six nucleotides from the 3' end of exon 7 of SMN
(i.e., nucleotide residues 49 to 54 from the 5' end of exon 7 of
SMN). In certain embodiments, the fragment of the nucleic acid
residues of exon 6 of SMN comprises a minimum of the first two
nucleotides from the 3' end of exon 6 of SMN. In other embodiments,
the fragment of exon 6 of SMN comprises a minimum of the first
three nucleotides from the 3' end of exon 6 of SMN. In certain
embodiments, an internal start codon (e.g., an ATG) of the nucleic
acid residues of exon 6 of SMN or a fragment thereof is used as a
start codon for the nucleic acid construct. In other embodiments,
the nucleic acid construct comprises a start codon upstream (5') to
the nucleic acid residues of exon 6 of SMN or a fragment thereof.
In some embodiments, the nucleic acid construct comprises one or
more nucleotide sequences encoding one or more amino acid sequences
(e.g., peptides or polypeptides), wherein said one or more
nucleotide sequences are upstream (5') of the nucleic acid residues
of exon 6 of SMN or a fragment thereof, and wherein the first codon
of each of the one or more nucleotide sequences, the first codon of
the nucleic acid residues of exon 6 of SMN or a fragment thereof,
and the first codon of the fragment of the nucleic acid residues of
exon 8 of SMN are in frame with one another in the mRNA transcript
transcribed from the nucleic acid construct. In some embodiments,
the first codon of the nucleic acid residues of exon 6 of SMN or a
fragment thereof, the first codon of the fragment of the nucleic
acid residues of exon 7 of SMN, and the first codon of the fragment
of exon 8 of SMN are in frame with each other in the mRNA
transcript transcribed from the nucleic acid construct. In certain
embodiments, the one or more nucleotide sequences encoding amino
acid sequences (e.g., peptides or polypeptides) upstream of the
nucleic acid residues of exon 6 of SMN or a fragment thereof
contains a start codon. In accordance with such embodiments, the
first start codon and the stop codon upstream of the reporter gene
coding sequence in the mRNA transcript are in the same contiguous
open reading frame.
[0298] In another embodiment, a nucleic acid construct comprises,
in 5' to 3' order: (a) a start codon; (b) the nucleic acid residues
of exon 7 of SMN, wherein any number of nucleotides are inserted
after the 48th nucleotide residue from the 5' end of exon 7 of SMN
(i.e., before the 6th nucleotide from the 3' end of exon 7 of SMN)
as long as the native stop codon of exon 7 of SMN is inactivated
and any additional stop codon is not generated; (c) a fragment of
the nucleic acid residues of exon 8 of SMN; and (d) a reporter gene
coding sequence lacking a start codon, wherein: (i) the reporter
gene coding sequence is fused to the fragment of the nucleic acid
residues of exon 8 of SMN such that the first codon of the reporter
gene coding sequence and the first codon of the fragment are out of
frame with each other in the mRNA transcript transcribed from the
nucleic acid construct and there is a stop codon in the region of
the mRNA transcript corresponding to the fragment of the nucleic
acid residues of exon 8 of SMN (i.e., upstream of the reporter gene
coding sequence); (ii) in the mRNA transcript transcribed from the
nucleic acid construct, the first start codon and the stop codon
upstream of the reporter gene coding sequence are in the same
contiguous open reading frame without any interruption by, e.g., a
stop codon. In some embodiments the first codon of the nucleic acid
residues of exon 7 of SMN, and the first codon of the fragment of
exon 8 of SMN are in frame with each other in the mRNA transcript
transcribed from the nucleic acid construct. In a specific
embodiment, the first codon of the fragment of the nucleic acid
residues of exon 8 of SMN and the first codon of the reporter gene
coding sequence are out of frame with each other by one nucleotide
in the mRNA transcript transcribed from the nucleic acid construct.
In one embodiment, the fragment of the nucleic acid residues of
exon 8 of SMN consists of the first 21 nucleotides from the 5' end
of exon 8 of SMN. In another embodiment, the fragment of the
nucleic acid residues of exon 8 of SMN consists of the first 23
nucleotides from the 5' end of exon 8 of SMN. In a specific
embodiment, a single nucleotide residue is inserted after the 48th
nucleotide residue from the 5' end of exon 7 of SMN (i.e., before
the 6th nucleotide from the 3' end of exon 7 of SMN). In some
embodiments, the nucleic acid construct comprises one or more
nucleotide sequences encoding one or more amino acid sequence
(e.g., peptides or polypeptides), wherein said one or more
nucleotide sequences are upstream (5') of the nucleic acid residues
of exon 7 of SMN, and wherein the first codon of each of the one or
more nucleotide sequences, the first codon of the nucleic acid
residues of exon 7 of SMN, and the first codon of the fragment of
the nucleic acid residues of exon 8 of SMN are in frame with one
another in the mRNA transcript transcribed from the nucleic acid
construct. In accordance with such embodiments, the first start
codon and the stop codon upstream of the reporter gene coding
sequence in the mRNA transcript are in the same contiguous open
reading frame without any interruption by, e.g., a stop codon.
[0299] In another embodiment, a nucleic acid construct comprises,
in 5' to 3' order: (a) the nucleic acid residues of exon 6 of SMN
or a fragment thereof; (b) the nucleic acid residues of exon 7 of
SMN, wherein any number of nucleotides are inserted after the 48th
nucleotide residue from the 5' end of exon 7 of SMN (i.e., before
the 6th nucleotide from the 3' end of exon 7 of SMN) as long as the
native stop codon of exon 7 of SMN is inactivated and any
additional stop codon is not generated; (c) a fragment of the
nucleic acid residues of exon 8 of SMN; and (d) a reporter gene
coding sequence lacking a start codon, wherein: (i) the reporter
gene coding sequence is fused to the fragment of the nucleic acid
residues of exon 8 of SMN such that the first codon of the reporter
gene coding sequence and the first codon of the fragment are out of
frame with each other in the mRNA transcript transcribed from the
nucleic acid construct and there is a stop codon in the region of
the mRNA transcript corresponding to the fragment of the nucleic
acid residues of exon 8 of SMN (i.e., upstream of the reporter gene
coding sequence); (ii) in the mRNA transcript transcribed from the
nucleic acid construct the first start codon and the stop codon
upstream of the reporter gene coding sequence are in the same
contiguous open reading frame without any interruption by, e.g., a
stop codon; and (iii) the fragment of the nucleic acid residues of
exon 6 of SMN comprises any number of nucleotides of exon 6 of SMN
so long as in the mRNA transcript transcribed from the nucleic acid
construct in the mRNA transcript transcribed from the nucleic acid
construct the first start codon and the stop codon upstream of the
reporter gene coding sequence are in the same contiguous open
reading frame without any interruption by, e.g., a stop codon. In
some embodiments, in the mRNA transcript transcribed from the
nucleic acid construct, the regions of the mRNA transcript
corresponding to the fragment of the nucleic acid residues of exon
6 of SMN and the nucleic acid residues of exon 7 of SMN do not
contain a stop codon. In some embodiments, the first codon of the
nucleic acid residues of exon 6 of SMN or a fragment thereof, the
first codon of the nucleic acid residues of exon 7 of SMN, and the
first codon of the fragment of exon 8 of SMN are in frame with each
other in the mRNA transcript transcribed from the nucleic acid
construct. In a specific embodiment, the first codon of the
fragment of the nucleic acid residues of exon 8 of SMN and the
first codon of the reporter gene coding sequence are out of frame
with each other by one nucleotide in the mRNA transcript
transcribed from the nucleic acid construct. In one embodiment, the
fragment of the nucleic acid residues of exon 8 of SMN consists of
the first 21 nucleotides from the 5' end of exon 8 of SMN. In
another embodiment, the fragment of the nucleic acid residues of
exon 8 of SMN consists of the first 23 nucleotides from the 5' end
of exon 8 of SMN. In a specific embodiment, a single nucleotide
residue is inserted after the 48th nucleotide residue from the 5'
end of exon 7 of SMN (i.e., before the 6th nucleotide from the 3'
end of exon 7 of SMN). In certain embodiments, an internal start
codon (e.g., an ATG) in the nucleic acid residues of exon 6 of SMN
or a fragment thereof is used as a start codon for the nucleic acid
construct. In other embodiments, the nucleic acid construct
comprises a start codon upstream (5') to the nucleic acid residues
of exon 6 of SMN or a fragment thereof. In some embodiments, the
nucleic acid construct comprises one or more nucleotide sequences
encoding one or more amino acid sequence (e.g., peptides or
polypeptides), wherein said one or more nucleotide sequences are
upstream (5') of the nucleic acid residues of exon 6 of SMN or a
fragment thereof, and wherein the first codon of each of the one or
more nucleotide sequences, the first codon of the nucleic acid
residues of exon 6 of SMN or a fragment thereof, and the first
codon of the fragment of the nucleic acid residues of exon 8 of SMN
are in frame with one another in the mRNA transcript transcribed
from the nucleic acid construct. In certain embodiments, the one or
more nucleotide sequences encoding amino acid sequences (e.g.,
peptides or polypeptides) upstream of the nucleic acid residues of
exon 6 of SMN or a fragment thereof contains a start codon. In
accordance with such embodiments, the first start codon and the
stop codon upstream of the reporter gene coding sequence in the
mRNA transcript are in the same contiguous open reading frame.
[0300] In another embodiment, a nucleic acid construct comprises,
in 5' to 3' order: (a) a start codon; (b) the nucleic acid residues
of exon 7 of SMN, wherein any number of nucleotides are inserted
after the 48th nucleotide residue from the 5' end of exon 7 of SMN
(i.e., before the 6th nucleotide from the 3' end of exon 7 of SMN)
as long as the native stop codon of exon 7 of SMN is inactivated
and any additional stop codon is not generated; (c) the nucleic
acid residues of intron 7 of SMN or a fragment thereof, wherein the
fragment of the nucleic acid residues of intron 7 comprises any
number of nucleotides of intron 7 of SMN required for a functional,
minimum intron; (d) a fragment of the nucleic acid residues of exon
8 of SMN; and (e) a reporter gene coding sequence lacking a start
codon, wherein: (i) the reporter gene coding sequence is fused to
the fragment of the nucleic acid residues of exon 8 of SMN such
that the first codon of the reporter gene coding sequence and the
first codon of the fragment are out of frame with each other in the
mRNA transcript transcribed from the nucleic acid construct and
there is a stop codon in the region of the mRNA transcript
corresponding to the fragment of the nucleic acid residues of exon
8 of SMN (i.e., upstream of the reporter gene coding sequence);
(ii) in the mRNA transcript transcribed from the nucleic acid
construct, the first start codon and the stop codon upstream of the
reporter gene coding sequence are in the same contiguous open
reading frame without any interruption by, e.g., a stop codon. In
some embodiments, the first codon of the nucleic acid residues of
exon 7 of SMN and the first codon of the fragment of exon 8 of SMN
are in frame with each other in the mRNA transcript transcribed
from the nucleic acid construct. In a specific embodiment, the
first codon of the fragment of the nucleic acid residues of exon 8
of SMN and the first codon of the reporter gene coding sequence are
out of frame with each other by one nucleotide in the mRNA
transcript transcribed from the nucleic acid construct. In one
embodiment, the fragment of the nucleic acid residues of exon 8 of
SMN consists of the first 21 nucleotides from the 5' end of exon 8
of SMN. In another embodiment, the fragment of the nucleic acid
residues of exon 8 of SMN consists of the first 23 nucleotides from
the 5' end of exon 8 of SMN. In a specific embodiment, a single
nucleotide residue is inserted after the 48th nucleotide residue
from the 5' end of exon 7 of SMN (i.e., before the 6th nucleotide
from the 3' end of exon 7 of SMN). In some embodiments, the nucleic
acid construct comprises one or more nucleotide sequences encoding
one or more amino acid sequence (e.g., peptides or polypeptides),
wherein said one or more nucleotide sequences are upstream (5') of
the nucleic acid residues of exon 7 of SMN, and wherein the first
codon of each of the one or more nucleotide sequences, the first
codon of the nucleic acid residues of exon 7 of SMN, and the first
codon of the fragment of the nucleic acid residues of exon 8 of SMN
are in frame with one another in the mRNA transcript transcribed
from the nucleic acid construct. In accordance with such
embodiments, the first start codon and the stop codon upstream of
the reporter gene coding sequence in the mRNA transcript are in the
same contiguous open reading frame without any interruption by,
e.g., a stop codon.
[0301] In another embodiment, a nucleic acid construct comprises,
in 5' to 3' order: (a) the nucleic acid residues of exon 6 of SMN
or a fragment thereof; (b) the nucleic acid residues of exon 7 of
SMN, wherein any number of nucleotides are inserted after the 48th
nucleotide residue from the 5' end of exon 7 of SMN (i.e., before
the 6th nucleotide from the 3' end of exon 7 of SMN) as long as the
native stop codon of exon 7 of SMN is inactivated and any
additional stop codon is not generated; (c) the nucleic acid
residues of intron 7 of SMN or a fragment thereof, wherein the
fragment of the nucleic acid residues of intron 7 comprises any
number of nucleotides of intron 7 of SMN required for a functional,
minimum intron; (d) a fragment of the nucleic acid residues of exon
8 of SMN; and (e) a reporter gene coding sequence lacking a start
codon, wherein: (i) the reporter gene coding sequence is fused to
the fragment of the nucleic acid residues of exon 8 of SMN such
that the first codon of the reporter gene coding sequence and the
first codon of the fragment are out of frame with each other in the
mRNA transcript transcribed from the nucleic acid construct and
there is a stop codon in the region of the mRNA transcript
corresponding to the fragment of the nucleic acid residues of exon
8 of SMN (i.e., upstream of the reporter gene coding sequence); and
(ii) in the mRNA transcript transcribed from the nucleic acid
construct, the first start codon and the stop codon upstream from
the reporter gene coding sequence are in the same contiguous open
reading frame without any interruption by, e.g., a stop codon. In a
specific embodiment, the first codon of the fragment of the nucleic
acid residues of exon 8 of SMN and the first codon of the reporter
gene coding sequence are out of frame with each other by one
nucleotide in the mRNA transcript transcribed from the nucleic acid
construct. In one embodiment, the fragment of the nucleic acid
residues of exon 8 of SMN consists of the first 21 nucleotides from
the 5' end of exon 8 of SMN. In another embodiment, the fragment of
the nucleic acid residues of exon 8 of SMN consists of the first 23
nucleotides from the 5' end of exon 8 of SMN. In a specific
embodiment, a single nucleotide residue is inserted after the 48th
nucleotide residue from the 5' end of exon 7 of SMN (i.e., before
the 6th nucleotide from the 3' end of exon 7 of SMN). In certain
embodiments, an internal start codon (e.g., an ATG) of the nucleic
acid residues of exon 6 of SMN or a fragment thereof is used as a
start codon for the nucleic acid construct. In other embodiments,
the nucleic acid construct comprises a start codon upstream (5') to
the nucleic acid residues of exon 6 of SMN or a fragment thereof.
In some embodiments, the nucleic acid construct comprises one or
more nucleotide sequences encoding one or more amino acid sequence
(e.g., peptides or polypeptides), wherein said one or more
nucleotide sequences are upstream (5') of the nucleic acid residues
of exon 6 of SMN or a fragment thereof, and wherein the first codon
of each of the one or more nucleotide sequences, the first codon of
the nucleic acid residues of exon 6 of SMN or a fragment thereof,
and the first codon of the fragment of the nucleic acid residues of
exon 8 of SMN are in frame with one another in the mRNA transcript
transcribed from the nucleic acid construct. In certain
embodiments, the one or more nucleotide sequences encoding amino
acid sequences (e.g., peptides or polypeptides) upstream of the
nucleic acid residues of exon 6 of SMN or a fragment thereof
contains a start codon. In accordance with such embodiments, the
first start codon and the stop codon upstream of the reporter gene
coding sequence in the mRNA transcript are in the same contiguous
open reading frame.
[0302] In another embodiment, a nucleic acid construct comprises,
in 5' to 3' order: (a) the nucleic acid residues of exon 6 of SMN
or a fragment thereof; (b) the nucleic acid residues of intron 6 of
SMN or a fragment thereof, wherein the fragment of the nucleic acid
residues of intron 6 of SMN comprises any number of nucleotides of
intron 6 of SMN required for a functional, minimum intron; (c) the
nucleic acid residues of exon 7 of SMN, wherein any number of
nucleotides are inserted after the 48th nucleotide residue from the
5' end of exon 7 of SMN (i.e., before the 6th nucleotide from the
3' end of exon 7 of SMN) as long as the native stop codon of exon 7
of SMN is inactivated and any additional stop codon is not
generated; (d) the nucleic acid residues of intron 7 of SMN or a
fragment thereof, wherein the fragment of the nucleic acid residues
of intron 7 comprises any number of nucleotides of intron 7 of SMN
required for a functional, minimum intron; (e) a fragment of the
nucleic acid residues of exon 8 of SMN; and (f) a reporter gene
coding sequence lacking a start codon, wherein: (i) the fragment of
the nucleic acid residues of exon 6 of SMN comprises a minimum of
the nucleotides of exon 6 of SMN required for splicing; (ii) the
reporter gene coding sequence is fused to the fragment of the
nucleic acid residues of exon 8 of SMN such that the open reading
frames of the reporter gene coding sequence and the fragment are
out of frame with each other in the mRNA transcript transcribed
from the nucleic acid construct and there is a stop codon in the
region of the mRNA transcript corresponding to the fragment of the
nucleic acid residues of exon 8 of SMN (i.e., upstream of the
reporter gene coding sequence); and (iii) in the mRNA transcript
transcribed from the nucleic acid construct the first start codon
and the stop codon upstream of the reporter gene coding sequence
are in the same contiguous open reading frame without any
interruption by, e.g., a stop codon. In a specific embodiment, the
first codon of the fragment of the nucleic acid residues of exon 8
of SMN and the first codon of the reporter gene coding sequence are
out of frame with each other by one nucleotide in the mRNA
transcript transcribed from the nucleic acid construct. In one
embodiment, the fragment of the nucleic acid residues of exon 8 of
SMN consists of the first 21 nucleotides from the 5' end of exon 8
of SMN. In another embodiment, the fragment of the nucleic acid
residues of exon 8 of SMN consists of the first 23 nucleotides from
the 5' end of exon 8 of SMN. In a specific embodiment, a single
nucleotide residue is inserted after the 48th nucleotide residue
from the 5' end of exon 7 of SMN (i.e., before the 6th nucleotide
from the 3' end of exon 7 of SMN). In certain embodiments, the
fragment of the nucleic acid residues of exon 6 of SMN comprises a
minimum of the first three nucleotides from the 3' end of exon 6 of
SMN. In other embodiments, the fragment of exon 6 of SMN comprises
a minimum of the first three nucleotides from the 3' end of exon 6
of SMN. In certain embodiments, an internal start codon (e.g., an
ATG) of the nucleic acid residues of exon 6 of SMN or a fragment
thereof is used as a start codon for the nucleic acid construct. In
some embodiments, the nucleic acid construct comprises one or more
nucleotide sequences encoding one or more amino acid sequence
(e.g., peptides or polypeptides), wherein said one or more
nucleotide sequences are upstream (5') of the nucleic acid residues
of exon 6 of SMN or a fragment thereof, and wherein the first codon
of each of the one or more nucleotide sequences, the first codon of
the nucleic acid residues of exon 6 of SMN or a fragment thereof,
the first codon of the nucleic acid residues of exon 7 of SMN, and
the first codon of the fragment of the nucleic acid residues of
exon 8 of SMN are in frame with one another in the mRNA transcript
transcribed from the nucleic acid construct. In certain
embodiments, the one or more nucleotide sequences encoding amino
acid sequences (e.g., peptides or polypeptides) upstream of the
nucleic acid residues of exon 6 of SMN or a fragment thereof
contains a start codon. In accordance with such embodiments, the
first start codon and the stop codon upstream of the reporter gene
coding sequence in the mRNA transcript are in the same contiguous
open reading frame.
[0303] In another embodiment, a nucleic acid construct comprises,
in 5' to 3' order: (a) a start codon; (b) the nucleic acid residues
of exon 7 of SMN, wherein a single guanine residue is inserted
after the 48th nucleotide residue from the 5' end of exon 7 of SMN
(i.e., before the 6th nucleotide from the 3' end of exon 7 of SMN);
(c) the nucleic acid residues of intron 7 of SMN or a fragment
thereof, wherein the fragment of the nucleic acid residues of
intron 7 comprises any number of nucleotides of intron 7 of SMN
required for a functional, minimum intron; (d) a fragment of the
nucleic acid residues of exon 8 of SMN; and (e) a reporter gene
coding sequence lacking a start codon, wherein: (i) the reporter
gene coding sequence is fused to the fragment of the nucleic acid
residues of exon 8 of SMN such that the first codon of the reporter
gene coding sequence and the first codon of the fragment are out of
frame with each other in the mRNA transcript transcribed from the
nucleic acid construct and there is a stop codon in the region of
the mRNA transcript corresponding to the fragment of the nucleic
acid residues of exon 8 of SMN (i.e., upstream of the reporter gene
coding sequence); (ii) in the mRNA transcript transcribed from the
nucleic acid construct, the first start codon and the stop codon
upstream of the reporter gene coding sequence are in the same
contiguous open reading frame without any interruption by, e.g., a
stop codon. In some embodiments, the first codon of the nucleic
acid residues of exon 7 of SMN and the first codon of the fragment
of exon 8 of SMN are in frame with each other in the mRNA
transcript transcribed from the nucleic acid construct. In a
specific embodiment, the first codon of the fragment of the nucleic
acid residues of exon 8 of SMN and the first codon of the reporter
gene coding sequence are out of frame with each other by one
nucleotide in the mRNA transcript transcribed from the nucleic acid
construct. In one embodiment, the fragment of the nucleic acid
residues of exon 8 of SMN consists of the first 21 nucleotides from
the 5' end of exon 8 of SMN. In another embodiment, the fragment of
the nucleic acid residues of exon 8 of SMN consists of the first 23
nucleotides from the 5' end of exon 8 of SMN. In some embodiments,
the nucleic acid construct comprises one or more nucleotide
sequences encoding one or more amino acid sequence (e.g., peptides
or polypeptides), wherein said one or more nucleotide sequences are
upstream (5') of the nucleic acid residues of exon 7 of SMN, and
wherein the first codon of each of the one or more nucleotide
sequences, the first codon of the nucleic acid residues of exon 7
of SMN, and the first codon of the fragment of the nucleic acid
residues of exon 8 of SMN are in frame with one another in the mRNA
transcript transcribed from the nucleic acid construct. In
accordance with such embodiments, the first start codon and the
stop codon upstream of the reporter gene coding sequence in the
mRNA transcript are in the same contiguous open reading frame
without any interruption by, e.g., a stop codon.
[0304] In another embodiment, a nucleic acid construct comprises,
in 5' to 3' order: (a) the nucleic acid residues of exon 6 of SMN
or a fragment thereof; (b) the nucleic acid residues of exon 7 of
SMN, wherein a single guanine residue is inserted after the 48th
nucleotide residue from the 5' end of exon 7 of SMN (i.e., before
the 6th nucleotide from the 3' end of exon 7 of SMN); (c) the
nucleic acid residues of intron 7 of SMN or a fragment thereof,
wherein the fragment of intron 7 of SMN comprises any number of
nucleotides of intron 7 of SMN required for a functional, minimum
intron; (d) a fragment of the nucleic acid residues of exon 8 of
SMN; and (e) a reporter gene coding sequence lacking a start codon,
wherein (i) the reporter gene coding sequence is fused to the
fragment of the nucleic acid residues of exon 8 of SMN such that
the first codon of the reporter gene coding sequence and the first
codon of the fragment are out of frame with each other in the mRNA
transcript transcribed from the nucleic acid construct; (ii) the
production of the mRNA transcript generates a stop codon in the
region of the mRNA transcript that corresponds to the fragment of
the nucleic acid residues of exon 8 of SMN (i.e., upstream of the
reporter gene coding sequence); and (iii) in the mRNA transcript
transcribed from the nucleic acid construct, the first start codon
and the stop codon upstream from the reporter gene coding sequence
are in the same contiguous open reading frame without any
interruption by, e.g., a stop codon. In some embodiments, the first
codon of the nucleic acid residues of exon 6 of SMN or a fragment
thereof, the first codon of the nucleic acid residues of exon 7 of
SMN, and the first codon of the fragment of exon 8 of SMN are in
frame with each other in the mRNA transcript transcribed from the
nucleic acid construct. In a specific embodiment, the first codon
of the fragment of the nucleic acid residues of exon 8 of SMN and
the first codon of the reporter gene coding sequence are out of
frame with each other by one nucleotide in the mRNA transcript
transcribed from the nucleic acid construct. In one embodiment, the
fragment of the nucleic acid residues of exon 8 of SMN consists of
the first 21 nucleotides from the 5' end of exon 8 of SMN. In
another embodiment, the fragment of the nucleic acid residues of
exon 8 of SMN consists of the first 23 nucleotides from the 5' end
of exon 8 of SMN. In certain embodiments, an internal start codon
(e.g., an ATG) in the nucleic acid residues of exon 6 of SMN or a
fragment thereof is used as a start codon for the nucleic acid
construct. In other embodiments, the nucleic acid construct
comprises a start codon upstream (5') to the nucleic acid residues
of exon 6 of SMN or a fragment thereof. In some embodiments, the
nucleic acid construct comprises one or more nucleotide sequences
encoding one or more amino acid sequence (e.g., peptides or
polypeptides), wherein said one or more nucleotide sequences are
upstream (5') of the nucleic acid residues of exon 6 of SMN or a
fragment thereof, and wherein the first codon of each of the one or
more nucleotide sequences, the first codon of the nucleic acid
residues of exon 6 of SMN or a fragment thereof, the first codon of
the nucleic acid residues of exon 7 of SMN, and the first codon of
the fragment of the nucleic acid residues of exon 8 of SMN are in
frame with one another in the mRNA transcript transcribed from the
nucleic acid construct. In certain embodiments, the one or more
nucleotide sequences encoding amino acid sequences (e.g., peptides
or polypeptides) upstream of the nucleic acid residues of exon 6 of
SMN or a fragment thereof contains a start codon. In accordance
with such embodiments, the first start codon and the stop codon
upstream of the reporter gene coding sequence in the mRNA
transcript are in the same contiguous open reading frame.
[0305] In another embodiment, a nucleic acid construct comprises,
in 5' to 3' order: (a) the nucleic acid residues of exon 6 of SMN
or a fragment thereof; (b) the nucleic acid residues of intron 6 of
SMN or a fragment thereof, wherein the fragment of the nucleic acid
residues of intron 6 of SMN comprises any number of nucleotides of
intron 6 required for a functional, minimum intron; (c) the nucleic
acid residues of exon 7 of SMN, wherein a single guanine residue is
inserted after the 48th nucleotide residue from the 5' end of exon
7 of SMN (i.e., before the 6th nucleotide from the 3' end of exon 7
of SMN); (d) the nucleic acid residues of intron 7 of SMN or a
fragment thereof, wherein the fragment of intron 7 of SMN comprises
any number of nucleotides of intron 7 of SMN required for a
functional, minimum intron; (e) a fragment of the nucleic acid
residues of exon 8 of SMN; and (f) a reporter gene coding sequence
lacking a start codon, wherein (i) the fragment of the nucleic acid
residues of exon 6 of SMN comprises a minimum of the nucleotides of
exon 6 of SMN required for splicing; (ii) the reporter gene coding
sequence is fused to the fragment of the nucleic acid residues of
exon 8 of SMN such that the first codon of the reporter gene coding
sequence and the first codon of the fragment are out of frame with
each other in the mRNA transcript transcribed from the nucleic acid
construct; (iii) the production of the mRNA transcript generates a
stop codon in the region of the mRNA transcript that corresponds to
the fragment of the nucleic acid residues of exon 8 of SMN (i.e.,
upstream of the reporter gene coding sequence); and (iv) in the
mRNA transcript transcribed from the nucleic acid construct, the
first start codon and the stop codon upstream from the reporter
gene coding sequence are in the same contiguous open reading frame
without any interruption by, e.g., a stop codon. In a specific
embodiment, the first codon of the fragment of the nucleic acid
residues of exon 8 of SMN and the first codon of the reporter gene
coding sequence are out of frame with each other by one nucleotide
in the mRNA transcript transcribed from the nucleic acid construct.
In one embodiment, the fragment of the nucleic acid residues of
exon 8 of SMN consists of the first 21 nucleotides from the 5' end
of exon 8 of SMN. In another embodiment, the fragment of the
nucleic acid residues of exon 8 of SMN consists of the first 23
nucleotides from the 5' end of exon 8 of SMN. In certain
embodiments, the fragment of the nucleic acid residues of exon 6 of
SMN comprises a minimum of the first two nucleotides from the 3'
end of exon 6 of SMN. In other embodiments, the fragment of exon 6
of SMN comprises a minimum of the first three nucleotides from the
3' end of exon 6 of SMN. In certain embodiments, an internal start
codon (e.g., an ATG) in the nucleic acid residues of exon 6 of SMN
or a fragment thereof is used as a start codon for the nucleic acid
construct. In other embodiments, the nucleic acid construct
comprises a start codon upstream (5') to the nucleic acid residues
of exon 6 of SMN or a fragment thereof. In some embodiments, the
nucleic acid construct comprises one or more nucleotide sequences
encoding one or more amino acid sequence (e.g., peptides or
polypeptides), wherein said one or more nucleotide sequences are
upstream (5') of the nucleic acid residues of exon 6 of SMN or a
fragment thereof, and wherein the first codon of each of the one or
more nucleotide sequences, the first codon of the nucleic acid
residues of exon 6 of SMN or a fragment thereof, the first codon of
the nucleic acid residues of exon 7 of SMN, and the first codon of
the fragment of the nucleic acid residues of exon 8 of SMN are in
frame with one another in the mRNA transcript transcribed from the
nucleic acid construct. In certain embodiments, the one or more
nucleotide sequences encoding amino acid sequences (e.g., peptides
or polypeptides) upstream of the nucleic acid residues of exon 6 of
SMN or a fragment thereof contains a start codon. In accordance
with such embodiments, the first start codon and the stop codon
upstream of the reporter gene coding sequence in the mRNA
transcript are in the same contiguous open reading frame.
[0306] In another embodiment, a nucleic acid construct comprises,
in 5' to 3' order: (a) a start codon; (b) a minimum of one
nucleotide; (c) a fragment of the nucleic acid residues of exon 7
of SMN, wherein the fragment of the nucleic acid residues of exon 7
of SMN comprises a minimum of the first six nucleotides from the 3'
end of exon 7 of SMN (i.e., nucleotide residues 49 to 54 from the
5' end of exon 7 of SMN) and wherein a single guanine residue is
inserted into the fragment of the nucleic acid residues of exon 7
of SMN at the location that corresponds to the location in exon 7
of SMN that is after the 48th nucleotide from the 5' end of exon 7
of SMN; (d) the nucleic acid residues of intron 7 of SMN or a
fragment thereof, wherein the fragment of the nucleic acid residues
of intron 7 of SMN comprises any number of nucleotides of intron 7
required for a functional, minimum intron; (e) a fragment of the
nucleic acid residues of exon 8 of SMN; and (f) a reporter gene
coding sequence lacking a start codon, wherein (i) the reporter
gene coding sequence is fused to the fragment of the nucleic acid
residues of exon 8 of SMN such that the first codon of the reporter
gene coding sequence and the first codon of the fragment are out of
frame with each other in the mRNA transcript transcribed from the
nucleic acid construct; (ii) the production of the mRNA transcript
generates a stop codon in the region of the mRNA transcript that
corresponds to the fragment of the nucleic acid residues of exon 8
of SMN (i.e., upstream of the reporter gene coding sequence); and
(iii) in the mRNA transcript transcribed from the nucleic acid
construct, the first start codon and the stop codon upstream from
the reporter gene coding sequence are in the same contiguous open
reading frame without any interruption by, e.g., a stop codon. In
some embodiments, the first codon of the fragment of the nucleic
acid residues of exon 7 of SMN and the first codon of the fragment
of exon 8 of SMN are in frame with each other in the mRNA
transcript transcribed from the nucleic acid construct. In a
specific embodiment, the first codon of the fragment of the nucleic
acid residues of exon 8 of SMN and the first codon of the reporter
gene coding sequence are out of frame with each other by one
nucleotide in the mRNA transcript transcribed from the nucleic acid
construct. In one embodiment, the fragment of the nucleic acid
residues of exon 8 of SMN consists of the first 21 nucleotides from
the 5' end of exon 8 of SMN. In another embodiment, the fragment of
the nucleic acid residues of exon 8 of SMN consists of the first 23
nucleotides from the 5' end of exon 8 of SMN. In certain
embodiments, the nucleic acid construct comprises a start codon
upstream (5') to the minimum of one nucleotide.
[0307] In another embodiment, a nucleic acid construct comprises,
in 5' to 3' order: (a) a start codon; (b) a fragment of the nucleic
acid residues of exon 7 of SMN, wherein the fragment of the nucleic
acid residues of exon 7 of SMN comprises a minimum of the first six
nucleotides from the 3' end of exon 7 of SMN (i.e., nucleotide
residues 49 to 54 from the 5' end of exon 7 of SMN), wherein a
single guanine residue is inserted into the fragment of the nucleic
acid residues of exon 7 of SMN at the location that corresponds to
the location in exon 7 of SMN that is after the 48th nucleotide
from the 5' end of exon 7 of SMN, and wherein the fragment of the
nucleic acid residues of exon 7 of SMN comprises any number of
nucleotides of exon 7 required for splicing; (c) the nucleic acid
residues of intron 7 of SMN or a fragment thereof, wherein the
fragment of the nucleic acid residues of intron 7 of SMN comprises
any number of nucleotides of intron 7 required for a functional,
minimum intron; (d) a fragment of the nucleic acid residues of exon
8 of SMN; and (e) a reporter gene coding sequence lacking a start
codon, wherein (i) the reporter gene coding sequence is fused to
the fragment of the nucleic acid residues of exon 8 of SMN such
that the first codon of the reporter gene coding sequence and the
first codon of the fragment are out of frame with each other in the
mRNA transcript transcribed from the nucleic acid construct; (ii)
the production of the mRNA transcript generates a stop codon in the
region of the mRNA transcript that corresponds to the fragment of
the nucleic acid residues of exon 8 of SMN (i.e., upstream of the
reporter gene coding sequence); and (iii) in the mRNA transcript
transcribed from the nucleic acid construct, the first start codon
and the stop codon upstream from the reporter gene coding sequence
are in the same contiguous open reading frame without any
interruption by, e.g., a stop codon. In a specific embodiment, the
first codon of the fragment of the nucleic acid residues of exon 8
of SMN and the first codon of the reporter gene coding sequence are
out of frame with each other by one nucleotide in the mRNA
transcript transcribed from the nucleic acid construct. In one
embodiment, the fragment of the nucleic acid residues of exon 8 of
SMN consists of the first 21 nucleotides from the 5' end of exon 8
of SMN. In another embodiment, the fragment of the nucleic acid
residues of exon 8 of SMN consists of the first 23 nucleotides from
the 5' end of exon 8 of SMN. In certain embodiments, the nucleic
acid construct comprises a start codon upstream (5') to the
fragment of the nucleic acid residues of exon 7 of SMN.
[0308] In a specific embodiment, a nucleic acid construct
comprises, in 5' to 3' order: (a) a start codon; (b) a minimum of
one nucleotide; (c) a fragment of the nucleic acid residues of exon
7 of SMN, wherein the fragment of the nucleic acid residues of exon
7 of SMN consists of the first six nucleotides from the 3' end of
exon 7 of SMN (i.e., nucleotide residues 49 to 54 from the 5' end
of exon 7 of SMN) and wherein a single guanine residue is inserted
upstream (5') of the fragment of the nucleic acid residues of exon
7 of SMN; (d) the nucleic acid residues of intron 7 of SMN or a
fragment thereof, wherein the fragment of the nucleic acid residues
of intron 7 of SMN comprises any number of nucleotides of intron 7
required for a functional, minimum intron; (e) a fragment of the
nucleic acid residues of exon 8 of SMN; and (f) a reporter gene
coding sequence lacking a start codon, wherein (i) the reporter
gene coding sequence is fused to the fragment of the nucleic acid
residues of exon 8 of SMN such that the first codon of the reporter
gene coding sequence and the first codon of the fragment are out of
frame with each other in the mRNA transcript transcribed from the
nucleic acid construct; (ii) the production of the mRNA transcript
generates a stop codon in the region of the mRNA transcript that
corresponds to the fragment of the nucleic acid residues of exon 8
of SMN (i.e., upstream of the reporter gene coding sequence); and
(iii) in the mRNA transcript transcribed from the nucleic acid
construct, the first start codon and the stop codon upstream from
the reporter gene coding sequence are in the same contiguous open
reading frame without any interruption by, e.g., a stop codon. In a
specific embodiment, the first codon of the fragment of the nucleic
acid residues of exon 8 of SMN and the first codon of the reporter
gene coding sequence are out of frame with each other by one
nucleotide in the mRNA transcript transcribed from the nucleic acid
construct. In one embodiment, the fragment of the nucleic acid
residues of exon 8 of SMN consists of the first 21 nucleotides from
the 5' end of exon 8 of SMN. In another embodiment, the fragment of
the nucleic acid residues of exon 8 of SMN consists of the first 23
nucleotides from the 5' end of exon 8 of SMN. In certain
embodiments, the nucleic acid construct comprises a start codon
upstream (5') to the minimum of one nucleotide.
[0309] In a specific embodiment, a nucleic acid construct
comprises, in 5' to 3' order: (a) a start codon; (b) a fragment of
the nucleic acid residues of exon 7 of SMN, wherein the fragment of
the nucleic acid residues of exon 7 of SMN consists of the first
six nucleotides from the 3' end of exon 7 of SMN (i.e., nucleotide
residues 49 to 54 from the 5' end of exon 7 of SMN), wherein a
single guanine residue is inserted upstream (5') of the fragment of
the nucleic acid residues of exon 7 of SMN, and wherein the
fragment of the nucleic acid residues of exon 7 of SMN comprises
any number of nucleotides of exon 7 required for splicing; (c) the
nucleic acid residues of intron 7 of SMN or a fragment thereof,
wherein the fragment of the nucleic acid residues of intron 7 of
SMN comprises any number of nucleotides of intron 7 required for a
functional, minimum intron; (d) a fragment of the nucleic acid
residues of exon 8 of SMN; and (e) a reporter gene coding sequence
lacking a start codon, wherein (i) the reporter gene coding
sequence is fused to the fragment of the nucleic acid residues of
exon 8 of SMN such that the first codon of the reporter gene coding
sequence and the first codon of the fragment are out of frame with
each other in the mRNA transcript transcribed from the nucleic acid
construct; (ii) the production of the mRNA transcript generates a
stop codon in the region of the mRNA transcript that corresponds to
the fragment of the nucleic acid residues of exon 8 of SMN (i.e.,
upstream of the reporter gene coding sequence); and (iii) in the
mRNA transcript transcribed from the nucleic acid construct, the
first start codon and the stop codon upstream from the reporter
gene coding sequence are in the same contiguous open reading frame
without any interruption by, e.g., a stop codon. In some
embodiments, the first codon of the fragment of the nucleic acid
residues of exon 7 of SMN and the first codon of the fragment of
exon 8 of SMN are in frame with each other in the mRNA transcript
transcribed from the nucleic acid construct. In a specific
embodiment, the first codon of the fragment of the nucleic acid
residues of exon 8 of SMN and the first codon of the reporter gene
coding sequence are out of frame with each other by one nucleotide
in the mRNA transcript transcribed from the nucleic acid construct.
In one embodiment, the fragment of the nucleic acid residues of
exon 8 of SMN consists of the first 21 nucleotides from the 5' end
of exon 8 of SMN. In another embodiment, the fragment of the
nucleic acid residues of exon 8 of SMN consists of the first 23
nucleotides from the 5' end of exon 8 of SMN. In certain
embodiments, the nucleic acid construct comprises a start codon
upstream (5') to the fragment of the nucleic acid residues of exon
7 of SMN.
[0310] In another embodiment, a nucleic acid construct comprises,
in 5' to 3' order: (a) the nucleic acid residues of exon 6 of SMN
or a fragment thereof; (b) a fragment of the nucleic acid residues
of exon 7 of SMN, wherein the fragment of the nucleic acid residues
of exon 7 of SMN comprises a minimum of the first six nucleotides
from the 3' end of exon 7 of SMN (i.e., nucleotide residues 49 to
54 from the 5' end of exon 7 of SMN) and wherein a single guanine
residue is inserted into the fragment of the nucleic acid residues
of exon 7 of SMN at the location that corresponds to the location
in exon 7 of SMN that is after the 48th nucleotide from the 5' end
of exon 7 of SMN; (c) the nucleic acid residues of intron 7 of SMN
or a fragment thereof, wherein the fragment of the nucleic acid
residues of intron 7 of SMN comprises any number of nucleotides of
intron 7 required for a functional, minimum intron; (d) a fragment
of the nucleic acid residues of exon 8 of SMN; and (e) a reporter
gene coding sequence lacking a start codon, wherein (i) the
reporter gene coding sequence is fused to the fragment of the
nucleic acid residues of exon 8 of SMN such that the first codon of
the reporter gene coding sequence and the first codon fragment are
out of frame with each other in the mRNA transcript transcribed
from the nucleic acid construct; (ii) the production of the mRNA
transcript generates a stop codon in the region of the mRNA
transcript that corresponds to the fragment of the nucleic acid
residues of exon 8 of SMN (i.e., upstream of the reporter gene
coding sequence); and (iii) in the mRNA transcript transcribed from
the nucleic acid construct, the first start codon and the stop
codon upstream from the reporter gene coding sequence are in the
same contiguous open reading frame without any interruption by,
e.g., a stop codon. In a specific embodiment, the first codon of
the fragment of the nucleic acid residues of exon 8 of SMN and the
first codon of the reporter gene coding sequence are out of frame
with each other by one nucleotide in the mRNA transcript
transcribed from the nucleic acid construct. In one embodiment, the
fragment of the nucleic acid residues of exon 8 of SMN consists of
the first 21 nucleotides from the 5' end of exon 8 of SMN. In
another embodiment, the fragment of the nucleic acid residues of
exon 8 of SMN consists of the first 23 nucleotides from the 5' end
of exon 8 of SMN. In certain embodiments, an internal start codon
(e.g., an ATG) in the nucleic acid residues of exon 6 of SMN or a
fragment thereof is used as a start codon for the nucleic acid
construct. In some embodiments, the nucleic acid construct
comprises a start codon upstream (5') of the nucleic acid residues
of exon 6 of SMN or a fragment thereof.
[0311] In another embodiment, a nucleic acid construct comprises,
in 5' to 3' order: (a) the nucleic acid residues of exon 6 of SMN
or a fragment thereof; (b) the nucleic acid residues of intron 6 of
SMN or a fragment thereof, wherein the fragment of the nucleic acid
residues of SMN comprises any number of nucleotides of intron 6 of
SMN for a functional, minimum intron; (c) a fragment of the nucleic
acid residues of exon 7 of SMN, wherein the fragment of the nucleic
acid residues of exon 7 of SMN comprises a minimum of the first six
nucleotides from the 3' end of exon 7 of SMN (i.e., nucleotide
residues 49 to 54 from the 5' end of exon 7 of SMN) and wherein a
single guanine residue is inserted into the fragment of the nucleic
acid residues of exon 7 of SMN at the location that corresponds to
the location in exon 7 of SMN that is after the 48th nucleotide
from the 5' end of exon 7 of SMN; (d) the nucleic acid residues of
intron 7 of SMN or a fragment thereof, wherein the fragment of the
nucleic acid residues of intron 7 of SMN comprises any number of
nucleotides of intron 7 required for a functional, minimum intron;
(e) a fragment of the nucleic acid residues of exon 8 of SMN; and
(f) a reporter gene coding sequence lacking a start codon, wherein
(i) the fragment of the nucleic acid residues of exon 6 of SMN
comprises a minimum of the nucleotides of exon 6 of SMN required
for splicing; (ii) the reporter gene coding sequence is fused to
the fragment of the nucleic acid residues of exon 8 of SMN such
that the first codon of the reporter gene coding sequence and the
first codon fragment are out of frame with each other in the mRNA
transcript transcribed from the nucleic acid construct; (iii) the
production of the mRNA transcript generates a stop codon in the
region of the mRNA transcript that corresponds to the fragment of
the nucleic acid residues of exon 8 of SMN (i.e., upstream of the
reporter gene coding sequence); and (iv) in the mRNA transcript
transcribed from the nucleic acid construct, the first start codon
and the stop codon upstream from the reporter gene coding sequence
are in the same contiguous open reading frame without any
interruption by, e.g., a stop codon. In a specific embodiment, the
first codon of the fragment of the nucleic acid residues of exon 8
of SMN and the first codon of the reporter gene coding sequence are
out of frame with each other by one nucleotide in the mRNA
transcript transcribed from the nucleic acid construct. In one
embodiment, the fragment of the nucleic acid residues of exon 8 of
SMN consists of the first 21 nucleotides from the 5' end of exon 8
of SMN. In another embodiment, the fragment of the nucleic acid
residues of exon 8 of SMN consists of the first 23 nucleotides from
the 5' end of exon 8 of SMN. In a specific embodiment, the fragment
of the nucleic acid residues of exon 7 of SMN comprises a minimum
of the first nucleotide from the 5' end of exon 7 of SMN and the
first two nucleotides from the 3' end of exon 7 of SMN (i.e.,
nucleotide residues 53 and 54 from the 5' end of exon 7 of SMN). In
another specific embodiment, the fragment of the nucleic acid
residues of exon 7 of SMN comprises a minimum of the first two or
six nucleotides from the 3' end of exon 7 of SMN (i.e., nucleotide
residues 49 to 54 from the 5' end of exon 7 of SMN). In another
specific embodiment, the fragment of the nucleic acid residues of
exon 7 of SMN comprises a minimum of the first six nucleotides from
the 3' end of exon 7 of SMN (i.e., nucleotide residues 49 to 54
from the 5' end of exon 7 of SMN). In certain embodiments, the
fragment of exon 6 of SMN comprises a minimum of the first two
nucleotides from the 3' end of exon 6 of SMN. In other embodiments,
the fragment of exon 6 of SMN comprises a minimum of the first
three nucleotides from the 3' end of exon 6 of SMN. In certain
embodiments, an internal start codon (e.g., an ATG) in the nucleic
acid residues of exon 6 of SMN or a fragment thereof is used as a
start codon for the nucleic acid construct. In other embodiments,
the nucleic acid construct comprises a start codon upstream (5') to
the nucleic acid residues of exon 6 of SMN or a fragment
thereof.
[0312] In certain embodiments, the term "a functional, minimum
intron" in the context of a fragment of the nucleic acid residues
of intron 6 of SMN or a fragment of the nucleic acid residues of
intron 7 of SMN refers to a fragment that comprises at least six
nucleotides of the 5' splice site of intron 6 or intron 7 of SMN
and three nucleotides plus the polypyrimidine tract and the
branch-point sequence of the 3' splice site of intron 6 or intron 7
of SMN. In one embodiment, the fragment comprises the minimal
number of nucleic acids required for an intron to permit the
retention of the nucleotides of the exons flanking the intron after
splicing. In one embodiment, the 3' splice site plus the
polypyrimidine tract and the branch-point sequence of the 3' splice
comprises about 40 nucleic acid residues of the 3' splice site of
intron 6 or intron 7 of SMN. In another embodiment, the 3' splice
site plus the polypyrimidine tract and the branch-point sequence of
the 3' splice comprises 20 nucleic acid residues of the 3' splice
site of intron 6 or intron 7 of SMN.
[0313] In certain embodiments, the term "minimum of the nucleotides
of exon 6 of SMN required for splicing" refers to a fragment of
exon 6 of SMN that permits removal of an intron via mRNA splicing
and maintains the complete sequence of the mRNA fragment included
(or encoded) in a nucleic acid construct. In a specific embodiment,
the fragment includes the intact 3' end of exon 6 of SMN. In
another embodiment, the fragment of exon 6 of SMN is at least 3, at
least 6, at least 9, or at least 12 nucleic acids long. In a
specific embodiment, the intact 3' end of the fragment of exon 6 of
SMN at least 6, at least 9, or at least 12 nucleic acids long.
[0314] In certain embodiments, the term "minimum of the nucleotides
of exon 7 of SMN required for splicing" refers to a fragment of
exon 7 of SMN that permits removal of an intron via mRNA splicing
and maintains the complete sequence of the mRNA fragment included
(or encoded) in a nucleic acid construct.
[0315] In certain aspects of the invention, an RNA transcript
transcribed from a nucleic acid construct described above is
utilized in the cell-based and cell-free screening assays to
identify or validate compounds that modulate ribosomal
frameshifting (e.g., programmed ribosomal frameshifting).
Techniques for the production of an RNA transcript (e.g., a
pre-mRNA transcript or a mRNA transcript) from a nucleic acid
construct are known o one of skill in the art. For example, a mRNA
transcript can be produced in a run-off transcription of a
linearized form of a nucleic acid construct described herein. In a
specific embodiment, the nucleic acid constructs described herein
comprise bacteriophage promoters (e.g., a T3, SP6 or T7
bacteriophage promoter) or any other suitable promoter that may be
used together with the respective RNA polymerase derived from the
corresponding bacteriophage. Techniques for performing run-off
transcription are well-known in the art. In a specific embodiment,
a mRNA transcript transcribed from a nucleic acid construct
described above is utilized in the cell-based and cell-free
screening assays to identify or validate compounds that modulate
programmed ribosomal frameshifting.
[0316] In certain embodiments, a nucleic acid construct described
herein is isolated. In some embodiments, an RNA transcript (e.g., a
pre-mRNA or mRNA transcript) described herein is isolated.
Screening Assays
Cell-Based Assays
[0317] In one aspect, the present invention provides a method for
the identification or validation of a compound that modulates
ribosomal frameshifting comprising: (a) contacting a compound with
a host cell containing a nucleic acid construct described herein;
and (b) detecting the activity of a fusion protein expressed from
the nucleic acid construct. A compound that increases/causes
ribosomal frameshifting will result in an increase in the activity
of the fusion protein expressed by the host cell compared with (i)
the activity of the fusion protein expressed by the host cell in
the absence of the compound, (ii) the activity of the fusion
protein expressed by the host cell in the presence of a negative
control, and/or (iii) against a previously determined reference
range for a negative control. In a specific embodiment, the
increase in the activity of the fusion protein is a statistically
significant increase. In contrast, a compound that does not
modulate or decreases ribosomal frameshifting will not increase or
not statistically significantly increase the level of activity of
the fusion protein expressed by the host cell compared to (i) the
level of activity of the fusion protein expressed by the host cell
in the absence of the compound, (ii) the level of activity of
fusion protein expressed by the host cell in the presence of a
negative control, and/or (iii) a previously determined reference
range for a negative control.
[0318] In some embodiments, in addition to, or as an alternative
to, detecting the activity of a fusion protein expressed from the
nucleic acid construct, the amount of the fusion protein can be
detected. In accordance with such embodiments, an increase in the
amount of the fusion protein expressed by the host cell in the
presence of the compound when compared to (i) a previously
determined reference range for a negative control, (ii) the amount
of the fusion protein expressed by the host cell in the absence of
the compound in such an assay, and/or (iii) the amount of the
fusion protein expressed by the host cell in the presence of a
negative control in such an assay indicates that a particular
compound increases ribosomal frameshifting. In a specific
embodiment, the increase in the amount of the fusion protein is a
statistically significant increase. In contrast, a compound that
does not modulate ribosomal frameshifting will not increase or not
statistically significantly increase the amount of the fusion
protein expressed by the host cell compared to (i) the amount of
the fusion protein expressed by the host cell in the absence of the
compound, (ii) the amount of fusion protein expressed by the host
cell in the presence of a negative control, and/or (iii) a
previously determined reference range for a negative control.
[0319] In certain embodiments, a method for the identification or
validation of a compound that modulates ribosomal frameshifting
does not use a nucleic acid construct described in Zhang, et al.,
2001, Gene Therapy, 8:1532-1538 (e.g., the nucleic acid construct
in FIG. 1 of Zhang, et al., 2001, Gene Therapy 8: 1532-1538).
[0320] In another aspect, the present invention provides a method
for the identification or validation of a compound that modulates
the efficiency of programmed ribosomal frameshifting comprising:
(a) contacting a compound with a host cell containing a nucleic
acid construct described herein; and (b) detecting the activity of
a fusion protein expressed from the nucleic acid construct. A
compound that modulates the efficiency of programmed ribosomal
frameshifting will result in an increase in the activity of the
fusion protein expressed by the host cell compared with (i) the
activity of the fusion protein expressed by the host cell in the
absence of the compound, (ii) the activity of the fusion protein
expressed by the host cell in the presence of a negative control,
and/or (iii) against a previously determined reference range for a
negative control. In a specific embodiment, the increase in the
activity of the fusion protein is a statistically significant
increase. In contrast, a compound that does not modulate the
efficiency of programmed ribosomal frameshifting will not increase
or not statistically significantly increase the level of activity
of the fusion protein expressed by the host cell compared to (i)
the level of activity of the fusion protein expressed by the host
cell in the absence of the compound, (ii) the level of activity of
fusion protein expressed by the host cell in the presence of a
negative control, and/or (iii) a previously determined reference
range for a negative control.
[0321] In some embodiments, in addition to, or as an alternative
to, detecting the activity of a fusion protein expressed from the
nucleic acid construct, the amount of the fusion protein can be
detected. In accordance with such embodiments, an increase in the
amount of the fusion protein expressed by the host cell in the
presence of the compound when compared to (i) a previously
determined reference range for a negative control, (ii) the amount
of the fusion protein expressed by the host cell in the absence of
the compound in such an assay, and/or (iii) the amount of the
fusion protein expressed by the host cell in the presence of a
negative control in such an assay indicates that a particular
compound modulates the efficiency of programmed ribosomal
frameshifting. In a specific embodiment, the increase in the amount
of the fusion protein is a statistically significant increase. In
contrast, a compound that does not modulate the efficiency of
programmed ribosomal frameshifting will not increase or not
statistically significantly increase the amount of the fusion
protein expressed by the host cell compared to (i) the amount of
the fusion protein expressed by the host cell in the absence of the
compound, (ii) the amount of fusion protein expressed by the host
cell in the presence of a negative control, and/or (iii) a
previously determined reference range for a negative control.
[0322] In one embodiment, a method for identifying or validating a
compound that modulates the efficiency of programmed ribosomal
frameshifting comprises: (a) expressing in a host cell a nucleic
acid construct described herein; (b) contacting said host cell with
a compound; and (c) detecting the activity or amount of a fusion
protein encoded by the nucleic acid construct, wherein a compound
that modulates the efficiency of programmed ribosomal frameshifting
is identified or validated if the activity or amount of the fusion
protein expressed by the host cell in the presence of a compound is
increased relative to a previously determined reference range for a
negative control, or relative to the activity or amount of the
fusion protein expressed by the host cell in the absence of said
compound or the presence of a negative control (e.g., PBS or DMSO).
In a specific embodiment, an increase in the activity or amount of
the fusion protein is a statistically significant increase.
[0323] In a specific embodiment, a method for identifying or
validating a compound that modulates ribosomal frameshifting
comprises: (a) contacting a compound with a host cell expressing a
nucleic acid construct described in Zhang, et al., 2001, Gene
Therapy, 8:1532-1538 (e.g., the nucleic acid construct in FIG. 1 of
Zhang, et al., 2001, Gene Therapy 8: 1532-1538); and (b) detecting
the activity or amount of a fusion protein encoded by the nucleic
acid construct, wherein a compound that modulates ribosomal
frameshifting is identified or validated if the activity or amount
of the fusion protein detected in the presence of the compound is
increased relative to the activity or amount of the fusion protein
detected in the presence of a negative control (e.g., 0.005%-1%
DMSO). In a specific embodiment, an increase in the activity or
amount of the fusion protein is a statistically significant
increase.
[0324] In another specific embodiment, a method for identifying or
validating a compound that modulates the efficiency of programmed
ribosomal frameshifting comprises: (a) contacting a compound with a
host cell expressing a nucleic acid construct described in Zhang,
et al., 2001, Gene Therapy, 8:1532-1538 (e.g., the nucleic acid
construct in FIG. 1 of Zhang, et al., 2001, Gene Therapy 8:
1532-1538); and (b) detecting the activity or amount of a fusion
protein encoded by the nucleic acid construct, wherein a compound
that modulates the efficiency of programmed ribosomal frameshifting
is identified or validated if the activity or amount of the fusion
protein detected in the presence of the compound is increased
relative to the activity or amount of the fusion protein detected
in the presence of a negative control (e.g., 0.005%-1% DMSO). In a
specific embodiment, an increase in the activity or amount of the
fusion protein is a statistically significant increase.
[0325] In certain embodiments, a method for identifying or
validating a compound that modulates the efficiency of programmed
ribosomal frameshifting does not comprise: (a) contacting a
compound with a host cell expressing a nucleic acid construct
described in Zhang, et al., 2001, Gene Therapy, 8:1532-1538 (e.g.,
the nucleic acid construct in FIG. 1 of Zhang, et al., 2001, Gene
Therapy 8: 1532-1538); and (b) detecting the activity or amount of
a fusion protein by the nucleic acid construct, wherein a compound
that modulates the efficiency of programmed ribosomal frameshifting
is identified or validated if the activity or amount of the fusion
protein detected in the presence of the compound is increased
relative to the activity or amount of the fusion protein detected
in the presence of a negative control (e.g., 0.005%-1% DMSO).
[0326] In another aspect, the invention provides a method for
identifying or validating a compound that modulates the efficiency
of programmed ribosomal frameshifting comprising: (a) contacting a
compound with a host cell engineered to contain a pre-mRNA or mRNA
transcript transcribed from a nucleic acid construct described
herein; and (b) detecting the activity or amount of a fusion
protein translated from the pre-mRNA or mRNA transcript, wherein a
compound that modulates the efficiency of programmed ribosomal
frameshifting is identified or validated if the activity or amount
of the fusion protein expressed by the host cell in the presence of
a compound is increased relative to a previously determined
reference range for a negative control, or relative to the activity
or amount of the fusion protein expressed by the host cell in the
absence of said compound or the presence of a negative control
(e.g., PBS or DMSO). In a specific embodiment, an increase in the
activity or amount of the fusion protein is a statistically
significant increase.
[0327] In one embodiment, the invention provides a method for
identifying or validating a compound that modulates the efficiency
of programmed ribosomal frameshifting comprising: (a) transfecting
into a cell a RNA transcript (e.g., pre-mRNA or mRNA transcript)
transcribed from a nucleic acid construct described herein; (b)
contacting said host cell with a compound; and (c) detecting the
activity or amount of a fusion protein translated from the RNA
transcript, wherein a compound that modulates the efficiency of
programmed ribosomal frameshifting is identified or validated if
the activity or amount of the fusion protein expressed by the host
cell in the presence of a compound is increased relative to a
previously determined reference range for a negative control, or
relative to the activity or amount of the fusion protein expressed
by the host cell in the absence of said compound or the presence of
a negative control (e.g., PBS or DMSO). In a specific embodiment,
an increase in the activity or amount of the fusion protein is a
statistically significant increase.
[0328] In a specific embodiment, a method for identifying or
validating a compound that modulates the efficiency of programmed
ribosomal frameshifting comprises: (a) contacting a compound with a
host cell engineered to contain a RNA transcript (e.g., pre-mRNA or
mRNA transcript) transcribed from a nucleic acid construct
described in Zhang, et al., 2001, Gene Therapy, 8:1532-1538 (e.g.,
the nucleic acid construct in FIG. 1 of Zhang, et al., 2001, Gene
Therapy 8: 1532-1538); and (b) detecting the activity or amount of
a fusion protein translated from the RNA transcript, wherein a
compound that modulates the efficiency of programmed ribosomal
frameshifting is identified or validated if the activity or amount
of the fusion protein detected in the presence of the compound is
increased relative to the activity or amount of the fusion protein
detected in the presence of a negative control (e.g., 0.005%-1%
DMSO). In a specific embodiment, an increase in the activity or
amount of the fusion protein is a statistically significant
increase.
[0329] In a specific embodiment, a method for identifying or
validating a compound that modulates the efficiency of programmed
ribosomal frameshifting does not comprise: (a) contacting a
compound with a host cell engineered to contain a RNA transcript
(e.g., pre-mRNA or mRNA transcript) transcribed from a nucleic acid
construct described in Zhang, et al., 2001, Gene Therapy,
8:1532-1538 (e.g., the nucleic acid construct in FIG. 1 of Zhang,
et al., 2001, Gene Therapy 8: 1532-1538); and (b) detecting the
activity or amount of a fusion protein translated from the RNA
transcript, wherein a compound that modulates the efficiency of
programmed ribosomal frameshifting is identified or validated if
the activity or amount of the fusion protein detected in the
presence of the compound is increased relative to the activity or
amount of the fusion protein detected in the presence of a negative
control (e.g., 0.005%-1% DMSO).
[0330] The step of contacting a compound with a host cell
containing the nucleic acid construct or RNA transcript may be
conducted under conditions approximating or mimicking physiologic
conditions. In a specific embodiment, a compound is added to the
cells in the presence of an appropriate growth medium for said
cells. In another embodiment, a compound is added to the cells in
the presence of an aqueous solution. In accordance with this
embodiment, the aqueous solution may comprise a buffer and a
combination of salts, preferably approximating or mimicking
physiologic conditions. Alternatively, the aqueous solution may
comprise a buffer, a combination of salts, and a detergent or a
surfactant. Examples of salts which may be used in the aqueous
solution include, but are not limited to, KCl, NaCl, and/or
MgCl.sub.2. The optimal concentration of each salt used in the
aqueous solution is dependent on the host cells and compounds used
and can be determined using routine experimentation.
[0331] A compound is contacted with a host cell containing the
nucleic acid construct or RNA transcript for a specific period of
time. For example, the compound may be contacted with the host cell
containing the nucleic acid construct or RNA transcript for a time
period of about 1 minute, 2 minutes, 3 minutes, 4, minutes, 5,
minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 1 hour, 2
hours, 3 hours, 4 hours, 5 hours, 10 hours, 15 hours, 20 hours, 1
day, 2 days, 3 days, 4 days, 5 days, or 1 week. In a specific
embodiment, contact is over a time period of about 12 hours to 15
hours, i.e., overnight.
[0332] In specific embodiments, a negative control (e.g., DMSO at
0.005-1.0%, or PBS, or another agent that is known to have no
effect on the expression of the fusion protein) and a positive
control (e.g., an agent that modulates the efficiency of programmed
ribosomal frameshifting) are included in the cell-based assays
described herein.
[0333] The amount or activity of a fusion protein described herein
may be detected by any technique well-known to one of skill in the
art. For example, techniques well-known to one of skill in the art
for detecting reporter proteins can be used to detect either or
both the amount or activity of fusion proteins. Methods for
detecting the amount or activity of a reporter protein will vary
with the reporter gene used. Assays for the various reporter genes
are well-known to one of skill in the art.
Cell-Free Assays
[0334] In one aspect, the present invention provides a method for
the identification or validation of a compound that modulates
ribosomal frameshifting comprising: (a) contacting a compound with
a cell-free extract and a RNA transcript (e.g., mRNA or pre-mRNA)
which is transcribed from a nucleic acid construct described
herein; and (b) detecting the amount or activity of the fusion
protein translated from the RNA. A compound that increases/causes
ribosomal frameshifting will result in an increase in the amount or
activity of the fusion protein translated from the RNA compared
with (i) the amount or activity of the fusion protein translated
from the RNA in the absence of the compound, (ii) the amount or
activity of the fusion protein translated from the RNA in the
presence of a negative control, and/or (iii) against a previously
determined reference range for a negative control. In a specific
embodiment, the increase in the amount or activity of the fusion
protein is a statistically significant increase. In contrast, a
compound that does not modulate or decreases ribosomal
frameshifting will not increase or not statistically significantly
increase the amount or activity of the fusion protein translated
from the RNA compared to (i) the amount or activity of the fusion
protein translated from the RNA in the absence of the compound,
(ii) the amount or activity of the fusion protein translated from
the RNA in the presence of a negative control, and/or (iii) against
a previously determined reference range for a negative control.
[0335] In one embodiment, the present invention provides a method
for identifying or validating a compound that modulates ribosomal
frameshifting comprising: (a) contacting a compound with a
cell-free extract and a RNA transcript (e.g., mRNA or pre-mRNA)
which is transcribed from a nucleic acid construct described
herein; and (b) detecting the amount or activity of the fusion
protein translated from the RNA, wherein a compound that increases
ribosomal frameshifting is identified or validated if the amount or
activity of the fusion protein detected in the presence of the
compound is increased relative to the amount or activity of the
fusion protein detected in the absence of the compound or presence
of a negative control (e.g., DMSO, PBS and the like), or relative
to a previously determined reference range that is the amount or
activity of the fusion protein obtained for a negative control. In
a specific embodiment, the increase in the activity or amount of
the fusion protein is a statistically significant change.
[0336] In another embodiment, a method for identifying or
validating a compound that modulates ribosomal frameshifting
comprises: (a) contacting a compound with a cell-free extract and a
RNA transcript (e.g., a pre-mRNA or mRNA transcript) which is
transcribed from a nucleic acid construct described in Zhang, et
al., 2001, Gene Therapy, 8:1532-1538 (e.g., the nucleic acid
construct in FIG. 1 of Zhang, et al., 2001, Gene Therapy,
8:1532-1538); and (b) detecting the activity or amount of a fusion
protein translated from the RNA transcript, wherein a compound that
modulates ribosomal frameshifting is identified or validated if the
activity or amount of the fusion protein detected in the presence
of a compound is increased relative to activity or amount of the
fusion protein detected in the presence of a negative control
(e.g., 0.005%-1% DMSO). In a specific embodiment, the increase in
the activity or amount of the fusion protein is a statistically
significant increase.
[0337] In another aspect, the present invention provides a method
for identifying or validating a compound that modulates the
efficiency of programmed ribosomal frameshifting comprising: (a)
contacting a compound with a cell-free extract and a RNA transcript
(e.g., mRNA or pre-mRNA) which is transcribed from a nucleic acid
construct described herein; and (b) detecting the amount or
activity of the fusion protein translated from the RNA, wherein a
compound that modulates the efficiency of programmed ribosomal
frameshifting is identified or validated if the amount or activity
of the fusion protein detected in the presence of the compound is
increased relative to the amount or activity of the fusion protein
detected in the absence of the compound or presence of a negative
control (e.g., DMSO, PBS and the like), or relative to a previously
determined reference range that is the amount or activity of the
fusion protein obtained for a negative control. In a specific
embodiment, the increase in the activity or amount of the fusion
protein is a statistically significant change.
[0338] Typically, the RNA transcript (e.g., mRNA or pre-mRNA) used
in the cell-free assay described herein is one that has been
produced using, e.g., in vitro run-off transcription. For example,
a RNA can be made in run-off transcription of a linearized form of
a nucleic acid construct described herein. Bacteriophage promoters
from a T3, SP6 or T7 bacteriophage or any other suitable promoter
may be used together with the respective RNA polymerase derived
from the corresponding bacteriophage.
[0339] In another embodiment, a method for identifying or
validating a compound that modulates the efficiency of programmed
ribosomal frameshifting comprises: (a) contacting a compound with a
cell-free extract and a RNA transcript (e.g., a pre-mRNA or mRNA
transcript) which is transcribed from a nucleic acid construct
described in Zhang, et al., 2001, Gene Therapy, 8:1532-1538 (e.g.,
the nucleic acid construct in FIG. 1 of Zhang, et al., 2001, Gene
Therapy, 8:1532-1538); and (b) detecting the activity or amount of
a fusion protein translated from the RNA transcript, wherein a
compound that modulates the efficiency of programmed ribosomal
frameshifting is identified or validated if the activity or amount
of the fusion protein detected in the presence of a compound is
increased relative to activity or amount of the fusion protein
detected in the presence of a negative control (e.g., 0.005%-1%
DMSO). In a specific embodiment, the increase in the activity or
amount of the fusion protein is a statistically significant
increase.
[0340] In certain embodiments, a method for the identification or
validation of a compound that modulates ribosomal frameshifting
does not use a cell-free extract and a RNA transcript (e.g., a
pre-mRNA or mRNA transcript) which is transcribed from a nucleic
acid construct described in Zhang, et al., 2001, Gene Therapy,
8:1532-1538 (e.g., the nucleic acid construct in FIG. 1 of Zhang,
et al., 2001, Gene Therapy, 8:1532-1538).
[0341] In some embodiments, a method for identifying or validating
a compound that modulates the efficiency of programmed ribosomal
frameshifting does not comprise: (a) contacting a compound with a
cell-free extract and a RNA transcript (e.g., a pre-mRNA or mRNA
transcript) which is transcribed from a nucleic acid construct
described in Zhang, et al., 2001, Gene Therapy, 8:1532-1538 (e.g.,
the nucleic acid construct in FIG. 1 of Zhang, et al., 2001, Gene
Therapy, 8:1532-1538); and (b) detecting the activity or amount of
a fusion protein translated from the RNA transcript, wherein a
compound that modulates the efficiency of programmed ribosomal
frameshifting is identified or validated if the activity or amount
of the fusion protein detected in the presence of a compound is
increased relative to activity or amount of the fusion protein
detected in the presence of a negative control (e.g., 0.005%-1%
DMSO).
[0342] In another aspect, the present invention provides a method
for identifying or validating a compound that modulates the
efficiency of programmed ribosomal frameshifting comprising: (a)
contacting a compound with a cell-free extract and a nucleic acid
construct described herein; and (b) detecting the amount or
activity of the fusion protein expressed from the nucleic acid
construct, wherein a compound that modulates the efficiency of
programmed ribosomal frameshifting is identified or validated if
the amount or activity of the fusion protein detected in the
presence of the compound is increased relative to the amount or
activity of the fusion protein detected in the absence of the
compound or presence of a negative control (e.g., DMSO, PBS and the
like), or relative to a previously determined reference range that
is the amount or activity of the fusion protein obtained for a
negative control. In accordance with this aspect, the cell-free
extract used comprises components necessary for in vitro
transcription, splicing, and translation. In a specific embodiment,
the increase in the activity or amount of the fusion protein is a
statistically significant increase.
[0343] In one embodiment, a method for identifying or validating a
compound that modulates the efficiency of programmed ribosomal
frameshifting comprises: (a) contacting a compound with a cell-free
extract and a nucleic acid construct described in Zhang, et al.,
2001, Gene Therapy, 8:1532-1538 (e.g., the nucleic acid construct
in FIG. 1 of Zhang, et al., 2001, Gene Therapy, 8:1532-1538); and
(b) detecting the activity or amount of a fusion protein expressed
from the nucleic acid construct, wherein a compound that modulates
the efficiency of programmed ribosomal frameshifting is identified
or validated if the activity or amount of the fusion protein
detected in the presence of a compound is increased relative to
activity or amount of the fusion protein detected in the presence
of a negative control (e.g., 0.005%-1% DMSO). In accordance with
this embodiment, the cell-free extract used comprises components
necessary for in vitro transcription, splicing, and translation. In
a specific embodiment, the increase in the activity or amount of
the fusion protein is a statistically significant increase.
[0344] In some embodiments, a method for identifying or validating
a compound that modulates the efficiency of programmed ribosomal
frameshifting does not comprise: (a) contacting a compound with a
cell-free extract and a nucleic acid construct described in Zhang,
et al., 2001, Gene Therapy, 8:1532-1538 (e.g., the nucleic acid
construct in FIG. 1 of Zhang, et al., 2001, Gene Therapy,
8:1532-1538); and (b) detecting the activity or amount of a fusion
protein expressed from the nucleic acid construct, wherein a
compound that modulates the efficiency of programmed ribosomal
frameshifting is identified or validated if the activity or amount
of the fusion protein detected in the presence of a compound is
increased relative to activity or amount of the fusion protein
detected in the presence of a negative control (e.g., 0.005%-1%
DMSO).
[0345] The step of contacting a compound with a cell-free extract
and a RNA transcript or a nucleic acid construct as described
herein may be conducted under conditions approximating or mimicking
physiologic conditions. In a specific embodiment, a compound is
added to the cell-free extract and nucleic acid construct or RNA
transcript in the presence of an aqueous solution. In accordance
with this embodiment, the aqueous solution may comprise a buffer
and a combination of salts, preferably approximating or mimicking
physiologic conditions. Alternatively, the aqueous solution may
comprise a buffer, a combination of salts, and a detergent or a
surfactant. Examples of salts which may be used in the aqueous
solution include, but not limited to, KCl, NaCl, and/or MgCl.sub.2.
The optimal concentration of each salt used in the aqueous solution
is dependent on the cell-free extract and compounds used and can be
determined using routine experimentation. A compound may be
contacted with a cell-free extract and a RNA transcript or a
nucleic acid construct for a specific period of time. For example,
a compound may be contacted with a cell-free extract and a RNA
transcript or a nucleic acid construct for a time period of about 2
minutes, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes,
45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 8 hours, 10 hours,
12 hours, 14 hours, 16 hours, 18 hours or 24 hours. In some
embodiments, the compound is contacted with a cell-free containing
a RNA transcript or a nucleic acid construct for a time period in a
range of from about 1 minute to about 2 hours, from about 1 minute
to about 1 hour, from about 1 minute to about 45 minutes, from
about 1 minute to about 30 minutes, or from about 1 minute to about
15 minutes
[0346] In specific embodiments, a negative control (e.g., DMSO at
0.005-1.0%, or PBS, or another agent that is known to have no
effect on the expression of the fusion protein) and a positive
control (e.g., an agent that modulates the efficiency of programmed
ribosomal frameshifting) are included in the cell-free assays
described herein.
Host Cells, Cell-Free Extracts, and Reporter Genes
[0347] Techniques for the production or use of the nucleic acid
constructs, the production or use of RNA, and production of host
cells and cell-free extracts will employ, unless otherwise
indicated, routine conventional techniques of molecular biology,
microbiology, and recombinant DNA manipulation and production.
[0348] In some embodiments, the nucleic acid constructs utilized in
the assays above may comprise one or more regulatory elements. Any
transcriptional regulatory element(s) known to those skilled in the
art are intended to be included within the scope of the present
invention for use in controlling transcription of a nucleic acid
construct. Non-limiting examples of the types of transcriptional
regulatory element(s) include a constitutive promoter, a
tissue-specific promoter or an inducible promoter. In a specific
embodiment, the transcription of a nucleic acid construct is
controlled, at least in part, by one or more mammalian (in some
embodiments, human) transcriptional regulatory element(s). In a
specific embodiment, the transcription of a nucleic acid construct
is controlled, at least in part, by a strong promoter, such as CMV.
The transcriptional regulatory elements may be operably linked to a
nucleic acid construct.
[0349] The nucleic acid constructs described herein may be part of
a vector that provides post-transcriptional regulatory elements.
The vector chosen will depend upon a variety of factors, including,
without limitation, the strength of the transcriptional regulatory
elements and the host cell to be used to express a nucleic acid
construct.
[0350] In a specific embodiment, the nucleic acid construct is a
part of CMV vector, such as pcDNA.TM. 3.1/Hygro (Invitrogen Corp.,
Carlsbad, Calif.). In other embodiments, the nucleic acid construct
is part of a T7 vector, a lac vector, a pCEP4 vector or a 5.0/FRT
vector.
[0351] Any reporter gene well-known to one of skill in the art may
be used in the nucleic acid constructs described herein to identify
or validate whether a compound causes ribosomal frameshifting.
Reporter genes refer to a nucleotide sequence encoding or coding
for a protein that is readily detectable either by its presence or
activity. In certain embodiments, the nucleotide sequence of the
reporter gene includes exons and introns. In other embodiments, the
nucleotide sequence of the reporter gene excludes introns. In
specific embodiments, the reporter gene coding sequence is used.
Reporter genes may be obtained and the nucleotide sequence of the
reporter gene determined by any method well-known to one of skill
in the art.
[0352] Examples of reporter genes include, but are not limited to,
nucleotide sequences encoding or coding for luciferase (e.g.,
firefly luciferase, renilla luciferase, and click beetle
luciferase), fluorescent protein (e.g., green fluorescent protein
("GFP"), yellow fluorescent protein, red fluorescent protein, cyan
fluorescent protein, and blue fluorescent protein),
beta-galactosidase (".beta.-gal"), beta-glucoronidase,
beta-lactamase, chloramphenicol acetyltransferase ("CAT"), and
alkaline phosphatase ("AP").
[0353] In a specific embodiment, a gene product of a reporter gene
utilized in the nucleic acid constructs is easily detected and the
activity of the gene product detected is not normally found in the
cell or organism of interest. In a specific embodiment, a reporter
gene utilized in the nucleic acid constructs is not, per se, SMN1
or SMN2.
[0354] Host cells containing a nucleic acid construct or RNA
transcript (e.g., a pre-mRNA or RNA transcript) may be produced
utilizing any technique known to one of skill in the art. For
example, cells may be transformed or transfected with a nucleic
acid construct described herein or a RNA transcript transcribed
from a nucleic acid construct described herein. In one embodiment,
the host cell is transiently transfected with the nucleic acid
construct. In an alternative embodiment, the host cell is stably
transfected with a nucleic acid construct. In certain embodiments,
more than one nucleic acid construct may be transfected into a host
cell. In one specific embodiment, the host cell is a mammalian
cell. In another specific embodiment, the host cell is a human
cell. In another embodiment, the host cells are primary cells
isolated from a tissue or other biological sample of interest. Host
cells that can be used in the methods of the present invention
include, but are not limited to, hybridomas, pre-B cells, HEK293
cells, HEK293T cells, HEK293H cells, HeLa cells, HepG2 cells, K562
cells, 3T3 cells, MCF7 cells, SkBr3 cells, COS cells, BT474 cells,
the human type I SMA fibroblast cell line GMOGM03813 or
neuroblastoma cells lines such as MC-IXC, SK-N-MC, SK-N-MC,
SK-N-DZ, SH-SY5Y, and BE(2)-C. In one embodiment, the host cells
are immortalized cell lines derived from a source, e.g., a tissue.
In one embodiment, the host cells are stem cells.
[0355] Transformation may be by any known method for introducing
polynucleotides into a host cell. The transformation procedure used
depends upon the host to be transformed. Such methods are
well-known to one of skill in the art.
[0356] Stable cell lines may be generated by introducing a nucleic
acid construct further comprising a selectable marker, allowing the
cells to grow for 1-2 days in an enriched medium, and then growing
the cells on a selective medium. The selectable marker in the
recombinant plasmid confers resistance to the selection and allows
cells to stably integrate the plasmid into their chromosomes and
grow to form foci which in turn can be cloned and expanded into
cell lines.
[0357] The invention also provides for the translation of a RNA
transcript from a nucleic acid constructs in a cell-free system. In
a specific embodiment, a cell-free extract provides the components
necessary for translation of a RNA transcript in vitro. Any
technique well-known to one of skill in the art may be used to
generate cell-free extracts for translation in vitro. For example,
the cell-free extracts for in vitro translation reactions can be
generated by centrifuging cells and clarifying the supernatant.
[0358] In some embodiments, a cell-free extract provides the
components necessary for in vitro transcription of nucleic acid
constructs and translation. In a specific embodiment, the cell-free
extract utilized is the in vitro transcription and translation
(TNT)-coupled reticulocyte lysate available from Promega. In
certain embodiments, a cell-free extract provides the components
necessary for in vitro transcription of nucleic acid constructs,
splicing, and translation.
[0359] The cell-free extract may be isolated from cells of any
species origin. For example, the cell-free extract may be isolated
from human cells (e.g., HeLa cells, RD cells, A204 cells), HEK293
cells, Vero cells, yeast, mouse cells (e.g., cultured mouse cells),
rat cells (e.g., cultured rat cells), Chinese hamster ovary (CHO)
cells, Xenopus oocytes, rabbit reticulocytes, primary cells, cancer
cells (e.g., undifferentiated cancer cells), cell lines, wheat
germ, rye embryo, or bacterial cell extract. In a specific
embodiment, the cells from which the cell-free extract is obtained
do not endogenously express SMN or SMNAEx7. In another embodiment,
the cell-free extract is an extract isolated from human cells. In a
further embodiment, the human cells that can be used in the methods
described herein, include, but are not limited to HeLa cells,
HEK293 cells, HEK293T cells, HEK293H cells, HeLa cells, HepG2
cells, K562 cells, 3T3 cells, MCF7 cells, SkBr3 cells, BT474 cells,
MC-IXC cells, SK-N-MC cells, SK-N-MC cells, SK-N-DZ cells, SH-SY5Y
cells, or BE(2)C.
Assays For Assessing the Effect of Compounds on the Efficiency of
Viral Programmed Ribosomal Frameshifting
[0360] Compounds that modulate ribosomal frameshifting may be used
to alter a protein translated from a particular gene, e.g., SMN2.
The protein (SMN.DELTA.Ex7) found in nature that is encoded by the
SMN2 gene lacks amino acid residues encoded by exon 7 of SMN2 and
includes amino acids encoded by exon 8, resulting in rapid
degradation of the SMN.DELTA.Ex7 protein. Compounds that modulate
ribosomal frameshifting may be used to cause frameshifts in SMN2
resulting in the production of stabilized SMNAEx7 proteins that
have an increased abundance or half-life relative to
naturally-occurring SMNAEx7 protein. Assays for use in the
generation of stabilized SMNAEx7 proteins are described in
co-pending U.S. provisional application No. 61/156,429, which is
incorporated by reference herein in its entirety.
[0361] Compounds identified or validated herein as modulating the
efficiency of programmed ribosomal frameshifting may modulate any
form of programmed ribosomal frameshifting.
[0362] In certain embodiments, compounds identified or validated as
capable of modulating the efficiency of programmed ribosomal
frameshifting in the assays described above can be assessed for
their ability to modulate the efficiency of viral programmed
ribosomal frameshifting using any technique known to one of skill
in the art.
[0363] In one embodiment, the ability of a compound to modulate the
efficiency of viral programmed ribosomal frameshifting is assessed
utilizing an assay which measures the ratio of viral proteins
encoded by viral genes expressed from overlapping reading frames in
which programmed ribosomal frameshifting regulates the expression
of the genes.
[0364] Constructs comprising the HIV-1 frameshift signal for use in
frameshifting assays are described in the art (see Biswas, et al.,
2004, J. Virol. 78:2082-2087; Dulude, et al., 2006, Virology,
345:127-136, both of which are incorporated by reference in their
entireties). Accordingly, compounds identified as modulating viral
ribosomal frameshifting using the assays described above can be
validated for their ability to modulate the efficiency of
programmed ribosomal frameshifting using such constructs.
[0365] For example, the ability of a compound to modulate the
efficiency of viral programmed ribosomal frameshifting can be
validated in a dual luciferase assay comprising: (a) contacting a
compound with a host cell expressing a nucleic acid construct
comprising in 5' to 3' order: (i) a promoter sequence; (ii) the
Renilla luciferase (rluc) gene; (iii) the HIV-1 frameshift signal
sequence; (iv) the firefly luciferase gene (flue); and (v) a
polyadenylation sequence; and (b) detecting the activity of Renilla
luciferase versus that of firefly luciferase, wherein a compound
that modulates the efficiency of programmed ribosomal frameshifting
is validated if (i) the activity of Renilla luciferase in the
presence of the compound is not changed relative to the activity of
Renilla luciferase detected in the absence of the compound; and
(ii) the activity of firefly luciferase detected in the presence of
the compound is changed relative to the activity of firefly
luciferase detected in the absence of the compound. In other words,
the ratio of Renilla luciferase to firefly luciferase is changed in
the presence of the compound relative to the absence of the
compound.
[0366] In certain embodiments, the ability of a compound to
modulate the efficiency of viral programmed ribosomal frameshifting
is assessed utilizing an in vitro or in vivo viral replication
assay in which the ratio of viral proteins is monitored as an
indication of the degree to which the efficiency of viral
programmed ribosomal frameshifting is modulated. In a specific
embodiment, a compound identified herein changes the ratio of viral
proteins in an in vitro or in vivo assay known to one of skill in
the art.
[0367] In specific embodiments, the ability of a compound to
modulate the efficiency of viral programmed ribosomal frameshifting
is assessed by measuring the affect of the compound on the ratio of
Gag to Gag-Pol polyproteins. Any technique known to one skilled in
the art can be used to measure a change in the ratio of Gag to
Gag-Pol polyproteins. In a specific embodiment, a change in the
ratio Gag to Gag-Pol polyproteins is assessed by the assay
described in Biswas, et al., 2004, J. Virol. 78:2082-2087, which is
incorporated herein by reference in its entirety. Briefly, the
assay comprises: (a) contacting a compound with an in vitro
transcription and translation (TNT)-coupled reticulocyte lysate
(Promega) containing a plasmid (pHIV) that contains HIV-1 coding
regions, including the Gag-Pol coding sequences with wild-type
HIV-1 ribosomal frameshift signals, in the presence of
[.sup.35S]-methionine; and (b) determining the ratio of the
[.sup.35S]-methionine-labeled p55.sup.gag to
[.sup.35S]-methionine-labeled p160.sup.gag-pol polyproteins
expressed from the plasmid exposed to said compound, wherein a
compound that affects the ratio of Gag to Gag-Pol polyproteins is
identified or validated if the ratio of Gag to Gag-Pol expressed
from the plasmid exposed to said compound is changed relative to
the ratio of Gag to Gag-Pol expressed from the plasmid in the
absence of said compound or in the presence of a negative control.
The amount of Gag to Gag-Pol polyproteins can be determined using
SDS-polyacrylamide gel separation and quantitated using
densitometry.
[0368] In certain embodiments, the ratio of Gag to Gag-Pol
expressed from the plasmid exposed to a compound is altered by at
least 20% (and in some embodiments, at least a 25%, at least a 30%,
at least a 40%, at least a 50%, at least a 75%, at least a 80%, at
least a 90% or at least a 95%) relative to the ratio of Gag to
Gag-Pol expressed from the plasmid not exposed to the compound. In
other embodiments, the ratio of Gag to Gag-Pol expressed from the
plasmid exposed to a compound is altered by 20% to 50%, 20% to 75%,
20% to 95%, 25% to 50%, 25% to 75%, 25% to 95%, 50% to 75%, 50% to
95% or 75% to 95% relative to the ratio of Gag to Gag-Pol expressed
from the plasmid not exposed to the compound.
[0369] In certain embodiments, a compound that modulates the
efficiency of programmed ribosomal frameshifting causes at least a
20% (and in some embodiments at least at 25%, at least a 30%, at
least a 40%, at least a 75% or at least a 90%) alteration in
programmed ribosomal frameshifting activity, as measured using an
assay described herein or known to one of skill in the art.
[0370] In a specific embodiment, a compound that modulates the
efficiency of programmed ribosomal frameshifting causes a reduction
in programmed ribosomal frameshift activity, as measured using an
assay described above. In certain embodiments, the assay is the
dual luciferase construct assay as described in Biswas, et al.,
2004, J. Virol. 78:2082-2087, wherein 100% is the baseline activity
measured in the absence of compounds. In an aspect of these
embodiments, the reduction in programmed ribosomal frameshifting is
at least a 20%, at least a 25%, at least a 30%, at least a 40%, at
least a 50%, at least a 75%, at least a 80%, at least a 90% or at
least a 95% reduction in programmed ribosomal frameshifting
activity.
[0371] In a specific embodiment, a compound that modulates the
efficiency of programmed ribosomal frameshifting activity results
in greatly disabled virus production as measured by a viral
infectivity assay. In another specific embodiment, a compound that
modulates the efficiency of programmed ribosomal frameshifting
activity results in the production of an attenuated virus or viral
particle. In another embodiment, a compound that modulates the
efficiency of programmed ribosomal frameshifting activity results
in completely abolished virus production as measured by a viral
infectivity assay.
Anti-Viral Activity of Compounds that Modulate Programmed Ribosomal
Frameshifting
Anti-Viral Assays
[0372] The antiviral activity of a compound that modulates the
efficiency of programmed ribosomal frameshifting can be detected
using any technique known to one of skill in the art or described
herein.
Viral Infectivity Assay
[0373] Any viral infectivity known to one skilled in the art can be
utilized to determine the ability of a compound that modulates
programmed ribosomal frameshifting to inhibit viral replication
and/or infectivity. In one embodiment, an HIV-1 single cycle
infectivity assay is used to determine the ability of a compound
that modulates the efficiency of programmed ribosomal frameshifting
to inhibit viral replication and/or infectivity. In a specific
embodiment, the HIV-1 infectivity assay described in Biswas, et
al., 2004, J. Virol. 78:2082-2087 or Dulude, et al., 2006,
Virology, 345:127-136 (each of which is incorporated by reference)
is utilized to determine the ability of a compound that modulates
the efficiency of programmed ribosomal frameshifting to inhibit
viral infectivity and/or replication. Briefly, this assay
comprises: (a) contacting a compound with a first cell(s) infected
with a viral particle produced by (i) transfecting a second cell
with 1) a Gag-Pol vector which provides the coding sequences for
all of the HIV-1 accessory proteins and the Gag and Gag-Pol
polyproteins, with the wild-type frameshift signal (pHIVgpwt), 2) a
plasmid that encodes a viral envelope protein, such as vesicular
stomatitis virus G envelope protein (VSV-G), which allows the
vector virus to be pseudotyped, and 3) a transducing vector that
contains cis-acting signals for vector propagation, including the
LTRs and the packaging signal, as well as a marker gene (such as a
green fluorescent protein, gfp) and an antibiotic resistance gene
(e.g., puromycin resistance gene, puro) for the detection and
analysis of infectivity, and (ii) harvesting the resulting viral
particles 72 hours post-transfection; and (b) using the antibiotic
(e.g., puromycin) to select for infectious units of the HIV-1
vector virus and scoring the number of infectious units by counting
marker-positive (e.g., GFP-positive), antibiotic-resistant cells by
a technique known to one of skill in the art, such as fluorescence
microscopy, wherein a compound that interferes with viral
replication and/or infectivity is identified or validated if the
titer of the virus produced in the presence of the compound is
changed relative to the titer of the virus produced in the absence
of the compound or in the presence of a negative control (e.g.,
DMSO or PBS).
Viral Cytopathic Effect (CPE) Assay
[0374] CPE is the morphological changes that cultured cells undergo
upon being infected by most viruses. These morphological changes
can be observed easily in unfixed, unstained cells by microscopy.
Forms of CPE, which can vary depending on the virus, include, but
are not limited to, rounding of the cells, appearance of inclusion
bodies in the nucleus and/or cytoplasm of infected cells, and
formation of syncytia, or polykaryocytes (large cytoplasmic masses
that contain many nuclei).
[0375] The CPE assay can provide a measure of the antiviral effect
of a compound that modulates the efficiency of programmed ribosomal
frameshifting. In a non-limiting example of such an assay,
compounds that modulate the efficiency of programmed ribosomal
frameshifting are serially diluted (e.g. 1000, 500, 100, 50, 10, 1
.mu.g/ml) and added to 3 wells containing a cell monolayer
(preferably mammalian cells at 80-100% confluent) of a 96-well
plate. Within 5 minutes, viruses are added and the plate sealed,
incubated at 37.degree. C. for the standard time period required to
induce near-maximal viral CPE (e.g., approximately 48 to 120 hours,
depending on the virus and multiplicity of infection). CPE is read
microscopically after a known positive control drug is evaluated in
parallel with the compounds being assayed. The data are expressed
as 50% effective concentrations or approximated virus-inhibitory
concentration, 50% endpoint (EC.sub.50) and cell-inhibitory
concentration, 50% endpoint (IC.sub.50). General selectivity index
("SI") is calculated as the IC.sub.50 divided by the EC.sub.50.
These values can be calculated using any method known in the art,
e.g., the computer software program MacSynergy II by M. N.
Prichard, K. R. Asaltine, and C. Shipman, Jr., University of
Michigan, Ann Arbor, Mich.
[0376] In one embodiment, a compound that modulates the efficiency
of viral programmed ribosomal frameshifting has an SI of greater
than 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11, or 12, or
13, or 14, or 15, or 20, or 21, or 22, or 23, or 24, or 25, or 30,
or 35, or 40, or 45, or 50, or 60, or 70, or 80, or 90, or 100, or
200, or 300, or 400, or 500, 1,000, or 10,000. In some embodiments,
a compound that modulates the efficiency of viral programmed
ribosomal frameshifting has an SI of greater than 10. In a specific
embodiment, compounds with an SI of greater than 10 are further
assessed in other in vitro and in vivo assays described herein or
others known in the art to characterize safety and efficacy.
Neutral Red (NR) Dye Uptake Assay
[0377] The NR Dye Uptake assay can be used to validate the CPE
assay. In a non-limiting example of such an assay, the same 96-well
microplates used for the CPE assay can be used. Neutral red is
added to the medium, and cells not damaged by virus take up a
greater amount of dye. The percentage of uptake indicating viable
cells is read on a microplate autoreader at dual wavelengths of 405
and 540 nm, with the difference taken to eliminate background. (See
McManus, et al., Appl. Environment. Microbiol. 31:35-38, 1976). An
EC.sub.50 is determined for samples with infected cells and
contacted with a compound that modulates the efficiency of
programmed ribosomal frameshifting, and an IC.sub.50 is determined
for samples with uninfected cells contacted with a compound that
modulates the efficiency of programmed ribosomal frameshifting.
Virus Yield Assay
[0378] Lysed cells and supernatants from infected cultures such as
those in the CPE assay can be used to assay for virus yield
(production of viral particles after the primary infection). In a
non-limiting example, these supernatants are serial diluted and
added onto monolayers of susceptible cells (e.g., Vero cells).
Development of CPE in these cells is an indication of the presence
of infectious viruses in the supernatant. The 90% effective
concentration (EC.sub.90), the test compound concentration that
inhibits virus yield by 1 log 10, is determined from these data
using known calculation methods in the art. In one embodiment, the
EC.sub.90 of a compound that modulates the efficiency of viral
programmed ribosomal frameshifting is at least 1.5 fold, 2 fold, 3
fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 20
fold, 30 fold, 40 fold, or 50 fold less than the EC.sub.90 of the
negative control sample.
Plaque Reduction Assay
[0379] In a non-limiting example of a plaque reduction assay, a
virus is diluted into various concentrations and added to each well
containing a monolayer of the target mammalian cells in triplicate.
The plates are then incubated for a period of time to achieve
effective infection of the control sample (e.g., 1 hour with
shaking every fifteen minutes). After the incubation period, an
equal amount of 1% agarose is added to an equal volume of each
compound dilution prepared in 2.times. concentration. In certain
embodiments, compounds that modulate the efficiency of programmed
ribosomal frameshifting at test concentrations between about 0.03
.mu.g/ml to about 100 .mu.g/ml can be tested with a final agarose
overlay concentration of 0.5%. The compound-agarose mixture is
applied to each well in 2 ml volume and the plates are incubated
for three days, after which the cells are stained with a 1.5%
solution of neutral red. At the end of the 4-6 hour incubation
period, the neutral red solution is aspirated, and plaques counted
using a stereomicroscope. Alternatively, a final agarose
concentration of 0.4% can be used. In other embodiments, the plates
are incubated for more than three days with additional overlays
being applied on day four and on day 8 when appropriate. In another
embodiment, the overlay medium is liquid rather than
semi-solid.
Virus Titer Assay
[0380] In virus titer assays, a monolayer of a target mammalian
cell line is infected with different amounts (e.g., multiplicity of
3 plaque forming units (pfu) or 5 pfu) of virus and subsequently
cultured in the presence or absence of various dilutions of
compounds that modulate the efficiency of programmed ribosomal
frameshifting (e.g., 0.1 .mu.g/mL, 1 .mu.g/mL, 5 .mu.g/mL, or 10
.mu.g/mL) to be tested. Infected cultures are harvested 48 hours or
72 hours post infection and titered by standard plaque assays known
in the art on the appropriate target cell line (e.g., Vero cells,
MRCS cells). In certain embodiments, culturing the infected cells
in the presence of the compounds reduces the yield of infectious
virus by at least 1.5 fold, 2, fold, 3, fold, 4 fold, 5 fold, 6
fold, 7 fold, 8 fold, 9 fold, 10 fold, 15 fold, 20 fold, 25 fold,
30 fold, 35 fold, 40 fold, 45 fold, 50 fold, 100 fold, 500 fold, or
1000 fold relative to culturing the infected cells in the absence
of compounds. In a specific embodiment, culturing the infected
cells in the presence of compounds that modulate the efficiency of
programmed ribosomal frameshifting reduces the PFU/mL by at least
10 fold relative to culturing the infected cells in the absence of
compounds that modulate the efficiency of programmed ribosomal
frameshifting.
[0381] In certain embodiments, culturing the infected cells in the
presence of compounds that modulate the efficiency of programmed
ribosomal frameshifting reduces the yield of infectious virus by at
least 0.5 log 10, 1 log 10, 1.5 log 10, 2 log 10, 2.5 log 10, 3 log
10, 3.5 log 10, 4 log 10, 4.5 log 10, 5 log 10, 5.5 log 10, 6 log
10, 6.5 log 10, 7 log 10, 7.5 log 10, 8 log 10, 8.5 log 10, or 9
log 10 relative to culturing the infected cells in the absence of
compounds. In a specific embodiment, culturing the infected cells
in the presence of compounds that modulate the efficiency of
programmed ribosomal frameshifting reduces the yield of infectious
virus by at least 1 log 10 or 2 log 10 relative to culturing the
infected cells in the absence of compounds. In another specific
embodiment, culturing the infected cells in the presence of
compounds that modulate the efficiency of programmed ribosomal
frameshifting reduces the yield of infectious virus by at least 2
log 10 relative to culturing the infected cells in the absence of
compounds.
Flow Cytometry Assay
[0382] Flow cytometry can be utilized to detect expression of virus
antigens in infected target cells cultured in the presence or
absence of compounds that modulate the efficiency of programmed
ribosomal frameshifting (See, e.g., McSharry et al., Clinical
Microbiology Rev., 1994, 7:576-604). In other embodiments,
intracellular viral antigens or viral nucleic acid can be detected
by flow cytometry with techniques known in the art.
Cell Lines for Antiviral Assays
[0383] In a specific embodiment, cells used in the antiviral assays
described herein are susceptible to infection with a virus. In some
embodiments, cell lines for use in antiviral assays are genetically
engineered to render them more suitable hosts for viral infection
or viral replication and more convenient substrates for rapidly
detecting virus-infected cells (See, e.g., Olivo, P. D., Clin.
Microbiol. Rev., 1996, 9:321-334). In some aspects, these cell
lines are available for testing the antiviral activity of a
compound that modulates the efficiency of programmed ribosomal
frameshifting on blocking any step of viral replication and
infectivity, such as, transcription, translation, pregenome
encapsidation, reverse transcription, particle assembly and
release.
Cytotoxicity Assays
[0384] Compounds that modulate the efficiency of programmed
ribosomal frameshifting may be tested for cytotoxicity in
mammalian, preferably human, cell lines. In certain embodiments,
cytotoxicity is assessed in one or more of the following cell
lines: U937, a human monocyte cell line; primary peripheral blood
mononuclear cells (PBMC); Huh7, a human hepatoblastoma cell line;
293T and 293H, human embryonic kidney cell lines; and THP-1,
monocytic cells; a HeLa cell line; fibroblasts or other cell types
isolated from SMA patients; SMA patient-derived cell lines, e.g.,
the GM03813 cell line; and neuroblastoma cells lines, such as
MC-IXC, SK-N-MC, SK-N-MC, SK-N-DZ, SH-SY5Y and BE(2)-C. In general,
many assays known to one skilled in the art can be used to assess
viability of cells or cell lines following exposure to a compound
and, thus, determine the cytotoxicity of the compound.
[0385] The toxicity and/or efficacy of a compound that modulates
the efficiency of programmed ribosomal frameshifting can be
determined by standard pharmaceutical procedures in cell cultures
or experimental animals, e.g., for determining the LD.sub.50 (the
dose lethal to 50% of the population) and the ED.sub.50 (the dose
therapeutically effective in 50% of the population). The dose ratio
between toxic and therapeutic effects is the therapeutic index and
it can be expressed as the ratio LD.sub.50/ED.sub.50. A compound
identified or validated in accordance with the invention that
exhibits large therapeutic indices is preferred. While a compound
identified or validated in accordance with the invention that
exhibits toxic side effects may be used, care should be taken to
design a delivery system that targets such agents to the site of
affected tissue in order to minimize potential damage to uninfected
cells and, thereby, reduces side effects.
[0386] The data obtained from the cell culture assays and animal
studies can be used in formulating a range of dosage of a compound
identified or validated in accordance with the invention for use in
humans. The dosage of such agents lies preferably within a range of
circulating concentrations that include the ED.sub.50 with little
or no toxicity. The dosage may vary within this range depending
upon the dosage form employed and the route of administration
utilized. For any agent used in the method of the invention, the
therapeutically effective dose can be estimated initially from cell
culture assays. A dose may be formulated in animal models to
achieve a circulating plasma concentration range that includes the
IC.sub.50 (i.e., the concentration of the test compound that
achieves a half-maximal inhibition of symptoms) as determined in
cell culture. Such information can be used to more accurately
determine useful doses in humans. Levels in plasma may be measured,
for example, by high-performance liquid chromatography.
Compositions
[0387] Any compound described herein may optionally be in the form
of a composition comprising the compound and an optional carrier,
excipient or diluent. Other embodiments provided herein include
pharmaceutical compositions comprising an effective amount of a
compound and a pharmaceutically acceptable carrier, excipient, or
diluent. The pharmaceutical compositions are suitable for
veterinary and/or human administration. The pharmaceutical
compositions provided herein can be in any form that allows for the
composition to be administered to a subject, and these
pharmaceutical compositions may be formulated for the route of
administration.
[0388] In a specific embodiment and in this context, the term
"pharmaceutically acceptable carrier, excipient or diluent" means a
carrier, excipient or diluent approved by a regulatory agency of
the Federal or a state government or listed in the U.S.
Pharmacopeia or other generally recognized pharmacopeia for use in
animals, and more particularly in humans. The term "carrier" refers
to a diluent, adjuvant (e.g., Freund's adjuvant (complete and
incomplete)), excipient, or vehicle with which the therapeutic is
administered. Such pharmaceutical carriers can be sterile liquids,
such as water and oils, including those of petroleum, animal,
vegetable or synthetic origin, such as peanut oil, soybean oil,
mineral oil, sesame oil and the like. Water is a specific carrier
when the pharmaceutical composition is administered intravenously.
Saline solutions and aqueous dextrose and glycerol solutions can
also be employed as liquid carriers, particularly for injectable
solutions.
[0389] Typical compositions and dosage forms comprise one or more
excipients. Suitable excipients are well-known to those skilled in
the art of pharmacy, and non limiting examples of suitable
excipients include starch, glucose, lactose, sucrose, gelatin,
malt, rice, flour, chalk, silica gel, sodium stearate, glycerol
monostearate, talc, sodium chloride, dried skim milk, glycerol,
propylene, glycol, water, ethanol and the like. Whether a
particular excipient is suitable for incorporation into a
pharmaceutical composition or dosage form depends on a variety of
factors well known in the art including, but not limited to, the
way in which the dosage form will be administered to a patient and
the specific active ingredients in the dosage form. The composition
or single unit dosage form, if desired, can also contain minor
amounts of wetting or emulsifying agents, or pH buffering agents.
Further provided herein are anhydrous pharmaceutical compositions
and dosage forms comprising one or more compounds of the present
invention. The compositions and single unit dosage forms can take
the form of solutions, suspensions, emulsion, tablets, pills,
capsules, powders, sustained-release formulations and the like.
[0390] Pharmaceutical compositions provided herein that are
suitable for oral administration can be presented as discrete
dosage forms, such as, but are not limited to, tablets (e.g.,
chewable tablets), caplets, capsules, and liquids (e.g., flavored
syrups). Such dosage forms contain predetermined amounts of active
ingredients, and may be prepared by methods of pharmacy well known
to those skilled in the art.
[0391] Because of their ease of administration, tablets and
capsules represent the most advantageous oral dosage unit forms, in
which case solid excipients are employed. In general,
pharmaceutical compositions and dosage forms are prepared by
uniformly and intimately admixing the active ingredients with
liquid carriers, finely divided solid carriers, or both, and then
shaping the product into the desired presentation if necessary.
[0392] Examples of excipients that can be used in oral dosage forms
provided herein include, but are not limited to, binders, fillers,
disintegrants, and lubricants.
Prophylactic and Therapeutic Methods
[0393] In certain embodiments, a compound identified or validated
using an assay described herein has utility as an antiviral agent.
In one embodiment, a compound identified or validated using an
assay described herein may be used to inhibit or reduce viral
replication. In another embodiment, a compound identified or
validated using an assay described herein may be used to inhibit or
reduce a viral infection. In another embodiment, a compound
identified or validated using an assay described herein may be used
to reduce viral titers in vitro or in vivo. In another embodiment,
a compound identified or validated using an assay described herein
may be used to do one or more of the following: treat a viral
infection, prevent a viral disease, or treat a viral disease.
[0394] In one embodiment, the invention provides a method for
treating a viral infection in a subject, comprising administering
to a subject in need thereof an effective amount of a compound or
pharmaceutical composition thereof, wherein the compound modulates
the efficiency of programmed ribosomal frameshifting as measured in
vitro or in cells by an increase in the amount or activity of a
fusion protein encoded by a nucleic acid construct or translated
from a RNA transcript (e.g., a mRNA transcript) transcribed from
the nucleic acid construct, and wherein the nucleic acid construct
comprises, in 5' to 3' order: (i) the nucleic acid residues of exon
6 of SMN; (ii) the nucleic acid residues of intron 6 of SMN; (iii)
the nucleic acid residues of exon 7 of SMN, wherein a single
guanine is inserted after the 48th nucleotide residue from the 5'
end of exon 7 of SMN (i.e., before the 6th nucleotide from the 3'
end of exon 7 of SMN); (iv) the nucleic acid residues of intron 7
of SMN; (v) a fragment of the nucleic acid residues of exon 8 of
SMN, wherein the fragment is composed of the first 23 nucleotides
from the 5' end of exon 8 of SMN; and (vi) a reporter gene lacking
a start codon, wherein the reporter gene is fused to the fragment
of the nucleic acid residues of exon 8 of SMN such that the
reporter gene and the fragment are out of frame with each other in
the mRNA transcript transcribed from the nucleic acid construct,
and wherein the production of the mRNA transcript generates a stop
codon in the region of the mRNA transcript that corresponds to the
fragment of the nucleic acid residues of exon 8 of SMN. In certain
embodiments, an internal start codon (e.g., an ATG) found in exon 6
is used as the start codon for the nucleic acid construct. In some
embodiments, the nucleic acid construct comprises a start codon 5'
to the nucleic acid residues of exon 6 of SMN.
[0395] In another embodiment, the invention provides a method for
preventing a viral disease in a subject, comprising administering
to a subject in need thereof an effective amount of a compound or
pharmaceutical composition thereof, wherein the compound modulates
the efficiency of programmed ribosomal frameshifting as measured in
vitro or in cells by an increase in the amount or activity of a
fusion protein encoded by a nucleic acid construct or translated
from a RNA transcript (e.g., a mRNA transcript) transcribed from
the nucleic acid construct, and wherein the nucleic acid construct
comprises, in 5' to 3' order: (i) the nucleic acid residues of exon
6 of SMN; (ii) the nucleic acid residues of intron 6 of SMN; (iii)
the nucleic acid residues of exon 7 of SMN, wherein a single
guanine is inserted after the 48th nucleotide residue from the 5'
end of exon 7 of SMN (i.e., before the 6th nucleotide from the 3'
end of exon 7 of SMN); (iv) the nucleic acid residues of intron 7
of SMN; (v) a fragment of the nucleic acid residues of exon 8 of
SMN, wherein the fragment is composed of the first 23 nucleotides
from the 5' end of exon 8 of SMN; and (vi) a reporter gene lacking
a start codon, wherein the reporter gene is fused to the fragment
of the nucleic acid residues of exon 8 of SMN such that the
reporter gene and the fragment are out of frame with each other in
the mRNA transcript transcribed from the nucleic acid construct,
and wherein the production of the mRNA transcript generates a stop
codon in the region of the mRNA transcript that corresponds to the
fragment of the nucleic acid residues of exon 8 of SMN. In certain
embodiments, an internal start codon (e.g., an ATG) found in exon 6
is used as the start codon for the nucleic acid construct. In some
embodiments, the nucleic acid construct comprises a start codon 5'
to the nucleic acid residues of exon 6 of SMN.
[0396] In another embodiment, the invention provides a method for
treating a viral disease in a subject, comprising administering to
a subject in need thereof an effective amount of a compound or
pharmaceutical composition thereof, wherein the compound modulates
the efficiency of programmed ribosomal frameshifting as measured in
vitro or in cells by an increase in the amount or activity of a
fusion protein encoded by a nucleic acid construct or translated
from a RNA transcript (e.g., a mRNA transcript) transcribed from
the nucleic acid construct, and wherein the nucleic acid construct
comprises, in 5' to 3' order: (i) the nucleic acid residues of exon
6 of SMN; (ii) the nucleic acid residues of intron 6 of SMN; (iii)
the nucleic acid residues of exon 7 of SMN, wherein a single
guanine is inserted after the 48th nucleotide residue from the 5'
end of exon 7 of SMN (i.e., before the 6th nucleotide from the 3'
end of exon 7 of SMN); (iv) the nucleic acid residues of intron 7
of SMN; (v) a fragment of the nucleic acid residues of exon 8 of
SMN, wherein the fragment is composed of the first 23 nucleotides
from the 5' end of exon 8 of SMN; and (vi) a reporter gene lacking
a start codon, wherein the reporter gene is fused to the fragment
of the nucleic acid residues of exon 8 of SMN such that the
reporter gene and the fragment are out of frame with each other in
the mRNA transcript transcribed from the nucleic acid construct,
and wherein the production of the mRNA transcript generates a stop
codon in the region of the mRNA transcript that corresponds to the
fragment of the nucleic acid residues of exon 8 of SMN. In certain
embodiments, an internal start codon (e.g., an ATG) found in exon 6
is used as the start codon for the nucleic acid construct. In some
embodiments, the nucleic acid construct comprises a start codon 5'
to the nucleic acid residues of exon 6 of SMN.
[0397] In another embodiment, the invention provides a method for
reducing or inhibiting a viral infection, comprising administering
to a subject in need thereof an effective amount of a compound or
pharmaceutical composition thereof, wherein the compound modulates
the efficiency of programmed ribosomal frameshifting as measured in
vitro or in cells by an increase in the amount or activity of a
fusion protein encoded by a nucleic acid construct or translated
from a RNA transcript (e.g., a mRNA transcript) transcribed from
the nucleic acid construct, and wherein the nucleic acid construct
comprises, in 5' to 3' order: (i) the nucleic acid residues of exon
6 of SMN; (ii) the nucleic acid residues of intron 6 of SMN; (iii)
the nucleic acid residues of exon 7 of SMN, wherein a single
guanine is inserted after the 48th nucleotide residue from the 5'
end of exon 7 of SMN (i.e., before the 6th nucleotide from the 3'
end of exon 7 of SMN); (iv) the nucleic acid residues of intron 7
of SMN; (v) a fragment of the nucleic acid residues of exon 8 of
SMN, wherein the fragment is composed of the first 23 nucleotides
from the 5' end of exon 8 of SMN; and (vi) a reporter gene lacking
a start codon, wherein the reporter gene is fused to the fragment
of the nucleic acid residues of exon 8 of SMN such that the
reporter gene and the fragment are out of frame with each other in
the mRNA transcript transcribed from the nucleic acid construct,
and wherein the production of the mRNA transcript generates a stop
codon in the region of the mRNA transcript that corresponds to the
fragment of the nucleic acid residues of exon 8 of SMN. In certain
embodiments, an internal start codon (e.g., an ATG) found in exon 6
is used as the start codon for the nucleic acid construct. In some
embodiments, the nucleic acid construct comprises a start codon 5'
to the nucleic acid residues of exon 6 of SMN.
[0398] In another embodiment, the invention provides a method for
reducing or inhibiting a viral infection, comprising administering
to a subject in need thereof an effective amount of a compound or
pharmaceutical composition thereof, wherein the compound modulates
the efficiency of programmed ribosomal frameshifting as measured in
vitro or in cells by an increase in the amount or activity of a
fusion protein encoded by a nucleic acid construct or translated
from a RNA transcript (e.g., a mRNA transcript) transcribed from
the nucleic acid construct, and wherein the nucleic acid construct
comprises, in 5' to 3' order: (i) the nucleic acid residues of exon
6 of SMN; (ii) the nucleic acid residues of intron 6 of SMN; (iii)
the nucleic acid residues of exon 7 of SMN, wherein a single
guanine is inserted after the 48th nucleotide residue from the 5'
end of exon 7 of SMN (i.e., before the 6th nucleotide from the 3'
end of exon 7 of SMN); (iv) the nucleic acid residues of intron 7
of SMN; (v) a fragment of the nucleic acid residues of exon 8 of
SMN, wherein the fragment is composed of the first 23 nucleotides
from the 5' end of exon 8 of SMN; and (vi) a reporter gene lacking
a start codon, wherein the reporter gene is fused to the fragment
of the nucleic acid residues of exon 8 of SMN such that the
reporter gene and the fragment are out of frame with each other in
the mRNA transcript transcribed from the nucleic acid construct,
and wherein the production of the mRNA transcript generates a stop
codon in the region of the mRNA transcript that corresponds to the
fragment of the nucleic acid residues of exon 8 of SMN. In certain
embodiments, an internal start codon (e.g., an ATG) found in exon 6
is used as the start codon for the nucleic acid construct. In some
embodiments, the nucleic acid construct comprises a start codon 5'
to the nucleic acid residues of exon 6 of SMN.
[0399] In another embodiment, the invention provides a method for
reducing or inhibiting a viral replication, comprising
administering to a subject in need thereof an effective amount of a
compound or pharmaceutical composition thereof, wherein the
compound modulates the efficiency of programmed ribosomal
frameshifting as measured in vitro or in cells by an increase in
the amount or activity of a fusion protein encoded by a nucleic
acid construct or translated from a RNA transcript (e.g., a mRNA
transcript) transcribed from the nucleic acid construct, and
wherein the nucleic acid construct comprises, in 5' to 3' order:
(i) the nucleic acid residues of exon 6 of SMN; (ii) the nucleic
acid residues of intron 6 of SMN; (iii) the nucleic acid residues
of exon 7 of SMN, wherein a single guanine is inserted after the
48th nucleotide residue from the 5' end of exon 7 of SMN (i.e.,
before the 6th nucleotide from the 3' end of exon 7 of SMN); (iv)
the nucleic acid residues of intron 7 of SMN; (v) a fragment of the
nucleic acid residues of exon 8 of SMN, wherein the fragment is
composed of the first 23 nucleotides from the 5' end of exon 8 of
SMN; and (vi) a reporter gene lacking a start codon, wherein the
reporter gene is fused to the fragment of the nucleic acid residues
of exon 8 of SMN such that the reporter gene and the fragment are
out of frame with each other in the mRNA transcript transcribed
from the nucleic acid construct, and wherein the production of the
mRNA transcript generates a stop codon in the region of the mRNA
transcript that corresponds to the fragment of the nucleic acid
residues of exon 8 of SMN. In certain embodiments, an internal
start codon (e.g., an ATG) found in exon 6 is used as the start
codon for the nucleic acid construct. In some embodiments, the
nucleic acid construct comprises a start codon 5' to the nucleic
acid residues of exon 6 of SMN.
[0400] In another embodiment, the invention provides a method for
inhibiting or reducing viral replication and/or viral infectivity
in a subject, comprising administering to a subject in need thereof
an effective amount of a compound or pharmaceutical composition
thereof, wherein the compound modulates the efficiency of
programmed ribosomal frameshifting as measured in vitro or in cells
by an increase in the amount or activity of a fusion protein
encoded by a nucleic acid construct or translated from a RNA
transcript (e.g., a mRNA transcript) transcribed from the nucleic
acid construct, and wherein the nucleic acid construct comprises,
in 5' to 3' order: (i) the nucleic acid residues of exon 6 of SMN;
(ii) the nucleic acid residues of intron 6 of SMN; (iii) the
nucleic acid residues of exon 7 of SMN, wherein a single guanine is
inserted after the 48th nucleotide residue from the 5' end of exon
7 of SMN (i.e., before the 6th nucleotide from the 3' end of exon 7
of SMN); (iv) the nucleic acid residues of intron 7 of SMN; (v) a
fragment of the nucleic acid residues of exon 8 of SMN, wherein the
fragment is composed of the first 23 nucleotides from the 5' end of
exon 8 of SMN; and (vi) a reporter gene lacking a start codon,
wherein the reporter gene is fused to the fragment of the nucleic
acid residues of exon 8 of SMN such that the reporter gene and the
fragment are out of frame with each other in the mRNA transcript
transcribed from the nucleic acid construct, and wherein the
production of the mRNA transcript generates a stop codon in the
region of the mRNA transcript that corresponds to the fragment of
the nucleic acid residues of exon 8 of SMN. In certain embodiments,
an internal start codon (e.g., an ATG) found in exon 6 is used as
the start codon for the nucleic acid construct. In some
embodiments, the nucleic acid construct comprises a start codon 5'
to the nucleic acid residues of exon 6 of SMN.
[0401] In another embodiment, the invention provides a method for
reducing viral titers in a subject, comprising administering to a
subject in need thereof an effective amount of a compound or
pharmaceutical composition thereof, wherein the compound modulates
the efficiency of programmed ribosomal frameshifting as measured in
vitro or in cells by an increase in the amount or activity of a
fusion protein encoded by a nucleic acid construct or translated
from a RNA transcript (e.g., a mRNA transcript) transcribed from
the nucleic acid construct, and wherein the nucleic acid construct
comprises, in 5' to 3' order: (i) the nucleic acid residues of exon
6 of SMN; (ii) the nucleic acid residues of intron 6 of SMN; (iii)
the nucleic acid residues of exon 7 of SMN, wherein a single
guanine is inserted after the 48th nucleotide residue from the 5'
end of exon 7 of SMN (i.e., before the 6th nucleotide from the 3'
end of exon 7 of SMN); (iv) the nucleic acid residues of intron 7
of SMN; (v) a fragment of the nucleic acid residues of exon 8 of
SMN, wherein the fragment is composed of the first 23 nucleotides
from the 5' end of exon 8 of SMN; and (vi) a reporter gene lacking
a start codon, wherein the reporter gene is fused to the fragment
of the nucleic acid residues of exon 8 of SMN such that the
reporter gene and the fragment are out of frame with each other in
the mRNA transcript transcribed from the nucleic acid construct,
and wherein the production of the mRNA transcript generates a stop
codon in the region of the mRNA transcript that corresponds to the
fragment of the nucleic acid residues of exon 8 of SMN. In certain
embodiments, an internal start codon (e.g., an ATG) found in exon 6
is used as the start codon for the nucleic acid construct. In some
embodiments, the nucleic acid construct comprises a start codon 5'
to the nucleic acid residues of exon 6 of SMN.
[0402] In another embodiment, the invention provides a method for
inhibiting or reducing viral replication and/or viral infectivity
comprising contacting a cell or a population of cells containing a
virus or provirus with an effective amount of a compound or a
pharmaceutical composition thereof, wherein the effective amount is
an amount effective to modulate the efficiency of programmed
ribosomal frameshifting as measured by, e.g., a dual luciferase
construct assay, such as described herein. In a specific
embodiment, the invention provides a method for inhibiting or
reducing viral replication and/or viral infectivity, comprising
administering to a subject in need thereof an effective amount of a
compound or a pharmaceutical composition thereof, wherein the
effective amount is an amount sufficient to alter the efficiency of
programmed ribosomal frameshifting by at least 20% (and in some
embodiments, at least 25%, at least 30%, at least 40%, at least
50%, at least 75%, at least 80%, at least 90% or at least 95%)
relative to a negative control as measured by, e.g., a dual
luciferase construct assay, such as described herein.
[0403] In another embodiment, the invention provides a method for
preventing or treating a viral disease in a subject, comprising
administering to a subject in need thereof an effective amount of a
compound or a pharmaceutical composition thereof, wherein the
effective amount is an amount effective to modulate the efficiency
of programmed ribosomal frameshifting as measured by, e.g., a dual
luciferase construct assay, such as described herein. In a specific
embodiment, the invention provides a method for preventing or
treating a viral disease in a subject, comprising administering to
a subject in need thereof an effective amount of a compound or
pharmaceutical composition thereof, wherein the effective amount is
an amount sufficient to alter the efficiency of programmed
ribosomal frameshifting by at least 20% (and in some embodiments,
at least 25%, at least 30%, at least 40%, at least 50%, at least
75%, at least 80%, at least 90%, or at least 95%) relative to a
negative control as measured by, e.g., a dual luciferase construct
assay, such as described herein.
[0404] In another embodiment, the invention provides a method for
inhibiting or reducing viral infection, comprising administering to
a subject in need thereof an effective amount of a compound or a
pharmaceutical composition thereof, wherein the effective amount is
an amount effective to modulate the efficiency of programmed
ribosomal frameshifting as measured by, e.g., a dual luciferase
construct assay. In a specific embodiment, the invention provides a
method for inhibiting or reducing viral infection, comprising
administering to a subject in need thereof an effective amount of a
compound or a pharmaceutical composition thereof, wherein the
effective amount is an amount sufficient to alter the efficiency of
programmed ribosomal frameshifting by at least 20% (and in some
embodiments, at least 25%, at least 30%, at least 40%, at least
50%, at least 75%, at least 80%, at least 90% or at least 95%)
relative to a negative control as measured by, e.g., a dual
luciferase construct assay, such as described herein.
[0405] A compound or a composition thereof may be used in
combination with another agent to treat a viral infection, or
prevent or treat a viral disease. In a specific embodiment, two or
more compounds may be used to treat a viral infection, or prevent
or treat a viral disease. In specific embodiments, a compound or a
composition thereof is the only active ingredient administered to
treat a viral infection, or prevent or treat a viral disease.
[0406] The effective amount of a compound to be used depends on a
number of factors, including but not limited to the type of viral
infection, type of viral disease, health and age of the patient,
and toxicity or side effects. The present invention encompasses
methods for treating viral infections, or preventing or treating
viral diseases for which no therapy is available. The present
invention also encompasses methods for treating viral infections,
or preventing or treating viral diseases as an alternative to
conventional therapies.
[0407] The present invention also provides methods of treating
viral infections, or preventing or treating viral diseases in a
subject in need thereof, said methods comprising administering to
the subject one or more of compounds in combination with one or
more additional therapies (e.g., agents). In one embodiment, one or
more compounds are administered to the subject in combination with
a supportive therapy, a pain relief therapy, or other therapy that
does not have an effect per se on the viral infection.
[0408] Two or more compounds may be administered to a subject to
treat a viral infection, or preventing or treating viral diseases
in any order. In addition, one or more compounds and one or more
other agents may be administered in any order to a subject to treat
a viral infection, or prevent or treat a viral disease.
[0409] A combination product of one or more compounds and one or
more additional agents can be administered sequentially or
concurrently. In a specific embodiment, a combination product of
the present invention may improve the prophylactic and/or
therapeutic effect of the compound and the agent by functioning
together to have an additive or synergistic effect. In another
embodiment, a combination product may reduce the side effects
associated with each compound and agent when taken alone.
[0410] The one or more compounds and one or more other agents of a
combination product can be administered to a subject in the same
pharmaceutical composition. Alternatively, the one or more
compounds and one or more other agents of a combination product can
be administered concurrently to a subject in separate
pharmaceutical compositions. The one or more compounds and one or
more other agents of a combination product may be administered to a
subject by the same or different routes of administration.
[0411] In certain embodiments, the present invention encompasses
the use of an attenuated virus or viral particle, which may be
produced, at least in part, utilizing a compound that modulates
viral programmed ribosomal frameshifting, for the prevention of a
viral disease. In some embodiments, a compound that modulates
programmed ribosomal frameshifting is contacted with a cell or a
population of cells containing a virus, provirus or nucleic acids
encoding or coding for certain components of a virus as part of the
process for production of an attenuated virus or viral particle. In
specific embodiments, an attenuated virus or viral particle may be
produced by a method comprising contacting a compound that
modulates programmed ribosomal frameshifting with a cell or
population of cells containing a virus or provirus, and harvesting
the resulting attenuated virus or viral particle. An attenuated
virus or viral particle produced utilizing a compound that
modulates programmed ribosomal frameshifting can be utilized as a
vaccine. In particular, the attenuated virus or viral particle can
be administered to a subject in an effective amount to prevent the
development of a viral disease.
Patient Population
[0412] In some embodiments, a compound or pharmaceutical
composition thereof is administered to a subject suffering from a
viral infection. In other embodiments, a compound or pharmaceutical
composition thereof is administered to a subject predisposed or
susceptible to a viral infection. In other embodiments, a compound
or pharmaceutical composition thereof is administered to a subject
to treat a viral disease. In other embodiments, a compound or
pharmaceutical composition thereof is administered to a subject to
prevent the development of a viral disease.
[0413] In a specific embodiment, the subject suffering from a viral
infection or a disease associated therewith is infected with a
virus listed in Table 1 or a provirus. In another embodiment, the
subject suffering from a viral infection or a disease associated
therewith is infected with human immunodeficiency virus type 1
(HIV-1), human immunodeficiency virus type 2 (HIV-2), feline
immunodeficiency virus (FIV), rous sarcoma virus (RSV), mouse
mammary tumor virus (MMTV), simian retrovirus type 1 (SRV-1), human
T cell leukemia virus type I (HTLV-I), human T cell Leukemia virus
type II (HTLV-II), infectious bronchitis virus (IBV), human
coronavirus (HCoV), hepatitis A virus (HAV), hepatitis B virus
(HBV), hepatitis C virus (HCV), human papilloma virus (HPV),
transmissible gastroenteritis virus (TGEV), berne virus (BEV),
equine arteritis virus (EAV), human astrovirus serotype-1 (HAst-1),
Giardia lamblia virus (GLV), Saccharomyces cerevisiae dsRNA virus
L-A (ScV/L-A), S. cerevisiae dsRNA virus L1 (ScV/L1), bacteriophage
T7, bacteriophage lambda, barley yellow dwarf virus (BYDV), beet
western yellows virus (BWYV), potato leaf roll virus (PLRV), severe
acute respiratory syndrome coronavirus (SARS-CoV), herpes simplex
virus (HSV), and red clover necrotic mosaic virus (RCNMV).
[0414] In another specific embodiment, the subject suffering from a
viral infection or a disease associated therewith is infected with
any of feline immunodeficiency virus (FIV), rous sarcoma virus
(RSV), mouse mammary tumor virus (MMTV), simian retrovirus type 1
(SRV-1), human T cell leukemia virus type I (HTLV-I), HTLV-II,
infectious bronchitis virus (IBV), human coronavirus (HCV),
transmissible gastroenteritis virus (TGEV), berne virus (BEV),
equine arteritis virus (EAV), human astrovirus serotype-1 (HAst-1),
Giardia lamblia virus (GLV), Saccharomyces cerevisiae dsRNA virus
L-A (ScV/L-A), S. cerevisiae dsRNA virus L1 (ScV/L1), bacteriophage
T7, bacteriophage lambda, barley yellow dwarf virus (BYDV), beet
western yellows virus (BWYV), potato leaf roll virus (PLRV), severe
acute respiratory syndrome coronavirus (SARS-CoV), herpes simplex
virus (HSV), and red clover necrotic mosaic virus (RCNMV).
[0415] In certain embodiments, the subject suffering from a viral
infection or a disease associated therewith is not infected with a
human immunodeficiency virus, hepatitis virus, or human
papillomavirus.
[0416] In certain embodiments, a compound or pharmaceutical
composition thereof is administered to a human that has an age in a
range of from about 0 months to about 6 months old, from about 6 to
about 12 months old, from about 6 to about 18 months old, from
about 18 to about 36 months old, from about 1 to about 5 years old,
from about 5 to about 10 years old, from about 10 to about 15 years
old, from about 15 to about 20 years old, from about 20 to about 25
years old, from about 25 to about 30 years old, from about 30 to
about 35 years old, from about 35 to about 40 years old, from about
40 to about 45 years old, from about 45 to about 50 years old, from
about 50 to about 55 years old, from about 55 to about 60 years
old, from about 60 to about 65 years old, from about 65 to about 70
years old, from about 70 to about 75 years old, from about 75 to
about 80 years old, from about 80 to about 85 years old, from about
85 to about 90 years old, from about 90 to about 95 years old or
from about 95 to about 100 years old.
[0417] In some embodiments, a compound or pharmaceutical
composition thereof is administered to a human infant. In other
embodiments, a compound or pharmaceutical composition thereof is
administered to a human toddler. In other embodiments, a compound
or pharmaceutical composition thereof is administered to a human
child. In other embodiments, a compound or pharmaceutical
composition thereof is administered to a human adult. In yet other
embodiments, a compound or pharmaceutical composition thereof is
administered to an elderly human.
[0418] In certain embodiments, a compound or pharmaceutical
composition thereof is administered a subject in an
immunocompromised state or immunosuppressed state or at risk for
becoming immunocompromised or immunosuppressed. In certain
embodiments, a compound or pharmaceutical composition thereof is
administered to a subject receiving or recovering from
immunosuppressive therapy.
[0419] In some embodiments, a compound or pharmaceutical
composition thereof is administered to a patient who is susceptible
to adverse reactions to conventional anti-viral therapies. In some
embodiments, a compound or pharmaceutical composition thereof is
administered to a patient who has proven refractory to anti-viral
therapies other than compounds, but are no longer on these
therapies. Among these patients are refractory patients, and
patients who are too young for conventional therapies.
[0420] In some embodiments, the subject being administered a
compound or pharmaceutical composition thereof has not received
therapy prior to the administration of the compound or
pharmaceutical composition thereof.
Mode of Administration
[0421] When administered to a patient, a compound is preferably
administered as a component of a composition that optionally
comprises a pharmaceutically acceptable carrier, excipient or
diluent. The composition can be administered orally, or by any
other convenient route, for example, by infusion or bolus
injection, by absorption through epithelial or mucocutaneous
linings (e.g., oral mucosa, rectal, and intestinal mucosa) and may
be administered together with another biologically active agent.
Administration can be systemic or local. Various delivery systems
are known, e.g., encapsulation in liposomes, microparticles,
microcapsules, capsules, and can be used to administer the
compound.
[0422] Methods of administration include but are not limited to
parenteral, intradermal, intramuscular, intraperitoneal,
intravenous, subcutaneous, intranasal, epidural, oral, sublingual,
intranasal, intracerebral, intravaginal, transdermal, rectally, by
inhalation, or topically, particularly to the ears, nose, eyes, or
skin. The mode of administration is left to the discretion of the
practitioner. In some instances, administration will result in the
release of a compound into the bloodstream. In a specific
embodiment, a compound is administered orally.
Dosage and Frequency of Administration
[0423] The amount of a compound that will be effective in the
treatment of a viral infection, or prevention or treatment of a
viral disease can be determined by standard clinical techniques. In
vitro or in vivo assays may optionally be employed to help identify
optimal dosage ranges. The precise dose to be employed will also
depend, e.g., on the route of administration, the type of viral
infection, type of viral disease, and the seriousness of the viral
infection, and should be decided according to the judgment of the
practitioner and each patient's or subject's circumstances.
[0424] Exemplary doses of a compound include milligram (mg) or
microgram (m) amounts per kilogram (Kg) of subject or sample weight
per day (e.g., from about 1 .mu.g per Kg to about 500 mg per Kg per
day, from about 5 .mu.g per Kg to about 100 mg per Kg per day, or
from about 10 .mu.g per Kg to about 100 mg per Kg per day. In
specific embodiments, a daily dose is at least 0.1 mg, 0.5 mg, 1.0
mg, 2.0 mg, 5.0 mg, 10 mg, 25 mg, 50 mg, 75 mg, 100 mg, 150 mg, 250
mg, 500 mg, 750 mg, or at least 1 g. In another embodiment, the
dosage is a unit dose of about 0.1 mg, 1 mg, 5 mg, 10 mg, 50 mg,
100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 500 mg, 550
mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg or more. In another
embodiment, the dosage is a unit dose that ranges from about 0.1 mg
to about 1000 mg, 1 mg to about 1000 mg, 5 mg to about 1000 mg,
about 10 mg to about 500 mg, about 150 mg to about 500 mg, about
150 mg to about 1000 mg, 250 mg to about 1000 mg, about 300 mg to
about 1000 mg, or about 500 mg to about 1000 mg. In one embodiment,
a subject is administered one or more doses of an effective amount
of a compound or a pharmaceutical composition thereof, wherein the
effective amount is not the same for each dose.
Combination Products
[0425] Additional agents that can be used in a combination product
with compounds of the present invention for the treatment of viral
infections, or prevention or treatment of viral diseases include,
but are not limited to, small molecules, synthetic drugs, peptides
(including cyclic peptides), polypeptides, proteins, nucleic acids
(e.g., DNA and RNA nucleotides including, but not limited to,
antisense nucleotide sequences, triple helices, RNAi, and
nucleotide sequences encoding biologically active proteins,
polypeptides or peptides), antibodies, synthetic or natural
inorganic molecules, mimetic agents, and synthetic or natural
organic molecules. Specific examples of such agents include, but
are not limited to, immunomodulatory agents (e.g., interferon),
anti-inflammatory agents (e.g., adrenocorticoids, corticosteroids
(e.g., beclomethasone, budesonide, flunisolide, fluticasone,
triamcinolone, methylprednisolone, prednisolone, prednisone,
hydrocortisone), glucocorticoids, steriods, and non-steriodal
anti-inflammatory drugs (e.g., aspirin, ibuprofen, diclofenac, and
COX-2 inhibitors), pain relievers, leukotreine antagonists (e.g.,
montelukast, methyl xanthines, zafirlukast, and zileuton),
beta2-agonists (e.g., albuterol, biterol, fenoterol, isoetharie,
metaproterenol, pirbuterol, salbutamol, terbutalin formoterol,
salmeterol, and salbutamol terbutaline), anticholinergic agents
(e.g., ipratropium bromide and oxitropium bromide), sulphasalazine,
penicillamine, dapsone, antihistamines, anti-malarial agents (e.g.,
hydroxychloroquine), anti-viral agents (e.g., nucleoside analogs
(e.g., zidovudine, acyclovir, gangcyclovir, vidarabine,
idoxuridine, trifluridine, and ribavirin), foscarnet, amantadine,
rimantadine, saquinavir, indinavir, ritonavir, and AZT) and
antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin,
erythomycin, penicillin, mithramycin, and anthramycin (AMC)).
[0426] Any therapy which is known to be useful, or which has been
used, will be used or is currently being used for the treatment of
viral infections, or prevention or treatment of viral diseases can
be used in combination with compounds in accordance with the
invention described herein.
Cell Culture Uses
[0427] The present invention provides for the use of compounds as
ingredients in cell culture-related products in which it is
desirable to have antiviral activity. In one embodiment, one or
more compounds is added to cell culture media. In certain
embodiments, compounds that prove too toxic or are not used in
subjects are added to cell culture-related products, such as
media.
EXAMPLES
Cryptic Splice Site
[0428] This example demonstrates that a cryptic splice site is
created when a guanine residue is inserted after nucleotide 48 of
exon 7 of SMN in a minigene construct comprising in 5' to 3' order:
(i) the nucleic acid residues of exon 6 of SMN, the nucleic acid
residues of intron 6 of SMN, the nucleic acid residues of exon 7 of
SMN, the nucleic acid residues of intron 7 of SMN, the first 23
nucleic acid residues of exon 8 of SMN; and (ii) a reporter gene
coding sequence fused in frame to the nucleic acid residues of exon
8 of SMN, wherein the reporter gene does not have a start codon. As
a result of the cryptic splice site, a deletion of the last seven
nucleotides of exon 7 occurs and a frameshift in the open reading
frame of the reporter gene is created.
Materials and Methods
Preparation of the Minigene Constructs
[0429] DNA corresponding to a region of the SMN2 gene starting from
the 5' end of exon 6 (ATAATTCCCCC) (SEQ ID NO: 1) and ending at
nucleic acid residue 23 of exon 8 (CAGCAC) (SEQ ID NO: 2) was
amplified by PCR using the following primers:
TABLE-US-00002 Forward primer: (SEQ ID NO: 3)
5'-CGCGGATCCATAATTCCCCCACCACCTC-3' Reverse primer: (SEQ ID NO: 4)
5'-CGCGGATCCGTGCTGCTCTATGCCAGCA-3'
[0430] The 5' end of each primer was designed to add a BamHI site
at both the 5' end of exon 6 (GGATCC) (SEQ ID NO:5) and the 3' end,
after the 23.sup.rd nucleotide, of exon 8. Using the BamHI
restriction sites, the PCR fragment was cloned into a derivative of
the original pcDNA 3.1/Hygro vector which was modified as disclosed
in United States Patent Publication US2005/0048549.
[0431] New UTRs were added to the modified vector using the HindIII
site and the BamHI site comprising a 5' deg UTR:
5'-TAGCTTCTTACCCGTACTCCACCGTTGGCAGCACGATCGCACGTCCCACGT
GAACCATTGGTAAACCCTG-3' (SEQ ID NO: 6) was cloned into the modified
pcDNA3.1/Hygro vector together with a start codon upstream of the
BamHI site; and
[0432] a 3'deg UTR:
5'-ATCGAAAGTACAGGACTAGCCTTCCTAGCAACCGCGGGCTGGGAGTCTGAGA
CATCACTCAAGATATATGCTCGGTAACGTATGCTCTAGCCATCTAACTATTCCCT
ATGTCTTATAGGG-3' (SEQ ID NO: 7) was cloned into the modified
pcDNA3.1/Hygro vector with a stop codon using the NotI site and the
XhoI site. In addition, a luciferase gene lacking its start codon
was cloned into the vector using the BamHI and NotI sites.
[0433] The resulting minigene comprises, in 5' to 3' order: the
5'-deg UTR, the start codon, six additional nucleotides forming a
BamHI site, the nucleic acid residues of exon 6, the nucleic acid
residues of intron 6 of SMN2, the nucleic acid residues of exon 7
of SMN2, the nucleic acid residues of intron 7 of SMN2, and the
first 23 nucleic acid residues of exon 8 of SMN2, an additional six
nucleotides forming a BamHI site and the luciferase gene lacking
the start codon.
[0434] A single guanine residue was inserted after nucleotide 48 of
exon 7 of SMN2 by site-directed mutagenesis. The minigene construct
produced is referred to as SMN2-G.
[0435] To generate the SMN1 version of the minigene, the sixth
nucleotide of exon 7 (a thymine residue) was changed to cytosine by
site directed mutagenesis. The resulting SMN1 minigene construct is
referred to as SMN1-G.
Results
[0436] SMN1 and SMN2 transcripts derived from minigenes containing
exon 6 through 8 and the intervening introns recapitulate the
splicing of their endogenous pre-mRNAs (Lorson, et al., 1999, Proc.
Natl. Acad. Sci. U.S.A. 96(11):6307-6311). An SMN2-alternative
splicing reporter construct which contains exons 6 to 8 and the
intervening introns followed by a luciferase reporter gene was
generated. Salient features of this construct are the lack of the
start codon in the luciferase gene, inactivation of the termination
codon (in the open reading frame that encodes the SMN protein) of
exon 7 by insertion of a guanine residue after nucleic acid 48 of
exon 7 and addition of a start codon (ATG) immediately upstream of
exon 6.
[0437] The luciferase reporter was designed to be out of frame if
exon 7 of SMN2 is removed during splicing of the pre-mRNA. In
addition, the 23 nucleic acids of exon 8 are read in a different
frame in the absence of exon 7, resulting in a stop codon in exon 8
in the mature mRNA transcript. Thus, the protein translated from an
RNA transcript lacking exon 7 will be a truncated SMN protein
lacking the luciferase portion encoded by the minigene construct.
In the presence of compounds that increase the inclusion of exon 7
of SMN2 into mRNA transcribed from the SMN2 gene, more transcripts
containing exon 7 were expected to be produced. In view of the
teaching in Zhang, et al., 2001, Gene Therapy, 8:1532-1538, the
presence of the additional guanine residue after nucleic acid
residue 48 of exon 7 of SMN2 was expected to cause the SMN2
sequences to be in frame with the luciferase coding region in the
spliced mRNA transcript containing exon 7. Thus, the resulting
protein expressed from this mRNA transcript was expected to be a
truncated SMN-luciferase fusion protein.
[0438] The DNA sequence of the minigene from the SMN2-G construct
is provided in FIG. 2.
[0439] An SMN1 version of the SMN2 minigene construct was also
generated in which the sixth nucleotide (T) of exon 7 was mutated
to C to maximize the likelihood of inclusion of exon 7 into the
transcript. Similar to the SMN2 minigene construct, the SMN1
minigene construct had a single guanine (SMN1-G) residue inserted
after nucleic residue 48 of exon 7. The SMN1-G construct was
expected to produce a truncated SMN-luciferase fusion protein
because the SMN1 transcript derived from the minigene was expected
to contain exon 7 and the SMN1 sequence was expected to be in frame
with the luciferase coding region due to the guanine residue insert
after nucleotide 48 of exon 7 of SMN1.
[0440] An increase in luciferase expression from the SMN1-G
minigene construct when compared to the SMN2-G minigene construct
was expected. However, the SMN1-G minigene construct did not
exhibit an increase in luciferase expression when it was compared
to the SMN2-G minigene construct.
[0441] In order to determine why constructs with a guanine insert
yielded results different from those expected, total RNA was
isolated from cells transiently transfected with the SMN1 or SMN2
versions of the minigenes. Total RNA was reverse transcribed to
produce the cDNA. The cDNA was then amplified by PCR with primers
specific for the minigene/reporter gene transcript. The first
primer annealed to the luciferase gene and the second primer to
exon 6. The PCR products were resolved on a 2% agarose gel.
[0442] RNA isolated from HEK293H cells transfected with the SMN2-G
minigene construct predominately showed a band corresponding to the
size of a transcript that lacks exon 7. Expression of the SMN1-G
minigene construct in transiently transfected HEK293H cells
resulted in the appearance of an additional band corresponding to
the transcript containing exon 7. The band corresponding to the
transcript containing exon 7 produced from the SMN1-G minigene
construct was isolated and cloned into a pCR-blunt vector
(Invitrogen). 20 clones containing the SMN1-G minigene fragment
were sequenced. All of the clones lacked seven nucleotides from the
inserted guanine residue to the last nucleotide of exon 7 (GTAAGGA)
(SEQ ID NO: 8), demonstrating that the inclusion of exon 7 for the
SMN1-G version of the minigene occurred through utilization of a
cryptic splice site generated by the G insertion. Indeed, the G
insertion resulted in generation of a sequence element (GTAAGG)
(SEQ ID NO: 9) reminiscent of the 5' end of intron 7 (GTAAGT) (SEQ
ID NO: 10). Therefore, the spliceosome preferentially used the 5'
splice site between the nucleotide residue 48 of exon 7 and the G
insertion (position 49). Utilization of the cryptic splice site
resulted in a frameshift of the open reading frame that starts at
the ATG immediately upstream of exon 6 of SMN as well as a stop
codon before the luciferase portion of the minigene. Therefore,
luciferase expression was substantially reduced from the SMN1-G
minigene construct when a part of exon 7 was included. Analogously,
the G insertion in the SMN2-G minigene construct creates a cryptic
splice site in exon 7 of SMN2. The resulting inclusion of a
fragment of exon 7 of SMN2 that lacks seven nucleotides at the 3'
end significantly reduces luciferase expression from the SMN2-G
minigene construct.
Compound that Modulates Programmed Ribosomal Frameshifting
[0443] This example demonstrates that Compound 1 modulates the
efficiency of viral programmed ribosomal frameshifting.
[0444] Materials and Methods
[0445] The ability of Compound 1 to modulate the efficiency of
viral programmed ribosomal frameshifting was measured using a dual
luciferase assay (as described in Biswas, et al., 2004, J. Virol.
78(4):2082-2087).
[0446] Results
[0447] The inhibition in luciferase activity in the presence of
Compound 1 relative to luciferase activity in the presence of a
negative control (0.5% DMSO) averaged approximately 5-fold at a
compound test concentration in a range of from approximately 5
.mu.M to approximately 130 .mu.M (FIG. 1).
[0448] The present invention is not to be limited in scope by the
specific embodiments described herein. Indeed, various
modifications of the invention and their equivalents, in addition
to those described herein will become apparent to those skilled in
the art from the foregoing description and accompanying figures.
Such modifications are intended to fall within the scope of the
appended claims.
[0449] Various patents, patent applications, and publications are
cited herein, the disclosures of which are incorporated by
reference in their entirety and for all purposes.
Sequence CWU 1
1
11111DNAArtificial SequenceDNA corresponding to a region of the
SMN2 gene starting from the 5' end of exon 6 1ataattcccc c
1126DNAArtificial SequenceDNA corresponding to a region of the SMN2
gene ending at nucleic acid residue 23 of exon 8 2cagcac
6328DNAArtificial SequenceForward Primer used to generate DNA
corresponding to a region of the SMN2 gene 3cgcggatcca taattccccc
accacctc 28428DNAArtificial SequenceReverse Primer used to generate
DNA corresponding to a region of the SMN2 gene 4cgcggatccg
tgctgctcta tgccagca 2856DNAArtificial SequenceBamHI site at the 5
prime end of exon 6 of the SMN2 gene 5ggatcc 6670DNAArtificial
Sequence5 prime deg UTR added to the modified vector using the
HindIII site and the BamHI site 6tagcttctta cccgtactcc accgttggca
gcacgatcgc acgtcccacg tgaaccattg 60gtaaaccctg 707119DNAArtificial
Sequence3 prime deg UTR added to the modified vector using the
HindIII site and the BamHI site 7tcgaaagtac aggactagcc ttcctagcaa
ccgcgggctg ggagtctgag acatcactca 60agatatatgc tcggtaacgt atgctctagc
catctaacta ttccctatgt cttataggg 11987DNAArtificial SequenceSeven
nucleotides from the inserted guanine residue to the last
nucleotide of exon 7 missing in the clones containing the SMN1-G
minigene fragment 8gtaagga 796DNAArtificial SequenceSequence
element generated through the G-insertion in the SMN1-G version of
the minigene 9gtaagg 6106DNAArtificial Sequence5' end of intron 7
of the SMN1-G version of the minigene 10gtaagt 6118266DNAArtificial
SequenceDNA sequence of the minigene from the SMN-G minigene
construct 11tagcttctta cccgtactcc accgttggca gcacgatcgc acgtcccacg
tgaaccattg 60gtaaaccctg atgggatcca taattccccc accacctccc atatgtccag
attctcttga 120tgatgctgat gctttgggaa gtatgttaat ttcatggtac
atgagtggct atcatactgg 180ctattatatg gtaagtaatc actcagcatc
ttttcctgac aatttttttg tagttatgtg 240actttgtttt gtaaatttat
aaaatactac ttgcttctct ctttatatta ctaaaaaata 300aaaataaaaa
aatacaactg tctgaggctt aaattactct tgcattgtcc ctaagtataa
360ttttagttaa ttttaaaaag ctttcatgct attgttagat tattttgatt
atacactttt 420gaattgaaat tatacttttt ctaaataatg ttttaatctc
tgatttgaaa ttgattgtag 480ggaatggaaa agatgggata atttttcata
aatgaaaaat gaaattcttt tttttttttt 540tttttttttg agacggagtc
ttgctctgtt gcccaggctg gagtgcaatg gcgtgatctt 600ggctcacagc
aagctctgcc tcctggattc acgccattct cctgcctcag cctcagaggt
660agctgggact acaggtgcct gccaccacgc ctgtctaatt ttttgtattt
ttttgtaaag 720acagggtttc actgtgttag ccaggatggt ctcaatctcc
tgaccccgtg atccacccgc 780ctcggccttc caagagaaat gaaatttttt
taatgcacaa agatctgggg taatgtgtac 840cacattgaac cttggggagt
atggcttcaa acttgtcact ttatacgtta gtctcctacg 900gacatgttct
attgtatttt agtcagaaca tttaaaatta ttttatttta ttttattttt
960tttttttttt tgagacggag tctcgctctg tcacccaggc tggagtacag
tggcgcagtc 1020tcggctcact gcaagctccg cctcccgggt tcacgccatt
ctcctgcctc agcctctccg 1080agtagctggg actacaggcg cccgccacca
cgcccggcta attttttttt atttttagta 1140gagacggggt ttcaccgtgg
tctcgatctc ctgacctcgt gatccacccg cctcggcctc 1200ccaaagtgct
gggattacaa gcgtgagcca ccgcgcccgg cctaaaatta tttttaaaag
1260taagctcttg tgccctgcta aaattatgat gtgatattgt aggcacttgt
atttttagta 1320aattaatata gaagaaacaa ctgacttaaa ggtgtatgtt
tttaaatgta tcatctgtgt 1380gtgcccccat taatattctt atttaaaagt
taaggccaga catggtggct tacaactgta 1440atcccaacag tttgtgaggc
cgaggcaggc agatcacttg aggtcaggag tttgagacca 1500gcctggccaa
catgatgaaa ccttgtctct actaaaaata ccaaaaaaaa tttagccagg
1560catggtggca catgcctgta atccgagcta cttgggaggc tgtggcagga
aaattgcttt 1620aatctgggag gcagaggttg cagtgagttg agattgtgcc
actgcactcc acccttggtg 1680acagagtgag attccatctc aaaaaaagaa
aaaggcctgg cacggtggct cacacctata 1740atcccagtac tttgggaggt
agaggcaggt ggatcacttg aggttaggag ttcaggacca 1800gcctggccaa
catggtgact actccatttc tactaaatac acaaaactta gcccagtggc
1860gggcagttgt aatcccagct acttgagagg ttgaggcagg agaatcactt
gaacctggga 1920ggcagaggtt gcagtgagcc gagatcacac cgctgcactc
tagcctggcc aacagagtga 1980gaatttgcgg agggaaaaaa aagtcacgct
tcagttgttg tagtataacc ttggtatatt 2040gtatgtatca tgaattcctc
attttaatga ccaaaaagta ataaatcaac agcttgtaat 2100ttgttttgag
atcagttatc tgactgtaac actgtaggct tttgtgtttt ttaaattatg
2160aaatatttga aaaaaataca taatgtatat ataaagtatt ggtataattt
atgttctaaa 2220taactttctt gagaaataat tcacatggtg tgcagtttac
ctttgaaagt atacaagttg 2280gctgggcaca atggctcacg cctgtaatcc
cagcactttg ggaggccagg gcaggtggat 2340cacgaggtca ggagatcgag
accatcctgg ctaacatggt gaaaccccgt ctctactaaa 2400agtacaaaaa
caaattagcc gggcatgttg gcgggcacct tttgtcccag ctgctcggga
2460ggctgaggca ggagagtggc gtgaacccag gaggtggagc ttgcagtgag
ccgagattgt 2520gccagtgcac tccagcctgg gcgacagagc gagactctgt
ctcaaaaaat aaaataaaaa 2580agaaagtata caagtcagtg gttttggttt
tcagttatgc aaccatcact acaatttaag 2640aacattttca tcaccccaaa
aagaaaccct gttaccttca ttttccccag ccctaggcag 2700tcagtacact
ttctgtctct atgaatttgt ctattttaga tattatatat aaacggaatt
2760atacgatatg tggtcttttg tgtctggctt ctttcactta gcatgctatt
ttcaagattc 2820atccatgctg tagaatgcac cagtactgca ttccttctta
ttgctgaata ttctgttgtt 2880tggttatatc acattttatc cattcatcag
ttcatggaca tttaggttgt ttttattttt 2940gggctataat gaataatgtt
gctatgaaca ttcgtttgtg ttctttttgt ttttttggtt 3000ttttgggttt
tttttgtttt gtttttgttt ttgagacagt cttgctctgt ctcctaagct
3060ggagtgcagt ggcatgatct tggcttactg caagctctgc ctcccgggtt
cacaccattc 3120tcctgcctca gcccgacaag tagctgggac tacaggcgtg
tgccaccatg cacggctaat 3180tttttgtatt tttagtagag atggggtttc
accgtgttag ccaggatggt ctcgatctcc 3240tgacctcgtg atctgcctgc
ctaggcctcc caaagtgctg ggattacagg cgtgagccac 3300tgcacctggc
cttaagtgtt tttaatacgt cattgcctta agctaacaat tcttaacctt
3360tgttctactg aagccacgtg gttgagatag gctctgagtc tagcttttaa
cctctatctt 3420tttgtcttag aaatctaagc agaatgcaaa tgactaagaa
taatgttgtt gaaataacat 3480aaaataggtt ataactttga tactcattag
taacaaatct ttcaatacat cttacggtct 3540gttaggtgta gattagtaat
gaagtgggaa gccactgcaa gctagtatac atgtagggaa 3600agatagaaag
cattgaagcc agaagagaga cagaggacat ttgggctaga tctgacaaga
3660aaaacaaatg ttttagtatt aatttttgac tttaaatttt ttttttattt
agtgaatact 3720ggtgtttaat ggtctcattt taataagtat gacacaggta
gtttaaggtc atatatttta 3780tttgatgaaa ataaggtata ggccgggcac
ggtggctcac acctgtaatc ccagcacttt 3840gggaggccga ggcaggcgga
tcacctgagg tcgggagtta gagactagcc tcaacatgga 3900gaaaccccgt
ctctactaaa aaaaatacaa aattaggcgg gcgtggtggt gcatgcctgt
3960aatcccagct actcaggagg ctgaggcagg agaattgctt gaacctggga
ggtggaggtt 4020gcggtgagcc gagatcacct cattgcactc cagcctgggc
aacaagagca aaactccatc 4080tcaaaaaaaa aaaaataagg tataagcggg
ctcaggaaca tcattggaca tactgaaaga 4140agaaaaatca gctgggcgca
gtggctcacg ccggtaatcc caacactttg ggaggccaag 4200gcaggcgaat
cacctgaagt cgggagttcc agatcagcct gaccaacatg gagaaaccct
4260gtctctacta aaaatacaaa actagccggg catggtggcg catgcctgta
atcccagcta 4320cttgggaggc tgaggcagga gaattgcttg aaccgagaag
gcggaggttg cggtgagcca 4380agattgcacc attgcactcc agcctgggca
acaagagcga aactccgtct caaaaaaaaa 4440aggaagaaaa atattttttt
aaattaatta gtttatttat tttttaagat ggagttttgc 4500cctgtcaccc
aggctggggt gcaatggtgc aatctcggct cactgcaacc tccgcctcct
4560gggttcaagt gattctcctg cctcagcttc ccgagtagct gtgattacag
ccatatgcca 4620ccacgcccag ccagttttgt gttttgtttt gttttttgtt
tttttttttt gagagggtgt 4680cttgctctgt cccccaagct ggagtgcagc
ggcgcgatct tggctcactg caagctctgc 4740ctcccaggtt cacaccattc
tcttgcctca gcctcccgag tagctgggac tacaggtgcc 4800cgccaccaca
cccggctaat ttttttgtgt ttttagtaga gatggggttt cactgtgtta
4860gccaggatgg tctcgatctc ctgacctttt gatccacccg cctcagcctc
cccaagtgct 4920gggattatag gcgtgagcca ctgtgcccgg cctagtcttg
tatttttagt agagtcggga 4980tttctccatg ttggtcaggc tgttctccaa
atccgacctc aggtgatccg cccgccttgg 5040cctccaaaag tgcaaggcaa
ggcattacag gcatgagcca ctgtgaccgg caatgttttt 5100aaatttttta
catttaaatt ttatttttta gagaccaggt ctcactctat tgctcaggct
5160ggagtgcaag ggcacattca cagctcactg cagccttgac ctccagggct
caagcagtcc 5220tctcacctca gtttcccgag tagctgggac tacagtgata
atgccactgc acctggctaa 5280tttttatttt tatttattta tttttttttg
agacagagtc ttgctctgtc acccaggctg 5340gagtgcagtg gtgtaaatct
cagctcactg cagcctccgc ctcctgggtt caagtgattc 5400tcctgcctca
acctcccaag tagctgggat tagaggtccc caccaccatg cctggctaat
5460tttttgtact ttcagtagaa acggggtttt gccatgttgg ccaggctgtt
ctcgaactcc 5520tgagctcagg tgatccaact gtctcggcct cccaaagtgc
tgggattaca ggcgtgagcc 5580actgtgccta gcctgagcca ccacgccggc
ctaattttta aattttttgt agagacaggg 5640tctcattatg ttgcccaggg
tggtgtcaag ctccaggtct caagtgatcc ccctacctcc 5700gcctcccaaa
gttgtgggat tgtaggcatg agccactgca agaaaacctt aactgcagcc
5760taataattgt tttctttggg ataactttta aagtacatta aaagactatc
aacttaattt 5820ctgatcatat tttgttgaat aaaataagta aaatgtcttg
tgaaacaaaa tgctttttaa 5880catccatata aagctatcta tatatagcta
tctatatcta tatagctatt ttttttaact 5940tcctttattt tccttacagg
gttttagaca aaatcaaaaa gaaggaaggt gctcacattc 6000cttaaatgta
aggagtaagt ctgccagcat tatgaaagtg aatcttactt ttgtaaaact
6060ttatggtttg tggaaaacaa atgtttttga acatttaaaa agttcagatg
ttagaaagtt 6120gaaaggttaa tgtaaaacaa tcaatattaa agaattttga
tgccaaaact attagataaa 6180aggttaatct acatccctac tagaattctc
atacttaact ggttggttgt gtggaagaaa 6240catactttca caataaagag
ctttaggata tgatgccatt ttatatcact agtaggcaga 6300ccagcagact
tttttttatt gtgatatggg ataacctagg catactgcac tgtacactct
6360gacatatgaa gtgctctagt caagtttaac tggtgtccac agaggacatg
gtttaactgg 6420aattcgtcaa gcctctggtt ctaatttctc atttgcagga
aatgctggca tagagcagca 6480cggatccgaa gacgccaaaa acataaagaa
aggcccggcg ccattctatc ctctagagga 6540tggaaccgct ggagagcaac
tgcataaggc tatgaagaga tacgccctgg ttcctggaac 6600aattgctttt
acagatgcac atatcgaggt gaacatcacg tacgcggaat acttcgaaat
6660gtccgttcgg ttggcagaag ctatgaaacg atatgggctg aatacaaatc
acagaatcgt 6720cgtatgcagt gaaaactctc ttcaattctt tatgccggtg
ttgggcgcgt tatttatcgg 6780agttgcagtt gcgcccgcga acgacattta
taatgaacgt gaattgctca acagtatgaa 6840catttcgcag cctaccgtag
tgtttgtttc caaaaagggg ttgcaaaaaa ttttgaacgt 6900gcaaaaaaaa
ttaccaataa tccagaaaat tattatcatg gattctaaaa cggattacca
6960gggatttcag tcgatgtaca cgttcgtcac atctcatcta cctcccggtt
ttaatgaata 7020cgattttgta ccagagtcct ttgatcgtga caaaacaatt
gcactgataa tgaattcctc 7080tggatctact gggttaccta agggtgtggc
ccttccgcat agaactgcct gcgtcagatt 7140ctcgcatgcc agagatccta
tttttggcaa tcaaatcatt ccggatactg cgattttaag 7200tgttgttcca
ttccatcacg gttttggaat gtttactaca ctcggatatt tgatatgtgg
7260atttcgagtc gtcttaatgt atagatttga agaagagctg tttttacgat
cccttcagga 7320ttacaaaatt caaagtgcgt tgctagtacc aaccctattt
tcattcttcg ccaaaagcac 7380tctgattgac aaatacgatt tatctaattt
acacgaaatt gcttctgggg gcgcacctct 7440ttcgaaagaa gtcggggaag
cggttgcaaa acgcttccat cttccaggga tacgacaagg 7500atatgggctc
actgagacta catcagctat tctgattaca cccgaggggg atgataaacc
7560gggcgcggtc ggtaaagttg ttccattttt tgaagcgaag gttgtggatc
tggataccgg 7620gaaaacgctg ggcgttaatc agagaggcga attatgtgtc
agaggaccta tgattatgtc 7680cggttatgta aacaatccgg aagcgaccaa
cgccttgatt gacaaggatg gatggctaca 7740ttctggagac atagcttact
gggacgaaga cgaacacttc ttcatagttg accgcttgaa 7800gtctttaatt
aaatacaaag gatatcaggt ggcccccgct gaattggaat cgatattgtt
7860acaacacccc aacatcttcg acgcgggcgt ggcaggtctt cccgacgatg
acgccggtga 7920acttcccgcc gccgttgttg ttttggagca cggaaagacg
atgacggaaa aagagatcgt 7980ggattacgtc gccagtcaag taacaaccgc
gaaaaagttg cgcggaggag ttgtgtttgt 8040ggacgaagta ccgaaaggtc
ttaccggaaa actcgacgca agaaaaatca gagagatcct 8100cataaaggcc
aagaagggcg gaaagtccaa attgcgcggc cgctaaatcg aaagtacagg
8160actagccttc ctagcaaccg cgggctggga gtctgagaca tcactcaaga
tatatgctcg 8220gtaacgtatg ctctagccat ctaactattc cctatgtctt ataggg
8266
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