U.S. patent application number 12/442354 was filed with the patent office on 2010-07-29 for compositions and methods related to protein displacement therapy for myotonic distrophy.
This patent application is currently assigned to University of Rochester. Invention is credited to Jill Miller, Robert Osborne, Krzysztof Sobezak, Maurice Swanson, Charles A. Thornton, Thurman Wheeler.
Application Number | 20100190689 12/442354 |
Document ID | / |
Family ID | 39201119 |
Filed Date | 2010-07-29 |
United States Patent
Application |
20100190689 |
Kind Code |
A1 |
Thornton; Charles A. ; et
al. |
July 29, 2010 |
COMPOSITIONS AND METHODS RELATED TO PROTEIN DISPLACEMENT THERAPY
FOR MYOTONIC DISTROPHY
Abstract
Disclosed are compositions and methods related to the
interaction of polyCUG and polyCCUG repeat RNA and proteins that
bind to these repetitive RNA sequences. Also disclosed are methods
of treating DM1 or DM2 comprising inhibiting the interaction of
poly(CUG).sup.exp or poly(CCUG).sup.exp RNA with muscleblind
proteins, or by causing improvement of spliceopathy in myotonic
dystrophy.
Inventors: |
Thornton; Charles A.;
(Rochester, NY) ; Wheeler; Thurman; (Rochester,
NY) ; Sobezak; Krzysztof; (Rochester, NY) ;
Osborne; Robert; (Rochester, NY) ; Miller; Jill;
(Rockport, NY) ; Swanson; Maurice; (Gainesville,
FL) |
Correspondence
Address: |
Ballard Spahr LLP
SUITE 1000, 999 PEACHTREE STREET
ATLANTA
GA
30309-3915
US
|
Assignee: |
University of Rochester
Rochester
NY
University of Florida Research Foundation, Inc.
Gainesville
FL
|
Family ID: |
39201119 |
Appl. No.: |
12/442354 |
Filed: |
September 21, 2007 |
PCT Filed: |
September 21, 2007 |
PCT NO: |
PCT/US07/20503 |
371 Date: |
March 5, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60826396 |
Sep 21, 2006 |
|
|
|
Current U.S.
Class: |
514/6.9 ;
435/325; 435/6.16; 514/44A; 536/24.5 |
Current CPC
Class: |
C12N 2310/3515 20130101;
G01N 33/6893 20130101; A61K 38/00 20130101; C12N 2310/3233
20130101; G01N 33/5008 20130101; C12Y 207/11001 20130101; A61K
47/543 20170801; A61P 43/00 20180101; G01N 2800/2878 20130101; G01N
33/6872 20130101; A61K 31/7036 20130101; A61K 31/5375 20130101;
A61P 21/04 20180101; G01N 2500/02 20130101; C12N 15/113 20130101;
C12N 2310/11 20130101; A61P 21/00 20180101; G01N 33/6887
20130101 |
Class at
Publication: |
514/8 ; 435/6;
514/44.A; 435/325; 536/24.5 |
International
Class: |
A61K 38/14 20060101
A61K038/14; C12Q 1/68 20060101 C12Q001/68; A61K 31/7088 20060101
A61K031/7088; C12N 5/00 20060101 C12N005/00; C07H 21/00 20060101
C07H021/00 |
Goverment Interests
[0002] This work was funded by National Institutes of Health Grant
No. NIH/AR46806, NIH/AR/NS48143 and NIH/NS48843, the government has
certain rights in the invention.
Claims
1. A method of screening for an agent that inhibits the interaction
of a protein and a ligand comprising the steps of a) capturing the
ligand to a substrate; b) admixing a labeled protein with the
ligand; c) contacting an agent with the mixture of step b; d)
determining the level of the label; and e) comparing the amount of
the label relative to a control; wherein a decrease in the level of
the label indicates an agent that inhibits the interaction.
2. The method of claim 1, wherein the protein is a polyCUG or
polyCCUG repeat RNA interacting protein or a protein that is
sequestered by polyCUG or polyCCUG repeats.
3. The method of claim 2, wherein the polyCUG or polyCCUG repeat
interacting protein or sequestered protein is MBNL1, MBNL2, or
MBNL3.
4. The method of claim 1, wherein the ligand is RNA comprising a
polyCUG or polyCCUG repeat.
5. The method of claim 4, wherein the polyCUG or polyCCUG repeat is
flanked by RNA sequence from the DMPK gene or sequence that permits
capture to a substrate.
6. The method of claim 4, wherein the polyCUG or polyCCUG repeat
ligand is bound to the substrate by binding to a complementary
nucleic acid which is bound to the substrate.
7. The method of claim 6, wherein the complementary nucleic acid is
biotinylated.
8. The method of claim 7, wherein the biotinylated nucleic acid is
bound to the substrate through the binding of biotin with
streptavidin bound to the substrate.
9. The method of claim 6, wherein the complementary nucleic acid is
an oligodeoxynucleotide (ODN).
10. The method of claim 1, wherein the substrate is a polystyrene
plate.
11. The method of claim 1, wherein the label is a fluorescent
label.
12. The method of claim 11, wherein the level of fluorescence is
determined by a fluorometer or fluorescence plate reader.
13. A method of screening for an agent that inhibits the
interaction of a protein and a ligand comprising the steps of a)
mixing a protein bound to a substrate with a labeled ligand; b)
contacting an agent with the mixture of step a; c) determining the
level of the label; and d) comparing the amount of the label
relative to a control; wherein a decrease in the level of the label
indicates an agent that inhibits the interaction.
14. The method of claim 13, wherein the ligand comprises polyCUG or
polyCCUG repeat RNA.
15. The method of claim 13, wherein the protein is a polyCUG or
polyCCUG repeat RNA interacting protein or a protein that is
sequestered by polyCUG or polyCCUG repeats.
16. The method of claim 15, wherein the polyCUG or polyCCUG repeat
interacting protein or sequestered protein is MBNL1, MBNL2, or
MBNL3.
17. The method of claim 16, wherein the substrate is a
nitrocellulose filter.
18. The method of claim 13, wherein the label is a fluorescent
label.
19. A method of screening for an agent that inhibits the
interaction of a first protein and a first recognition element on a
nucleic acid comprising the steps of a) administering an agent to a
cell comprising the first protein, a second protein, and a nucleic
acid comprising the first recognition element adjacent to a second
recognition element, wherein the first protein binds the first
recognition element and the second protein binds the second
recognition element; and b) detecting co-localization of the first
and second protein, wherein a decrease in co-localization of the
first and second protein relative to a control indicates an agent
that inhibits the interaction.
20. The method of claim 19, wherein the first protein is MBNL1,
MBNL2, or MBNL3 and the first recognition element is polyCUG repeat
RNA.
21. The method of claim 19, wherein the second protein is MS2 and
the second recognition element is an MS2 coat protein RNA
recognition element.
22. The method of claim 19, wherein at least one of the first and
second proteins comprises a donor fluorescent dye and at least one
of the first and second proteins comprises an acceptor dye, wherein
excitation of the donor fluorescent dye results in a fluorescent
emission that excites the acceptor dye if the first and second
proteins are co-localized.
23. The method of claim 19, wherein at least one of the first and
second proteins comprises a first half of a split beta
galactosidase protein and at least one of the first and second
proteins comprises a second half of the split beta galactosidase
protein, wherein hydrolysis of a beta galactosidase substrate
results in fluorescence of luminescence if the first and second
proteins are co-localized.
24. The method of claim 19, wherein at least one of the first and
second proteins comprises a first half of a split fluorescent
protein and at least one of the first and second proteins comprises
a second half of the split fluorescent protein, wherein excitation
of the split fluorescent protein results in a fluorescent emission
if the first and second proteins are co-localized.
25. The method of claim 24, wherein the split fluorescent protein
is Venus fluorescent protein (VFP).
26. A method of screening for an agent that improves spliceopathy
comprising the steps of a) introducing an agent into a cell
comprising of a splicing regulator, overexpressed polyCUG or
polyCCUG repeat RNA, and spliceopathy reporter construct, wherein
the reporter construct comprises a gene susceptible to polyCUG or
polyCCUG repeat induced spliceopathy flanked by one or more genes
encoding a labeled protein; and b) measuring the level of the
labeled protein; and c) comparing the ratio of labeled protein,
wherein an increase of labeled protein indicates an agent that
improves spliceopathy.
27. The method of claim 26, wherein the gene susceptible to polyCUG
or polyCCUG is flanked by genes encoding first and second labeled
protein, wherein the first and second proteins are differentially
labeled; and wherein the method further comprises d) comparing the
ratio of the first labeled protein to the second labeled protein,
wherein a high ratio indicates an agent that improves
spliceopathy.
28. The method of claim 27, wherein the first and second labeled
proteins are, YFP and Y.cndot.CFP, respectively.
29. The method of claim 26, wherein the gene susceptible to polyCUG
or polyCCUG is flanked by a gene encoding a single labeled protein,
and wherein an increase of the labeled protein indicates an agent
that improves spliceopathy.
30. The method of claim 29, wherein the labeled protein is GFP
31. The method of claim 26, wherein the splicing regulator is
MBLN1, MBNL2, MBNL3, CUG-BP1, or ETR-3.
32. The method of claim 26, wherein the spliceopathy susceptible
gene is SERCA1 exon 22 or the fetal exon of TNNT3.
33. The method of claims 26, wherein the level of fluorescence is
determined by fluorometry or Fluorescence Resonance Energy Transfer
(FRET).
34. A method of screening for an agent that improves spliceopathy
comprising the steps of a) introducing an agent into a cell
comprising of a splicing regulator, overexpressed polyCUG or
polyCCUG repeat RNA, and spliceopathy reporter construct, wherein
the reporter construct comprises a gene susceptible to polyCUG or
polyCCUG induced spliceopathy, flanked by the luciferase gene; and
b) measuring the luferase activity, wherein an increase of
luciferase activity indicates an agent that improves
spliceopathy.
35. A method treating myotonic dystrophy (DM) in a subject in need
thereof comprising administering to the subject an agent that
inhibits the interaction of MBNL1 with polyCUG.sup.exp mRNA.
36. The method of claim 35, wherein the DM is myotonic dystrophy
type 1.
37. The method of claim 35, wherein the agent is a morpholino.
38. The method of claim 37, wherein the morpholino is CAG25.
39. The method of claim 38, wherein the morpholino is set forth in
SEQ ID NO: 3.
40. The method of claim 37, wherein the morpholino is an antisense
oligonucleotide (AON).
41. The method of claim 37, wherein the morpholino is set forth in
SEQ ID NO: 4 or SEQ ID NO: 6.
42. The method of claim 35, wherein the agent is a PNA-CAG.
43. The method of claim 42, wherein the PNA-CAG consists of 2, 3,
4, or 5 CAG repeats.
44. The method of claim 35, wherein the agent is an aminoglycosidic
antibiotic compound.
45. A method treating myotonic dystrophy in a subject in need
thereof comprising administering to the subject an agent that
corrects spliceopathy.
46. The method of claim 45, wherein the sliceopathy results in
channelopathy.
47. The method of claim 45, wherein the agent is a morpholino.
48. The method of claim 47, wherein the morpholino is CAG25.
49. The method of claim 48, wherein the morpholino is set forth in
SEQ ID NO: 3.
50. The method of claim 47, wherein the morpholino targets the
chloride ion channel ClC-1.
51. The method of claim 50, wherein the morpholino is an antisense
oligonucleotide (AON).
52. The method of claim 50, wherein the morpholino is set forth in
SEQ ID NO: 4 or SEQ ID NO: 5.
53. The method of claim 45, wherein the agent is a peptide nucleic
acid (PNA)-CAG.
54. The method of claim 53, wherein the PNA-CAG consists of 2, 3,
4, or 5 CAG repeats.
55. The method of claim 45, wherein the agent is an aminoglycosidic
antibiotic compound.
56. The method of claim 55, wherein the antibiotic compound is
neomycin or gentamicin.
57. A cell comprising the first protein, a second protein, and a
nucleic acid comprising the first recognition element adjacent to a
second recognition element, wherein the first protein binds the
first recognition element and the second protein binds the second
recognition element, wherein at least one of the first and second
proteins comprises a first half of a split fluorescent protein and
at least one of the first and second proteins comprises a second
half of the split fluorescent protein, wherein excitation of the
split fluorescent protein results in a fluorescent emission if the
first and second proteins are co-localized.
58. A kit comprising a polystyrene plate, polyCUG.sup.exp mRNA, a
capture oligodeoxynucleotide (ODN), and a muscleblind protein,
wherein the muscleblind protein is labeled.
59. The kit of claim 58, wherein the muscleblind protein is MBNL1,
MBNL2, or MBNL3.
60. The kit of claim 58, wherein the muscleblind protein is
fluorescently labeled.
61. A kit comprising a nitrocellulose filter plate, labeled
polyCUG.sup.exp mRNA, and a muscleblind protein.
62. The kit of claim 61, wherein the muscleblind protein is MBNL1,
MBNL2, or MBNL3.
63. The kit of claim 61, wherein the polyCUG.sup.exp mRNA is
fluorescently labeled.
64. An antisense oligonucleotide as set forth in SEQ ID NO: 3.
65. An antisense oligonucleotide as set forth in SEQ ID NO: 4.
66. An antisense oligonucleotide as set forth in SEQ ID NO: 5.
67. The antisense oligonucleotide of claim 64, wherein the
antisense oligonucleotide has a peptide nucleic acid backbone.
68. A method of treating myotonic dystrophy in a subject in need
thereof comprising administering to the subject the peptide nucleic
acid of claims 67.
69. The antisense oligonucleotide of claims claim 64, wherein the
antisense oligonucleotide is a morpholino.
70. A method of treating myotonic dystrophy in a subject in need
thereof comprising administering to the subject the morpholino of
claims 69.
71. A method of screening for an agent that inhibits the
interaction of a protein and a ligand comprising the steps of a)
contacting an agent with the protein or labeled ligand; b) admixing
the protein with labeled ligand; c) determining the level of the
label bound to protein; and d) comparing the amount of the label
relative to a control; wherein a decrease in the level of the label
bound to protein relative to a control indicates an agent that
inhibits the interaction.
72. A method of screening for an agent that inhibits the
interaction of a protein and a ligand comprising the steps of a)
contacting an agent with the labeled protein or ligand; b) admixing
the protein with ligand; c) determining the level of the label; and
d) comparing the amount of the label relative to a control; wherein
a decrease in the level of the label bound to protein indicates an
agent that inhibits the interaction.
73. A method of screening for an agent that inhibits the
interaction of a protein and ligand comprising the steps of a)
admixing the protein and labeled ligand; b) contacting an agent
with the protein and labeled ligand; and c) comparing the amount of
ligand bound to protein relative to a control; wherein a decrease
in the level of the label bound to protein indicates an agent that
inhibits the interaction.
74. A method of screening for an agent that inhibits the
interaction of a protein and a ligand comprising the steps of a)
contacting the protein or labeled ligand with an agent; b) admixing
the protein and labeled ligand in the presence of agent; and c)
comparing the amount of ligand bound to protein relative to a
control; wherein a decrease in the level of the label bound to
protein indicates an agent that inhibits the interaction.
Description
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/826,396, filed on Sep. 21, 2006 which is
incorporated herein in its entirety.
I. BACKGROUND
[0003] Myotonic Dystrophy type 1 (DM1) is autosomal dominant and
characterized by progressive weakness, muscle wasting, myotonia,
and multisystem impairment (abnormal cardiac conduction,
neuropsychiatric impairment, cataracts). Disability occurs at an
early stage due to preferential involvement of hand muscles by
myotonia and weakness. Death from respiratory failure, aspiration,
or cardiac arrhythmia occurs at a median age of 55, usually after
several decades of severe disability. Presently there is no
treatment other than supportive care. DM1 is caused by expansion of
a CTG repeat in the 3' untranslated of DMPK, the gene encoding
dystrophia myotonica protein kinase. Individuals with small CTG
repeat expansions of 50-100 repeats generally have mild, late-onset
symptoms, whereas large expansions of a thousand or more repeats
are associated with severe disease in infancy. Associations between
repeat length and disease severity have been made with DNA isolated
from circulating blood cells. However, it was found that in many
tissues, including skeletal muscle and brain, somatic instability
of the expanded CTG repeat leads to much larger expansions, of
1,000 to 5,000 repeats, even in individuals with relatively short
expansions in circulating blood cells (Thornton C A, et al. Ann
Neurol 1994; 35:104-107). Myotonic dystrophy type 2 (DM2) is
similar to DM1, but less common and less severe. DM2 is caused by
expansion of a CCTG repeat in intron 1 of the ZNF9 gene, encoding a
nucleic acid binding protein.
II. SUMMARY
[0004] Disclosed are screening methods and compositions related to
Myotonic Dystrophy (DM) type 1 (DM1) and type 2 (DM2). In
particular, disclosed herein are methods of screening for compounds
effective in treating DM. Also disclosed are compositions capable
of treating DM.
[0005] Thus, in one aspect, disclosed herein are methods of using
antisense oligonucleotides as protein displacement therapy in
myotonic dystrophy.
[0006] Also disclosed are methods of high throughput screening to
find other compounds that can inhibit the interaction of CUG repeat
RNA with MBNL1 protein.
[0007] Also disclosed are methods of using antisense
oligonucleotides to cause exon skipping and correct the splicing
defects in myotonic dystrophy.
III. BRIEF DESCRIPTION OF THE DRAWINGS
[0008] The accompanying drawings, which are incorporated in and
constitute a part of this specification, illustrate several
embodiments and together with the description illustrate the
disclosed compositions and methods.
[0009] FIG. 1. (A) RT-PCR assay for SERCA1 exon 22 alternative
splicing in mouse muscle at postnatal day 2 (P2), P10, P20, and 6
months (Ad) shows postnatal transition to exon 22 inclusion. The
postnatal transition is absent in HSA.sup.LR transgenic and MBNL1
knockout mice. (B) Inclusion of exon 22 of SERCA1 leads to a
termination codon exon 22. Skipping of exon 22 leads to a
termination codon in exon 23. Note that the Ex22+ transcript is not
subject to nonsense mediated decay because the stop codon is within
55 nt from the final exon junction. (C) pSERF minigene spliceopathy
reporter construct. Human SERCA1 exon 22 and its flanking introns
have been included intact. PA, polyadenylation signal. (D)
Electroporation of pSERF in muscle in vivo shows increased exon 22
inclusion in WT compared to HSA.sup.LR transgenic mice (RT-PCR
splicing assay 4 days after electroporation).
[0010] FIG. 2. Synthesis, purification and fluorescent labeling of
MBNL1 protein and poly(CUG) transcript. (A) Coomassie-stained
SDS-gels of following samples: crude protein lysate of bacteria
expressing GST-MBNL1-41-del105 (truncated at C-terminal to remove
hydrophobic domain); supernatant and pellet after 14,000 g
centrifugation of crude protein; flow through after Ni-column
chromatography; four samples eluted from Ni-column with different
concentration of imidazole or EDTA. Next, eluate III was purified
on Glutathione Sepharose column (second gel). Fraction I and II are
washes with loading buffer; fractions are eluates with 10 mM
glutathione. Purity of protein after purification is >90%. (B)
Product of fluorescent labeling of both MBNL1-41-del105 with
fluorescein (left), and poly(CUG).sup.109 labeling at the 3' end by
incorporation of TAMRA conjugated to ATP. Note, that relative small
amounts of such protein and RNA, as low as 62.5 fmol, are detected
both in acrylamide gel (upper FluorImager scan) and microplate
(lower scan).
[0011] FIG. 3. Interaction of recombinant MBNL1 with
poly(CUG).sup.109. (A) In nitrocellulose filter binding assay, a
constant concentration of fluorescently-labeled poly(CUG).sup.109
[0.1 nM] is mixed with decreasing concentration (from 100 to 0.012
nM) of MBNL1-41 protein fused or not with GST. Unbound RNA washes
through filter, protein-bound RNA is retained. Note that protein
concentrations listed to the right correspond to two rows of wells
on the filter. (B) Comparison of saturation curve for full length
and C-terminus truncated GST-MBNL1-41. Note that neither presence
of GST-fusion partner nor deletion of 105 aa from C-terminus of
MBNL1 have significant influence on poly(CUG).sup.109 binding. (C)
Diagram showing experimental design for poly(CUG).sup.109
attachment assay. The capture oligodeoxynucleotide (ODN) is labeled
at the 5' end with biotin via a 12-carbon linker. Interaction of
biotin with streptavidin followed by hybridization of capture ODN
to the 3' end of poly(CUG)109 tethers the transcript to plates.
Poly(CUG)109-MBNL1 interaction is performed with excess of
fluorescently-labeled MBNL1 protein.
[0012] FIG. 4. Optimization of conditions for monitoring of
poly(CUG)109-MBNL1 interaction in microplate format. In upper
panel, gel mobility shift assay demonstrates interaction of
fluorescently-labeled poly(CUG)109 (0.5 pmole) with increasing
concentration of full-length, GST-cleaved recombinant MBNL1. Note
that increasing MBNL1 concentration influences both percentage of
poly(CUG)109 in complex with protein and the molecular weight of
complex (increasing number of MBNL1 molecules bound per
transcript). Lower panel shows the interaction in microplate
format, with fluorescence-labeled MBNL1 binding to unlabeled
poly(CUG)109 tethered to plate.
[0013] FIG. 5. Transfection with MBNL1, but not other RNA binding
proteins, drives splicing of TNNT3 from the DM1/fetal pattern to
the mature muscle (fetal exon exclusion, 100 bp) pattern.
[0014] FIG. 6. The transcription unit for MBNL1 is flanked by
insulators from the chicken .beta.-globin locus. These elements
have been shown to insulate transgene expression from adjacent
chromatin context, improving the uniformity of transgene expression
(Chung J H, et al. Proc Natl Acad Sci USA 1997; 94(2):575-580). The
attB element directs integration by phiC31 integrase. CBA, chicken
.beta. actin. Due to the presence of several monospecific
antibodies to Mbnl1, there was no need for a reporter gene or
epitope tag to monitor expression levels.
[0015] FIG. 7. pLLC7. CMV/C.beta.M, CMV enhancer coupled to chicken
beta actin promoter; triple stop, concatamer of three SV40
polyadenylation signals and transcription terminator elements
(Novak A, et al. Genesis 2000; 28(3-4):147-155); GFP/NEO, Bizyme
neomycin resistance-GFP fluorescence selection cassette (Hansen S
G, et al. Biotechniques 2002; 32(5):1178, 1180, 1182-1178); AttB,
integration signal for phiC31 integrase.
[0016] FIG. 8. Trimolecular fluorescence complementation assay for
compounds that inhibit MBNL1 binding to poly(CUG)exp. In the 3' UTR
of luc mRNA, poly(CUG)exp is interspersed with MS2 coat protein RNA
recognition elements (MS2REs). MS2 coat protein (MS2CP) and MBNL1
are expressed as fusions with the N and C terminal halves of split
Venus fluorescent protein (Nagai T, et al. Nat Biotechnol 2002;
20(1):87-90) (VFP), respectively. Assembly of MS2CP.cndot.VFPN and
MBNL1.cndot.VFPC on the chimeric transcript leads to VFP
fluorescence activity (Rackham O, Brown C M. EMBO J. 2004;
23(16):3346-3355). Inhibition of MBNL1-poly(CUG)exp interactions
causes loss of VFP fluorescence.
[0017] FIG. 9 shows a diagram of the transgene in HSALR transgenic
mice. To produce a transgenic mouse model of myotonic dystrophy, an
expanded CTG repeat was inserted downstream from the stop codon in
a DNA fragment containing the entire human skeletal actin gene.
This fragment was used to derive HSALR transgenic mice. It was
found that these transgenic mice express high levels of CUG
expansion RNA in skeletal muscle. They also develop myotonia, a
cardinal symptom of myotonic dystrophy, and histologic changes in
skeletal muscle that resemble myotonic dystrophy.
[0018] FIG. 10 shows that MBNL1 protein is sequestered in
ribonuclear foci of CUG repeat RNA in HSA.sup.LR transgenic mice.
This high power view of a section of skeletal muscle shows a single
nucleus at postnatal day 2. In the left panel, ribonuclear foci of
CUG expansion RNA are shown by fluorescence in situ hybridization.
In the center panel, the distribution of MBNL1 protein in the
nucleus is shown by immunofluorescence. The merged image on the
right shows that MBNL1 is sequestered in the ribonuclear foci of
CUG expansion RNA.
[0019] FIG. 11 shows that MBNL1 is required for normal
developmental regulation of alternative splicing for SERCA1 and
ZASP. The left panel shows reverse transcriptase-PCR (RT-PCR)
analysis of alternative splicing for SERCA1, the calcium reuptake
pump of the sarcoplasmic reticulum, and ZASP, a structural
component of the Z disc. In wild-type mice, alternative splicing of
SERCA1 exon 22 is developmentally regulated. At postnatal day 2
(P2), exon 22 is mainly skipped. By postnatal day 20 (P20), and
continuing in adults (Ad), exon 22 is mainly included. However,
this transition of alternative splicing fails to occur in mice
deficient for MBNL1 (Mbnl1.sup..DELTA.E3/.DELTA.E3). A similar
pattern of failure is seen for HSA.sup.LR transgenic mice. Exon 11
of ZASP shows an alternative splicing transition during the same
interval of postnatal development. The transition from exon 11
inclusion to exon 11 exclusion fails to occur in MBNL1 deficient or
HSA.sup.LR transgenic mice.
[0020] FIG. 12 shows a morpholino composed of CAG repeats displaced
MBNL1 protein from ribonuclear foci in HSA.sup.LR transgenic mice.
The morpholino was compromised entirely of CAG repeats and was 25
nucleotides in length (CAG25). CAG25 morpholino dissolved in
phosphate buffered saline was injected into tibialis anterior
muscle of HSA.sup.LR transgenic mice, followed by electroporation
to facilitate entry into muscle cells. The contralateral tibialis
anterior muscle was injected with saline alone, followed by
electroporation. The distribution of MBNL1 in tibialis anterior was
determined by immunofluorescence with anti-MBNL1 antibody A2764. In
muscle injected with saline, MBNL1 was sequestered in ribonuclear
foci (nuclei are counterstained with DAPI). In muscle injected with
CAG25 morpholino, MBNL1 was became more widely distributed
throughout the nucleus.
[0021] FIG. 13 shows the release of MBNL1 from ribonuclear foci
following treatment with CAG25 morpholino restored proper
regulation of alternative splicing for SERCA1 (3 weeks following
CAG25 injection). In 4 different HSA.sup.LR transgenic mice,
injection of CAG25 morpholino into tibialis anterior improved the
defect of SERCA1 alternative splicing. Morpholino-treated muscle is
indicated by "+". Results from tibialis anterior in the opposite
hindlimb, injected with saline alone, are indicated by "-". The
side of morpholino injection was randomly determined, and this
assignment remained blinded until after the splicing analysis was
completed. Het. dupl. indicates a heteroduplex PCR product.
[0022] FIG. 14 shows the release of MBNL1 from ribonuclear foci
following treatment with CAG25 morpholino improved regulation of
alternative splicing for ZASP (n=4 different HSA.sup.LR transgenic
mice, 3 weeks following CAG25 injection). Morpholino-treated muscle
is indicated by "+". Results from tibialis anterior in the opposite
hindlimb, injected with saline alone, are indicated by "-". The
side of morpholino injection was randomly determined, and this
assignment remained blinded until after the splicing analysis was
completed.
[0023] FIG. 15 shows that CAG25 morpholino treatment in HSA.sup.LR
transgenic mice improved the alternative splicing of ClC-1, the
muscle specific chloride ion channel. Exon 7a of ClC-1 shows
developmentally regulated alternative splicing, and it was
previously shown that MBNL1 is required for its normal regulation
(Kanadia et al, Science, 302:1978-1980, 2003). Adult wild-type (WT)
mice show low levels of exon 7a inclusion. In HSA.sup.LR transgenic
mice, the fraction of ClC-1 splice products that include exon 7a is
increased. Inclusion of exon 7a causes a frame shift and creates a
premature termination codon that truncates most of the ClC-1 coding
sequence. Of note, transcripts that include exon 7a have
accelerated degradation via nonsense mediated decay, therefore they
are underrepresented at steady state and on this gel. The higher
bands on the gel are other alternative splice products, as shown
(Mankodi et al, Molecular Cell 10:35-44, 2002.) In HSA.sup.LR
transgenic mice, injection of CAG morpholino into tibialis anterior
improved the defect of ClC-1 alternative splicing.
Morpholino-treated muscle is indicated by "+". Results from
tibialis anterior in the opposite hindlimb, injected with saline
alone, are indicated by "-".
[0024] FIG. 16 shows the expression of ClC-1 chloride channel at
the surface membrane is increased 3 weeks following injection of
CAG25 morpholino. Immunofluorescence for ClC-1 chloride channel is
shown in sections of tibialis anterior muscle from HSA.sup.LR
transgenic mice. Muscle fibers show mosaic expression of ClC-1 in
HSA.sup.LR muscle injected with saline. Some of these fibers are
completely lacking in ClC-1 protein. Treatment with CAG25
morpholino leads to increased expression ClC-1 protein at the
surface membrane of muscle fibers.
[0025] FIG. 17 uses Electromyography to show the improvement of
myotonia at 3 and 6 weeks after injection of CAG25 morpholino into
tibialis anterior of HSA.sup.LR transgenic mice. Myotonia is a
state of muscle hyperexcitability in which muscle fibers display
repetitive action potentials. Myotonia in the HSA.sup.LR transgenic
mouse model of myotonic dystrophy is caused by abnormal regulation
of alternative splicing for ClC-1 and subsequent reduction of
chloride ion channels in muscle fibers. A parallel abnormality of
ClC-1 alternative splicing exists in human myotonic dystrophy. In
this experiment, the vertical axis shows mean myotonia severity
score among 4 HSA.sup.LR transgenic mice at each timepoint. For
each mouse, CAG25 morpholino dissolved in saline was injected into
tibialis anterior muscle of one hindlimb, whereas saline alone was
injected into tibialis anterior on the opposite side. The side of
morpholino injection was randomly assigned. Electromyography was
performed by a blinded examiner. Severity of myotonia was graded on
a 4 point scale: 0=no myotonic, 1=occasional myotonic discharge
(fewer than 25% of needle insertions), 2=abundant myotonic
discharges (25-75% of needle insertions), and 3=florid myotonia
(myotonic discharges in nearly every needle insertion). Protein
displacement therapy with the CAG morpholino resulted in
significant reduction of myotonia at 3 and 6 weeks following
injection (p<0.001 both time points.)
[0026] FIG. 18 shows that filter binding screening assay identifies
compounds in the aminoglycoside family as having ability to inhibit
interaction of recombinant MBNL1 protein with (CUG).sub.109 RNA.
Among 9 aminoglycoside compounds tested, neomycin (left curve) and
gentamicin (right curve) showed the highest activity to inhibit
formation of MBNL1-poly(CUG).sup.exp RNA-protein complexes. The
order of addition was compound+(CUG).sub.109 RNA (incubate 5
minutes), followed by recombinant MBNL1 protein (15 minute
incubation), followed by application to filter.
[0027] FIG. 19 shows that CAG25 morpholino inhibits the interaction
of MBNL1 poly(CUG).sup.exp RNA in vitro. Interaction of recombinant
MBNL1 protein with (CUG).sub.109 RNA was examined by gel shift
assay, in the presence of increasing amounts of CAG25
morpholino.
[0028] FIG. 20 shows a screening assay for compounds that improve
spliceopathy could use readouts other than protein fluorescence.
SERCA1 exon 22 and flanking introns are used to generate a
spliceopathy reporter construct using luciferase. Point mutations
have been induced in SERCA1 exon 22 so that it no longer encodes a
termination codon. When this spliceopathy reporter construct is
transiently transfected in COS cells, the luciferase activity is
sensitive indicator of MBNL1 activity, as indicated by the
>10-fold upreglation of luciferase when cotransfected with small
amounts of expression construct for GFP-tagged MBNL1.
[0029] FIG. 21 shows the effects of antisense morpholino targeting
the 3' splice junction of ClC-1 exon 7a on splicing and myotonia in
HSA.sup.LR transgenic mice. Antisense morpholino targeting the 3'
splice junction of ClC-1 exon 7a was injected into tibialis
anterior muscle of HSA.sup.LR mice under general anesthesia
(sequence 5'-CCAGGCACGGTCTGCAACAGAGAAG-3' (SEQ ID NO: 4)). The
contralateral muscle was injected with morpholino having the
inverted sequence (5'-GAAGAGACAACGTCTGGCACGGACC-3'(SEQ ID NO: 5)).
Uptake into muscle fibers was enhanced by in vivo electroporation.
The determination of which side received the antisense morpholino
was randomized. 21 days later, myotonia was evaluated by
electromyography and muscle was harvested for RT-PCR analysis of
ClC-1 alternative splicing. Electromyography was blinded to the
randomization. A. RT-PCR analysis of alternative splicing shows
that exon 7a inclusion products (bands 2 and 4, see splicing
diagram in B) are decreased in muscle treated with antisense
morpholino but not by the inverted morpholino. Concurrently, the
antisense morpholino increases the fraction of splice products
encoding functional ClC-1 channels (band 1). Quantification in
graph C confirms that antisense morpholino caused a significant
reduction of exon 7a inclusion (p<0.0001). Treatment with the
antisense morpholino caused a marked reduction of myotonia in the
antisense-treated muscle (p<0.00001) (D).
[0030] FIG. 22 shows whole-cell voltage clamp from single muscle
fibers shows that treatment with antisense morpholino targeting
ClC-1 exon 7a restores normal chloride current density in
HSA.sup.LR transgenic mice. Antisense morpholino targeting the 3'
splice junction of ClC-1 exon 7a was injected into foot pad muscle
of HSA.sup.LR mice under general anesthesia. Uptake into muscle
fibers was enhanced by in vivo electroporation. The morpholino was
tagged with fluorescein. 4 days later, individual FDB muscle fibers
were isolated. Greater than 90% of fibers showed fluorescein
uptake, and only these fibers were studied. As a control, the
opposite footpad was injected with morpholino having the inverted
sequence. A. The upper panel shows ClC-1 currents at different
membrane potentials. The peak current density in HSA.sup.LR mice
(center panel) is much lower than in wild-type mice (left panel).
However, after morpholino treatment in HSA.sup.LR mice (right
panel), the current density is restored to levels that are similar
to wild-type mice. B. To quantify this effect, the graph in the
lower panel shows chloride current density in relation to membrane
potential. In fibers treated with inverted morpholino, or in
untreated HSA.sup.LR fibers, the current density is markedly
reduced (closed circles). The antisense morpholino (closed squares)
restores chloride current density to normal levels (open circles or
triangles).
[0031] FIG. 23 shows the design of antisense morpholinos. FIG. 23A
shows the inclusion of ClC-1 exon 7a induces a frame shift and
premature termination codon in exon 7. Annealing of antisense
morpholino to the 3' splice site of exon 7a in the ClC-1 pre-mRNA
is intended to prevent spliceosomal recognition of this exon. FIG.
23B shows the alignment of ClC-1 pre-mRNA (top strand) with
antisense morpholinos targeting the 3' or 5' splice sites of exon
7a is shown. The control morpholino is the 5'-3' invert of the 3'
splice site blocker. Exonic sequences are in upper case, intronic
sequences are in lower case.
[0032] FIG. 24 shows that antisense morpholino localizes
preferentially to muscle nuclei and restores ClC-1 expression at
the sarcolemma. FIGS. 24A-C show a cross-section of HSA.sup.LR
tibialis anterior (TA) muscle showing distribution of
3'-carboxyfluorescein-labeled antisense morpholino 3 weeks after
injection. The morpholino was complementary to the 3' splice site
of ClC-1 pre-mRNA. Muscle fibers are outlined by wheat germ
agglutinin (wga) and nuclei are highlighted by DAPI. FIGS. 24D and
24 E show brightfield (D) and fluorescence (E) images of a single
FDB fiber showing preferential nuclear localization of the
antisense morpholino (post injection day 5). FIGS. 24F and 24G show
that as compared to invert-treated control (F), immunofluorescence
shows an increase of sarcolemmal ClC-1 protein in HSA.sup.LR TA
muscle 3 weeks after treatment with antisense morpholino (G).
Bars=20 .mu.M.
[0033] FIG. 25 shows that antisense morpholino represses splicing
of ClC-1 exon 7a. FIG. 25A reveals that RT-PCR showed reduction of
exon 7a inclusion three weeks after injection of antisense (anti)
morpholino (antisense 1+antisense 2, 5 .mu.g each) into TA muscle
of HSA.sup.LR mice. Pairs of injected TA muscles from each mouse
are identified by "1, 2, 3." Muscle injected with control
morpholino (inv) (10 .mu.g) was not different from untreated
HSA.sup.LR muscle. HSA.sup.LR and WT mice have the same (FVB)
inbred strain background. FIG. 25B shows the inclusion of exon 7a
remained partially suppressed 8 weeks after injection of antisense
morpholino (20 .mu.g antisense 1 vs. 20 .mu.g invert control). FIG.
25C shows that ClC-1 antisense morpholino did not correct the
misregulated alternative splicing of Titin m-line exon 5. FIGS. 25D
and 25E show the percentage of ClC-1 splice products that include
exon 7a is shown at 3 (D) and 8 (E) weeks following morpholino
injection. Mean.+-.s.d.; n=3 per group; **P<0.001; *P=0.035
antisense-versus invert-treated controls; t-test. FIG. 25F shows
that the level of ClC-1 mRNA is increased 3 weeks after treatment
with antisense moropholino. ClC-1 mRNA level is expressed in
arbitrary units relative to housekeeping gene RNA polymerase II
transcription factor IIB. Mean.+-.s.d.; n=3 per group; *P=0.06 for
antisense vs invert-treated control; t-test.
[0034] FIG. 26 shows that antisense morpholino rescues ClC-1
channel function and reverses myotonia in skeletal muscle of
HSA.sup.LR mice. FIG. 26A shows that representative ClC-1 currents
obtained from flexor digitorum brevis (FDB) fibers isolated from
HSA.sup.LR mice electroporated with either invert (left) or
antisense (middle) morpholino and WT mice electroporated with
antisense morpholino (right). The dashed lines represent the zero
current level. Capacitative currents recorded from each fiber are
shown in the insert of each panel (scale bars: vertical, 3 nA;
horizontal, 4 ms). Superimposed traces (solid lines) of normalized
ClC-1 current deactivation at -100 mV in FDB fibers obtained from
invert-(circles) and antisense-treated (squares) HSA.sup.LR mice
and antisense-treated WT mice (triangles) fit with a second order
exponential (symbols) are shown in the insert to the left hand
panel. Note that accelerated ClC-1 deactivation kinetics of FDB
fibers obtained from HSA.sup.LR mice is normalized only following
treatment with antisense morpholino. FIG. 26B shows the voltage
dependence of average instantaneous ClC-1 current density recorded
from FDB fibers of 16-18 day old WT mice treated with invert
morpholino (open circles; n=11), WT mice treated with antisense
morpholino (open triangles; n=10), HSA.sup.LR mice treated with
invert morpholino (filled circles; n=12), and HSA.sup.LR mice
treated with antisense morpholino (filled squares, n=16). FIG. 26C
shows the average relative Po-V curves for the same experiments
shown in (B). Smooth curves through each dataset were generated
using a modified Boltzmann equation (Lueck, J. D., et al. (2007) J
Gen Physiol 129:79-94). FIG. 26D shows the average relative
contribution of the fast (A.sub.f/A.sub.total), slow
(A.sub.s/A.sub.total), and non-deactivating (C/A.sub.total)
components of ClC-1 current deactivation elicited from a voltage
step to -100 mV for the same experiments shown in (B).
Mean.+-.s.e.m.; *P<0.05 invert-treated HSA.sup.LR fibers
compared to each of the other experimental conditions; t-test.
FIGS. 26E and 26F shows that myotonia was significantly reduced 3
(E) and 8 (F) weeks following injection of antisense morpholino.
Mean.+-.s.d.; n=3 to 7 per group. Antisense morpholino was injected
into one TA, invert morpholino was injected into the contralateral
TA, and gastrocnemius muscle served as an untreated control.
**P<0.0001 for antisense-vs invert-treated control; ANOVA.
[0035] FIG. 27 shows antisense morpholino represses exon 7a
inclusion, restores ClC-1 protein expression, and rescues myotonia
in Mbnl1.sup..DELTA.E3/.DELTA.E3 mice. FIG. 27A indicates that
RT-PCR shows reduced inclusion of exon 7a at 3 weeks after
injection of antisense morpholino 1 (20 .mu.g antisense or invert
control). FIG. 27B shows Quantitation of splicing results shown in
(A) as mean.+-.s.d.; (n=3 per group); **P<0.001 antisense-versus
invert-treated control; t-test. FIGS. 27C and 27D shows that
immunofluorescence for ClC-1 is increased 3 weeks after injection
with antisense (D) as compared to invert-treated control (C).
Bar=20 .mu.M. FIG. 27E shows Myotonia in
Mbnl1.sup..DELTA.E3/.DELTA.E3 TA muscle is reduced 3 weeks after
treatment with antisense morpholino but not in muscle treated with
invert control. Mean.+-.s.d.; n=3 per group; **P<0.0001
antisense-versus invert-treated control; ANOVA.
[0036] FIG. 28 shows a Comparison of antisense morpholinos
targeting ClC-1 exon 7a. Antisense oligo was injected into tibialis
anterior (TA) muscle of HSALR mice and invert oligo (inv) (20
.mu.g) was injected into the contralateral TA. Tissue was obtained
3 weeks later for analysis of ClC-1 splicing by RT-PCR. Antisense
morpholino targeting the 3' splice site (antisense 1; 20 .mu.g)
induced a higher level of exon 7a skipping than antisense
morpholino directed against the 5' splice site (antisense 2; 20
.mu.g) Effects of antisense 1 alone (20 .mu.g) were similar to
co-injection of antisense 1 and 2 (10 .mu.g each) (n=3 each group;
2 from each group are shown).
[0037] FIG. 29 shows that antisense morpholino had no effect on the
formation of ribonuclear inclusions. Fluorescence in situ
hybridization and immunofluorescence demonstrate co-localization of
CUGexp RNA and MBNL1 protein in muscle nuclei (blue) 3 weeks after
injection of HSALR TA muscle with invert (a-c) and antisense (d-f)
morpholino.
[0038] FIG. 30 shows protein displacement therapy with peptide
nucleic acid (PNA) oligomers composed of CAG repeats. FIG. 30A
shows that PNA-CAG repeat oligos of lengths ranging from 2 to 5 CAG
repeats can invade (CUG).sub.109 hairpins and effectively interact
with expanded CUG repeat hairpin structures in vitro. FIG. 30B
shows that these PNA-CAG oligos can also inhibit the interaction of
(CUG).sub.109 RNA with MBNL1 protein in vitro.
[0039] FIG. 31 shows screening for compounds that inhibit
interaction of MBNL1 protein and CUG expansion RNA: fluorescence
anisotropy assay shows interaction of CUG expansion RNA with
recombinant MBNL1 protein in vitro. Fluorescein-labeled
(CUG).sub.36 RNA (2 nM) was incubated with MBNL1 protein (100 nM)
and anisotropy was measured at time points ranging from 1 to 90
minutes. Increasing values for fluorescence anisotropy indicate
interaction of fluorescein-labeled (CUG).sub.36 transcript with
MBNL1 protein. Values are averages from 4 experiments and error
bars shows SD.
[0040] FIG. 32 shows a fluorescence anisotropy assay to screen for
compounds that inhibit interaction of CUG repeat RNA with
recombinant MBNL1 protein. Fluorescein-labeled (CUG).sub.36
transcript (2 nM) was incubated first with aminoglycoside compound
(10 or 50 .mu.M) and then with excess amount of recombinant MBNL1
protein (100 nM). To calculate the fraction of CUG repeat RNA that
remains bound to MBNL1 protein ("% bound CUG.sup.exp", vertical
axis), results are expressed as the percentage of maximal
fluorescence anisotropy in assays from which aminoglycosides were
omitted. Among the compounds tested, neomycin showed the strongest
inhibition of MBNL1 binding to CUG repeat RNA. Values are the
average+/-SD from three measurements.
[0041] FIG. 33 shows a diagram of enzymatic complementation assay
to screen for compounds that inhibit interaction of CUG repeat RNA
with recombinant MBNL1 protein. (CUG)109 transcripts are tethered
to the surface of a streptavidin-coated microtiter plate using a
capture oligonucleotide that is biotinylated. The capture oligo
anneals to complementary sequence at the 3' end of the CUG repeat
RNA. Recombinant human MBNL1 is expressed as a fusion with the PL
fragment of beta-galactosidase. PL is a 55 amino acid fragment of
beta-galactosidase. Preliminary experiments determined that fusion
of MBNL1 with the PL fragment did not inhibit the binding of MBNL1
protein to CUG repeat RNA. After incubation with test compound,
unbound MBNL1-PL is washed away (panel B). Next, the complementing
fragment of beta-galactosidase is added to determine the amount of
MBNL1-PL that continues to interact with (CUG)109 RNA and thereby
is retained on the microtiter plate. The binding of complementing
fragment of beta-galactosidase to PL reconstitutes its enzymatic
activity. This activity is then determined by adding substrate to
provide a fluorescence or chemiluminescence signal from active
beta-galactosidase.
[0042] FIG. 34 shows Enzymatic complementation assay to screen for
compounds that inhibit interaction of CUG repeat RNA with
recombinant MBNL1 protein. Operation of the beta-galactosidase
enzymatic complementation assay was demonstrated using two kinds of
inhibitors. On the left panel, excess soluble (CUG).sub.109 RNA was
added to the assay reaction. The soluble (CUG).sub.109 RNA binds to
MBNL1-PL protein and prevents its retention on the microtiter
plate, reflected by reduced beta-galactosidase activity (expressed
on the vertical axis in terms of relative luminescence activity).
On the right panel, compounds having the ability to intercalate
into CUG-repeat-RNA-hairpins (EtBr, ethidium bromide; or SybrGreen
stain) were added at the indicated concentrations. Both compounds
reduce the amount of MBNL1-PL retained on plate, reflected by
reduced beta-galactosidase activity.
[0043] FIG. 35 shows that injection of peptide nucleic acid (PNA)
comprised of CAG repeats caused reduction of electromyographic
myotonia in HSALR transgenic mouse model of myotonic dystrophy.
PNA-(CAG).sub.6mer or PNA-(CAG).sub.9mer (i.e., 2 or 3 CAG repeats)
was injected into tibialis anterior muscle on a single occasion.
Myotonia was assessed by electromyography 3 weeks following the
intramuscular injection. As control, vehicle alone (phosphate
buffered saline) was injected in the tibialis anterior muscle of
the contralateral limb. All mice had robust action myotonia prior
to treatment. Assignment as to which limb received PNA vs control
was randomized, and EMG analysis was performed blinded to this
assignment.
IV. DETAILED DESCRIPTION
[0044] Before the present compounds, compositions, articles,
devices, and/or methods are disclosed and described, it is to be
understood that they are not limited to specific synthetic methods
or specific recombinant biotechnology methods unless otherwise
specified, or to particular reagents unless otherwise specified, as
such may, of course, vary. It is also to be understood that the
terminology used herein is for the purpose of describing particular
embodiments only and is not intended to be limiting.
A. DEFINITIONS
[0045] As used in the specification and the appended claims, the
singular forms "a," "an" and "the" include plural referents unless
the context clearly dictates otherwise. Thus, for example,
reference to "a pharmaceutical carrier" includes mixtures of two or
more such carriers, and the like.
[0046] Ranges can be expressed herein as from "about" one
particular value, and/or to "about" another particular value. When
such a range is expressed, another embodiment includes from the one
particular value and/or to the other particular value. Similarly,
when values are expressed as approximations, by use of the
antecedent "about," it will be understood that the particular value
forms another embodiment. It will be further understood that the
endpoints of each of the ranges are significant both in relation to
the other endpoint, and independently of the other endpoint. It is
also understood that there are a number of values disclosed herein,
and that each value is also herein disclosed as "about" that
particular value in addition to the value itself. For example, if
the value "10" is disclosed, then "about 10" is also disclosed. It
is also understood that when a value is disclosed that "less than
or equal to" the value, "greater than or equal to the value" and
possible ranges between values are also disclosed, as appropriately
understood by the skilled artisan. For example, if the value "10"
is disclosed the "less than or equal to 10" as well as "greater
than or equal to 10" is also disclosed. It is also understood that
the throughout the application, data is provided in a number of
different formats, and that this data, represents endpoints and
starting points, and ranges for any combination of the data points.
For example, if a particular data point "10" and a particular data
point 15 are disclosed, it is understood that greater than, greater
than or equal to, less than, less than or equal to, and equal to 10
and 15 are considered disclosed as well as between 10 and 15.
[0047] In this specification and in the claims which follow,
reference will be made to a number of terms which shall be defined
to have the following meanings:
[0048] "Optional" or "optionally" means that the subsequently
described event or circumstance may or may not occur, and that the
description includes instances where said event or circumstance
occurs and instances where it does not.
[0049] "Primers" are a subset of probes which are capable of
supporting some type of enzymatic manipulation and which can
hybridize with a target nucleic acid such that the enzymatic
manipulation can occur. A primer can be made from any combination
of nucleotides or nucleotide derivatives or analogs available in
the art which do not interfere with the enzymatic manipulation.
[0050] "Probes" are molecules capable of interacting with a target
nucleic acid, typically in a sequence specific manner, for example
through hybridization. The hybridization of nucleic acids is well
understood in the art and discussed herein. Typically a probe can
be made from any combination of nucleotides or nucleotide
derivatives or analogs available in the art.
[0051] Throughout this application various reference is made to
polyCUG repeat RNA or poly(CUG).sup.exp RNA. It is understood and
herein contemplated that these terms can be used interchangeably
throughout the description and the claims. Likewise, reference is
made throughout the application to polyCCUG repeat RNA or
poly(CCUG).sup.exp. It is also understood and herein contemplated
that these terms can be used interchangeably throughout the
description and the claims.
[0052] Throughout this application, various publications are
referenced. The disclosures of these publications in their
entireties are hereby incorporated by reference into this
application in order to more fully describe the state of the art to
which this pertains. The references disclosed are also individually
and specifically incorporated by reference herein for the material
contained in them that is discussed in the sentence in which the
reference is relied upon.
[0053] Myotonic dystrophy type 1 (DM1), the most common form of
muscular dystrophy in adults (frequency .about.1 in 7,400), is a
genetic disease affecting muscle, heart, and brain. DM1 involves a
novel disease mechanism in which mRNA from the mutant gene ha a
direct toxic effect. The toxic gain-of-function of the mutant mRNA
is entirely independent of the protein it encodes.
[0054] DM1 is caused by expansion of a CTG repeat in the 3'
untranslated region of DMPK, the gene encoding DM protein
kinase((1)). A novel RNA-mediated disease process has recently been
elucidated in DM1. Transcripts from the mutant DMPK allele contain
an expanded CUG repeat, designated here as poly(CUG).sup.exp. The
repeat-bearing transcripts are not exported from the nucleus
(Taneja K L, et al. J Cell Biol 1995; 128(6):995-1002; Davis B M,
et al. Proc Natl Acad Sci USA 1997; 94(14):7388-7393). Instead,
they accumulate in nuclear foci. Here, proteins are recruited to
the foci, and some of the proteins bind to CUG.sup.exp RNA, for
example, proteins in the muscleblind (MBNL) family (Miller J W, et
al. EMBO J 2000; 19(17):4439-4448). One of the functions of MBNL is
to regulate alternative splicing of pre-mRNA. However,
sequestration of MBNL proteins in nuclear foci leads to abnormal
regulation of alternative splicing for a select group of pre-mRNAs
(Kanadia R N, et al. Science 2003; 302(5652):1978-1980; (6). This
is a result of the depletion of MBNL from other regions of the
nucleus. This regulatory defect is referred to herein as
"spliceopathy." Symptoms of DM1, such as, myotonia, are directly
attributable to the effect on splicing regulation (Mankodi A, et
al. Mol Cell 2002; 35-44) (ie., spliceopathy). Disclosed herein is
evidence that spliceopathy in DM1 muscle can be explained, to a
significant extent, by sequestration of a splicing factors in the
MBNL family, including muscleblind 1 (MBNL1), muscleblind 2
(MBNL2), and muscleblind 3 (MBNL3). Splicing factors in the MBNL
family are also sequestered on nuclear foci of polyCCUG expanded
repeats in DM2. For example, it is disclosed herein that (1) MBNL1
is markedly depleted from the nucleoplasm in DM1 muscle cells, as
it is recruited into ribonuclear foci; (2) expression of
poly(CUG).sup.exp and ablation of MBNL1 have equivalent effects on
splicing regulation in mouse skeletal muscle; (3) spliceopathy in
DM1 is remarkably similar to that observed in
poly(CUG).sup.exp-expressing or MBNL1 knockout mice (Lin X, et al.
Hum Mol Genet. 2006); and (4) spliceopathy in
poly(CUG).sup.exp-expressing mice is corrected by overexpression of
MBNL1. Therefore, poly(CUG).sup.exp and its interaction with MBNL1
are valid targets for therapy.
[0055] Among neurogenetic disorders, the possibility of developing
effective treatment through the use of high throughput screens for
therapeutic agents is especially attractive in the case of DM1. The
DM1 mutation does not lead to an absence of essential protein, nor
does it create a deleterious effect of mutant protein. Instead, the
fundamental problem is mislocalization of MBNL1 and a consequence
is the abnormal expression in adult tissue of splice isoforms that
are normally expressed in neonatal (immature) tissue (Lin X, et al.
Hum Mol Genet. 2006). The findings of ribonuclear foci in
presymptomatic DM1 patients (Mankodi A, et al. Hum Mol Genet. 2001;
10:2165-2170), and in phenotypically normal transgenic mice that
have low poly(CUG).sup.exp expression (Mankodi A, et al. Science
2000; 289(5485):1769-1773), indicate that subthreshold accumulation
of poly(CUG).sup.exo has no discernable effects on muscle function.
Thus, modest reduction of poly(CUG).sup.exp or partial release of
MBNL1 from ribonuclear foci translates into large therapeutic
effects. By focusing on pharmacotherapy, whole-body therapeutic
effects can be achieved as well as the prevention of disease
progression. However, based on the character of the disease
process, it also disclosed that reversal of phenotype can be
achieved. For example, in skeletal muscle, a tissue with great
intrinsic regenerative capacity, DM1 produces mainly fiber atrophy
with little fibrosis or necrosis, an eminently reversible lesion.
In addition, DM1 is a "composite" disease, in which distinct facets
of the phenotype can be parsed to effects of spliceopathy on
different transcripts, thereby impacting many different pathways.
In many cases, the spliceopathy can result in functional impairment
rather than irreversible cell degeneration. For example, myotonia
in DM1 is a functional defect that results from chloride
channelopathy (Mankodi A, et al. Mol Cell 2002; 35-44), and the
data indicate that it is reversible in a transgenic mouse model
either by AAV-mediated overexpression of MBNL1 or antisense
oligonucleotides that target the mis-spliced exon. By a similar
logic, the insulin resistance resulting from abnormal splicing of
insulin receptor is also likely to be reversible (Savkur R S, et
al. Nat Genet. 2001; 29(1):40-47). It is understood herein that by
correcting spliceopathy associated with DM1, the disease can be
treated. For example, by inhibiting the interaction of MBNL1 with
poly(CUG).sup.exp, MBNL1 is free to resume its effect on
alternatively sliced transcripts, or by inhibiting sequestration of
other CUG interacting protein, their normal functions can be
restored. One method of inhibiting the interaction of MBNL1 with
poly(CUG).sup.exp is by displacing bound MBNL1 with another
molecule.
B. METHODS OF SCREENING
[0056] Disclosed herein are methods of screening for an agent that
inhibits the interaction of a protein and a ligand comprising the
steps of a) capturing the ligand to a substrate; b) admixing a
labeled protein with the ligand; c) contacting an agent with the
mixture of step b; d) determining the level of the label; and e)
comparing the amount of the label relative to a control; wherein a
decrease in the level of the label indicates an agent that inhibits
the interaction. Thus for example, disclosed herein are methods
[0057] It is understood that the proteins of the method can be any
polyCUG or polyCCUG interacting protein or a protein that is
sequestered by polyCUG or polyCCUG repeats. Examples of the protein
of the method are members of the Muscleblind family of RNA binding
proteins which include MBNL1, MBNL2, and MBNL3. Mucleblind proteins
play a significant role in the regulation of alternative splicing.
During the DM1 disease process MBNL proteins are sequestered in the
ribonuclear foci through interaction with polyCUG.sup.exp or
polyCCUG.sup.exp RNA leading to a disregulation of alternative
splicing function
[0058] "Inhibit," "inhibiting," and "inhibition" mean to decrease
an activity, response, condition, disease, or other biological
parameter. This can include but is not limited to the complete
ablation of the activity, response, condition, or disease. This may
also include, for example, a 10% reduction in the activity,
response, condition, or disease as compared to the native or
control level. Thus, the reduction can be a 10, 20, 30, 40, 50, 60,
70, 80, 90, 100%, or any amount of reduction in between as compared
to native or control levels.
[0059] The methods disclosed herein refer to a ligand bound to a
substrate. It is understood that "ligand" can refer to any protein,
polypeptide, peptide, amino acid, or nucleotide chain (including,
for example, all forms of DNA and RNA) capable of being bound by a
protein. Thus, for example, disclosed herein are ligands wherein
the ligand is polyCUG or polyCCUG repeat RNA. Therefore, disclosed
herein are methods of screening for an agent that inhibits the
interaction of a protein and a ligand comprising the steps of a)
capturing the ligand to a substrate; b) admixing a labeled protein
with the ligand; c) contacting an agent with the mixture of step b;
d) determining the level of the label; and e) comparing the amount
of the label relative to a control; wherein a decrease in the level
of the label indicates an agent that inhibits the interaction; and
wherein the ligand is polyCUG mRNA or polyCCUG repeat RNA.
[0060] It is also understood that those of skill in the art will
recognize that the assay will not lose effectiveness by reversing
the order of the protein-ligand interaction. Thus, those of skill
in the art will recognize that a method comprising a bound protein
and labeled ligand will also be effective. Therefore, disclosed
herein are methods of screening for an agent that inhibits the
interaction of a protein and a ligand comprising the steps of a)
mixing a protein bound to a substrate with a labeled ligand; b)
contacting an agent with the mixture of step a; c) determining the
level of the label; and d) comparing the amount of the label
relative to a control; wherein a decrease in the level of the label
indicates an agent that inhibits the interaction. Similarly it is
understood that contacting the agent with the protein or ligand of
the method before mixing the protein and ligand will also be
effective. Thus, disclosed herein are A method in which agent is
contacted with ligand or protein prior to step a, in order to
prevent the interaction, will also be effective. Therefore
disclosed herein are methods of screening for an agent that
inhibits the interaction of a protein and a ligand comprising the
steps of a) contacting an agent with the protein or labeled ligand;
b) admixing the protein with labeled ligand; c) determining the
level of the label; and d) comparing the amount of the label
relative to a control; wherein a decrease in the level of the label
bound to protein indicates an agent that inhibits the
interaction.
[0061] Substrate refers to a solid support structure to which a
molecule can be bound. The substrate can include any solid
material. This includes materials such as acrylamide, agarose,
cellulose, nitrocellulose, glass, gold, polystyrene, polyethylene
vinyl acetate, polypropylene, polymethacrylate, polyethylene,
polyethylene oxide, polysilicates, polycarbonates, teflon,
fluorocarbons, nylon, silicon rubber, polyanhydrides, polyglycolic
acid, polylactic acid, polyorthoesters, functionalized silane,
polypropylfumerate, collagen, glycosaminoglycans, and polyamino
acids. Substrates can have any useful form including thin film,
membrane, bottles, dishes, fibers, gels, woven fibers, shaped
polymers, particles, beads, microparticles, or a combination.
Substrates can be porous or non-porous. A chip is a rectangular or
square small piece of material. Useful forms for substrates are
sheets, films, and chips. A useful form for a substrate is a
microtiter dish. Such dish can be, for example, a polystyrene dish
or a polystyrene dish with nitrocellulose bottoms.
[0062] It is contemplated that any method known to the art can be
used to bind the ligand or protein of the method to the substrate.
Such binding can occur directly, for example, by contacting the
protein to a substrate or indirectly through the use of GST or like
molecules. Additionally, binding to the substrate can occur via a
multiple binding reactions. For example, a substrate may be coated
with streptavidin to which a biotinylated ligand, protein, or
intermediary oligonucleotide may be bound. When a biotinylated
oligonucleotide is bound to streptavidin, a complementary ligand
can bind to the oligonucleotide. For example, an intermediary
oligonucleotide can comprise biotinylated oligodeoxynucleotide
(ODN). Alternatively, the ligand can be flanked by an RNA sequence
from the DMPK gene or other sequence that permits capture to a
substrate.
[0063] The disclosed methods can utilize any means of detecting a
labeled moiety known in the art. Herein, a "label" or a "detectable
moiety" is a composition detectable by spectroscopic,
photochemical, biochemical, immunochemical, or chemical means. For
example, useful labels include .sup.32P, fluorescent dyes,
electron-dense reagents, enzymes (e.g., as commonly used in an
ELISA or ELISPOT), biotin, digoxigenin, or haptens and proteins
which can be made detectable, e.g., by incorporating a radiolabel
into the peptide or used to detect antibodies specifically reactive
with the peptide. Thus, for example the method of detection can
comprise, for example, anisotropy.
[0064] As used herein, a label can include a fluorescent dye, a
member of a binding pair, such as biotin/streptavidin, a metal
(e.g., gold), or an epitope tag that can specifically interact with
a molecule that can be detected, such as by producing a colored
substrate or fluorescence. Substances suitable for detectably
labeling proteins include fluorescent dyes (also known herein as
fluorochromes and fluorophores) and enzymes that react with
colorometric substrates (e.g., horseradish peroxidase). The use of
fluorescent dyes is generally preferred in the practice of the
invention as they can be detected at very low amounts. Furthermore,
in the case where multiple antigens are reacted with a single
array, each antigen can be labeled with a distinct fluorescent
compound for simultaneous detection. Labeled spots on the array are
detected using a fluorimeter, the presence of a signal indicating
an antigen bound to a specific antibody.
[0065] Fluorophores are compounds or molecules that luminesce.
Typically fluorophores absorb electromagnetic energy at one
wavelength and emit electromagnetic energy at a second wavelength.
Examples of fluorophores include, but are not limited to, 1,5
IAEDANS; 1,8-ANS; 4-Methylumbelliferone;
5-carboxy-2,7-dichlorofluorescein; 5-Carboxyfluorescein (5-FAM);
5-Carboxynapthofluorescein; 5-Carboxytetramethylrhodamine
(5-TAMRA); 5-Hydroxy Tryptamine (5-HAT); 5-ROX
(carboxy-X-rhodamine); 6-Carboxyrhodamine 6G; 6-CR 6G; 6-JOE;
7-Amino-4-methylcoumarin; 7-Aminoactinomycin D (7-AAD);
7-Hydroxy-4-I methylcoumarin; 9-Amino-6-chloro-2-methoxyacridine
(ACMA); ABQ; Acid Fuchsin; Acridine Orange; Acridine Red; Acridine
Yellow; Acriflavin; Acriflavin Feulgen SITSA; Aequorin
(Photoprotein); AFPs--AutoFluorescent Protein--(Quantum
Biotechnologies) see sgGFP, sgBFP; Alexa Fluor 350.TM.; Alexa Fluor
430.TM.; Alexa Fluor 488.TM.; Alexa Fluor 532.TM.; Alexa Fluor
546.TM.; Alexa Fluor 568.TM.; Alexa Fluor 594.TM.; Alexa Fluor
633.TM.; Alexa Fluor 647.TM.; Alexa Fluor 660.TM.; Alexa Fluor
680.TM.; Alizarin Complexon; Alizarin Red; Allophycocyanin (APC);
AMC, AMCA-S; Aminomethylcoumarin (AMCA); AMCA-X; Aminoactinomycin
D; Aminocoumarin; Anilin Blue; Anthrocyl stearate; APC-Cy7;
APTRA-BTC; APTS; Astrazon Brilliant Red 4G; Astrazon Orange R;
Astrazon Red 6B; Astrazon Yellow 7 GLL; Atabrine;
ATTO-TAG.TM.CBQCA; ATTO-TAG.TM.FQ; Auramine; Aurophosphine G;
Aurophosphine; BAO 9 (Bisaminophenyloxadiazole); BCECF (high pH);
BCECF (low pH); Berberine Sulphate; Beta Lactamase; BFP blue
shifted GFP (Y66H); Blue Fluorescent Protein; BFP/GFP FRET; Bimane;
Bisbenzemide; Bisbenzimide (Hoechst); bis-BTC; Blancophor FFG;
Blancophor SV; BOBO.TM.-1; BOBO.TM.-3; Bodipy492/515;
Bodipy493/503; Bodipy500/510; Bodipy; 505/515; Bodipy 530/550;
Bodipy 542/563; Bodipy 558/568; Bodipy 564/570; Bodipy 576/589;
Bodipy 581/591; Bodipy 630/650-X; Bodipy 650/665-X; Bodipy 665/676;
Bodipy Fl; Bodipy FL ATP; Bodipy Fl-Ceramide; Bodipy R6G SE; Bodipy
TMR; Bodipy TMR-X conjugate; Bodipy TMR-X, SE; Bodipy TR; Bodipy TR
ATP; Bodipy TR-X SE; BO-PRO.TM.-1; BO-PRO.TM.-3; Brilliant
Sulphoflavin FF; BTC; BTC-5N; Calcein; Calcein Blue; Calcium
Crimson-; Calcium Green; Calcium Green-1 Ca.sup.2+Dye; Calcium
Green-2 Ca.sup.2+; Calcium Green-5N Ca.sup.2+; Calcium Green-C18
Ca.sup.2+; Calcium Orange; Calcofluor White; Carboxy-X-rhodamine
(5-ROX); Cascade Blue.TM.; Cascade Yellow; Catecholamine; CCF2
(GeneBlazer); CFDA; CFP (Cyan Fluorescent Protein); CFP/YFP FRET;
Chlorophyll; Chromomycin A; Chromomycin A; CL-NERF; CMFDA;
Coelenterazine; Coelenterazine cp; Coelenterazine f; Coelenterazine
fcp; Coelenterazine h; Coelenterazine hcp; Coelenterazine ip;
Coelenterazine n; Coelenterazine O; Coumarin Phalloidin;
C-phycocyanine; CPM I Methylcoumarin; CTC; CTC Formazan; Cy2.TM.;
Cy3.1 8; Cy3.5.TM.; Cy3.TM.; Cy5.1 8; Cy5.5.TM.; Cy5.TM.; Cy7.TM.;
Cyan GFP; cyclic AMP Fluorosensor (FiCRhR); Dabcyl; Dansyl; Dansyl
Amine; Dansyl Cadaverine; Dansyl Chloride; Dansyl DHPE; Dansyl
fluoride; DAPI; Dapoxyl; Dapoxyl 2; Dapoxyl 3'DCFDA; DCFH
(Dichlorodihydrofluorescein Diacetate); DDAO; DHR (Dihydrorhodamine
123); Di-4-ANEPPS; Di-8-ANEPPS (non-ratio); DiA (4-Di 16-ASP);
Dichlorodihydrofluorescein Diacetate (DCFH); DiD-Lipophilic Tracer;
DiD (DilC18(5)); DIDS; Dihydrorhodamine 123 (DHR); Dil (DilC18(3));
1Dinitrophenol; DiO (DiOC18(3)); DiR; DiR (DilC18(7)); DM-NERF
(high pH); DNP; Dopamine; DsRed; DTAF; DY-630-NHS; DY-635-NHS;
EBFP; ECFP; EGFP; ELF 97; Eosin; Erythrosin; Erythrosin ITC;
Ethidium Bromide; Ethidium homodimer-1 (EthD-1); Euchrysin;
EukoLight; Europium (111) chloride; EYFP; Fast Blue; FDA; Feulgen
(Pararosaniline); FIF (Formaldehyd Induced Fluorescence);
fluorescein isothiocyanate (FITC); Flazo Orange; Fluo-3; Fluo-4;
Fluorescein (FITC); Fluorescein Diacetate; Fluoro-Emerald;
Fluoro-Gold (Hydroxystilbamidine); Fluor-Ruby; Fluor X; FM
1-43.TM.; FM 4-46; Fura Red.TM. (high pH); Fura Red.TM./Fluo-3;
Fura-2; Fura-2/BCECF; Genacryl Brilliant Red B; Genacryl Brilliant
Yellow 10GF; Genacryl Pink 3G; Genacryl Yellow 5GF; GeneBlazer;
(CCF2); GFP (S65T); GFP red shifted (rsGFP); GFP wild type` non-UV
excitation (wtGFP); GFP wild type, UV excitation (wtGFP); GFPuv;
Gloxalic Acid; Granular blue; Haematoporphyrin; Hoechst 33258;
Hoechst 33342; Hoechst 34580; HPTS; Hydroxycoumarin;
Hydroxystilbamidine (FluoroGold); Hydroxytryptamine; Indo-1, high
calcium; Indo-1 low calcium; Indodicarbocyanine (DiD);
Indotricarbocyanine (DiR); Intrawhite Cf; JC-1; JO JO-1; JO-PRO-1;
LaserPro; Laurodan; LDS 751 (DNA); LDS 751 (RNA); Leucophor PAF;
Leucophor SF; Leucophor WS; Lissamine Rhodamine; Lissamine
Rhodamine B; Calcein/Ethidium homodimer; LOLO-1; LO-PRO-1; Lucifer
Yellow; Lyso Tracker Blue; Lyso Tracker Blue-White; Lyso Tracker
Green; Lyso Tracker Red; Lyso Tracker Yellow; LysoSensor Blue;
LysoSensor Green; LysoSensor Yellow/Blue; Mag Green; Magdala Red
(Phloxin B); Mag-Fura Red; Mag-Fura-2; Mag-Fura-5; Mag-lndo-1;
Magnesium Green; Magnesium Orange; Malachite Green; Marina Blue; I
Maxilon Brilliant Flavin 10 GFF; Maxilon Brilliant Flavin 8 GFF;
Merocyanin; Methoxycoumarin; Mitotracker Green FM; Mitotracker
Orange; Mitotracker Red; Mitramycin; Monobromobimane;
Monobromobimane (mBBr-GSH); Monochlorobimane; MPS (Methyl Green
Pyronine Stilbene); NBD; NBD Amine; Nile Red; Nitrobenzoxedidole;
Noradrenaline; Nuclear Fast Red; i Nuclear Yellow; Nylosan
Brilliant lavin E8G; Oregon Green.TM.; Oregon Green.TM. 488; Oregon
Green.TM. 500; Oregon Green.TM. 514; Pacific Blue; Pararosaniline
(Feulgen); PE-Cy5; PE-Cy7; PerCP; PerCP-Cy5.5; PE-TexasRed (Red
613); Phloxin B (Magdala Red); Phorwite AR; Phorwite BKL; Phorwite
Rev; Phorwite RPA; Phosphine 3R; PhotoResist; Phycoerythrin B [PE];
Phycoerythrin R [PE]; PKH26 (Sigma); PKH67; PMIA; Pontochrome Blue
Black; POPO-1; POPO-3; PO-PRO-1; PO-I PRO-3; Primuline; Procion
Yellow; Propidium lodid (P1); PyMPO; Pyrene; Pyronine; Pyronine B;
Pyrozal Brilliant Flavin 7GF; QSY 7; Quinacrine Mustard; Resorufin;
RH 414; Rhod-2; Rhodamine; Rhodamine 110; Rhodamine 123; Rhodamine
5 GLD; Rhodamine 6G; Rhodamine B; Rhodamine B 200; Rhodamine B
extra; Rhodamine BB; Rhodamine BG; Rhodamine Green; Rhodamine
Phallicidine; Rhodamine: Phalloidine; Rhodamine Red; Rhodamine WT;
Rose Bengal; R-phycocyanine; R-phycoerythrin (PE); rsGFP; S65A;
S65C; S65L; S65T; Sapphire GFP; SBFI; Serotonin; Sevron Brilliant
Red 2B; Sevron Brilliant Red 4G; Sevron I Brilliant Red B; Sevron
Orange; Sevron Yellow L; sgBFP.TM. (super glow BFP); sgGFP.TM.
(super glow GFP); SITS (Primuline; Stilbene Isothiosulphonic Acid);
SNAFL calcein; SNAFL-1; SNAFL-2; SNARF calcein; SNARF1; Sodium
Green; SpectrumAqua; SpectrumGreen; SpectrumOrange; Spectrum Red;
SPQ (6-methoxy-N-(3 sulfopropyl) quinolinium); Stilbene;
Sulphorhodamine B and C; Sulphorhodamine Extra; SYTO 11; SYTO 12;
SYTO 13; SYTO 14; SYTO 15; SYTO 16; SYTO 17; SYTO 18; SYTO 20; SYTO
21; SYTO 22; SYTO 23; SYTO 24; SYTO 25; SYTO 40; SYTO 41; SYTO 42;
SYTO 43; SYTO 44; SYTO 45; SYTO 59; SYTO 60; SYTO 61; SYTO 62; SYTO
63; SYTO 64; SYTO 80; SYTO 81; SYTO 82; SYTO 83; SYTO 84; SYTO 85;
SYTOX Blue; SYTOX Green; SYTOX Orange; Tetracycline;
Tetramethylrhodamine (TRITC); Texas Red.TM.; Texas Red-X.TM.
conjugate; Thiadicarbocyanine (DiSC3); Thiazine Red R; Thiazole
Orange; Thioflavin 5; Thioflavin S; Thioflavin TON; Thiolyte;
Thiozole Orange; Tinopol CBS (Calcofluor White); TIER; TO-PRO-1;
TO-PRO-3; TO-PRO-5; TOTO-1; TOTO-3; TriColor (PE-Cy5); TRITC
TetramethylRodaminelsoThioCyanate; True Blue; Tru Red; Ultralite;
Uranine B; Uvitex SFC; wt GFP; WW 781; X-Rhodamine; XRITC; Xylene
Orange; Y66F; Y66H; Y66W; Yellow GFP; YFP; YO-PRO-1; YO-PRO3;
YOYO-1; YOYO-3; Sybr Green; Thiazole orange (interchelating dyes);
semiconductor nanoparticles such as quantum dots; or caged
fluorophore (which can be activated with light or other
electromagnetic energy source), or a combination thereof.
[0066] A modifier unit such as a radionuclide can be incorporated
into or attached directly to any of the compounds described herein
by halogenation. Examples of radionuclides useful in this
embodiment include, but are not limited to, tritium, iodine-125,
iodine-131, iodine-123, iodine-124, astatine-210, carbon-11,
carbon-14, nitrogen-13, fluorine-18. In another aspect, the
radionuclide can be attached to a linking group or bound by a
chelating group, which is then attached to the compound directly or
by means of a linker. Examples of radionuclides useful in this
aspect include, but are not limited to, Tc-99m, Re-186, Ga-68,
Re-188, Y-90, Sm-153, Bi-212, Cu-67, Cu-64, and Cu-62.
Radiolabeling techniques such as these are routine in the
radiopharmaceutical industry.
[0067] The radiolabeled compounds are useful as imaging agents to
diagnose neurological disease (e.g., a neurodegenerative disease)
or a mental condition or to follow the progression or treatment of
such a disease or condition in a mammal (e.g., a human). The
radiolabeled compounds described herein can be conveniently used in
conjunction with imaging techniques such as positron emission
tomography (PET) or single photon emission computerized tomography
(SPECT).
[0068] Labeling can be either direct or indirect. In direct
labeling, the detecting antibody (the antibody for the molecule of
interest) or detecting molecule (the molecule that can be bound by
an antibody to the molecule of interest) include a label. Detection
of the label indicates the presence of the detecting antibody or
detecting molecule, which in turn indicates the presence of the
molecule of interest or of an antibody to the molecule of interest,
respectively. In indirect labeling, an additional molecule or
moiety is brought into contact with, or generated at the site of,
the immunocomplex. For example, a signal-generating molecule or
moiety such as an enzyme can be attached to or associated with the
detecting antibody or detecting molecule. The signal-generating
molecule can then generate a detectable signal at the site of the
immunocomplex. For example, an enzyme, when supplied with suitable
substrate, can produce a visible or detectable product at the site
of the immunocomplex. ELISAs use this type of indirect
labeling.
[0069] As another example of indirect labeling, an additional
molecule (which can be referred to as a binding agent) that can
bind to either the molecule of interest or to the antibody (primary
antibody) to the molecule of interest, such as a second antibody to
the primary antibody, can be contacted with the immunocomplex. The
additional molecule can have a label or signal-generating molecule
or moiety. The additional molecule can be an antibody, which can
thus be termed a secondary antibody. Binding of a secondary
antibody to the primary antibody can form a so-called sandwich with
the first (or primary) antibody and the molecule of interest. The
immune complexes can be contacted with the labeled, secondary
antibody under conditions effective and for a period of time
sufficient to allow the formation of secondary immune complexes.
The secondary immune complexes can then be generally washed to
remove any non-specifically bound labeled secondary antibodies, and
the remaining label in the secondary immune complexes can then be
detected. The additional molecule can also be or include one of a
pair of molecules or moieties that can bind to each other, such as
the biotin/avadin pair. In this mode, the detecting antibody or
detecting molecule should include the other member of the pair.
[0070] Other modes of indirect labeling include the detection of
primary immune complexes by a two step approach. For example, a
molecule (which can be referred to as a first binding agent), such
as an antibody, that has binding affinity for the molecule of
interest or corresponding antibody can be used to form secondary
immune complexes, as described above. After washing, the secondary
immune complexes can be contacted with another molecule (which can
be referred to as a second binding agent) that has binding affinity
for the first binding agent, again under conditions effective and
for a period of time sufficient to allow the formation of immune
complexes (thus forming tertiary immune complexes). The second
binding agent can be linked to a detectable label or
signal-genrating molecule or moiety, allowing detection of the
tertiary immune complexes thus formed. This system can provide for
signal amplification.
[0071] Immunoassays that involve the detection of as substance,
such as a protein or an antibody to a specific protein, include
label-free assays, protein separation methods (i.e.,
electrophoresis), solid support capture assays, or in vivo
detection. Label-free assays are generally diagnostic means of
determining the presence or absence of a specific protein, or an
antibody to a specific protein, in a sample. Protein separation
methods are additionally useful for evaluating physical properties
of the protein, such as size or net charge. Capture assays are
generally more useful for quantitatively evaluating the
concentration of a specific protein, or antibody to a specific
protein, in a sample. Finally, in vivo detection is useful for
evaluating the spatial expression patterns of the substance, i.e.,
where the substance can be found in a subject, tissue or cell.
[0072] Provided that the concentrations are sufficient, the
molecular complexes ([Ab-Ag]n) generated by antibody-antigen
interaction are visible to the naked eye, but smaller amounts may
also be detected and measured due to their ability to scatter a
beam of light. The formation of complexes indicates that both
reactants are present, and in immunoprecipitation assays a constant
concentration of a reagent antibody is used to measure specific
antigen ([Ab-Ag]n), and reagent antigens are used to detect
specific antibody ([Ab-Ag]n). If the reagent species is previously
coated onto cells (as in hemagglutination assay) or very small
particles (as in latex agglutination assay), "clumping" of the
coated particles is visible at much lower concentrations. A variety
of assays based on these elementary principles are in common use,
including Ouchterlony immunodiffusion assay, rocket
immunoelectrophoresis, and immunoturbidometric and nephelometric
assays. The main limitations of such assays are restricted
sensitivity (lower detection limits) in comparison to assays
employing labels and, in some cases, the fact that very high
concentrations of analyte can actually inhibit complex formation,
necessitating safeguards that make the procedures more complex.
Some of these Group 1 assays date right back to the discovery of
antibodies and none of them have an actual "label" (e.g. Ag-enz).
Other kinds of immunoassays that are label free depend on
immunosensors, and a variety of instruments that can directly
detect antibody-antigen interactions are now commercially
available. Most depend on generating an evanescent wave on a sensor
surface with immobilized ligand, which allows continuous monitoring
of binding to the ligand. Immunosensors allow the easy
investigation of kinetic interactions and, with the advent of
lower-cost specialized instruments, may in the future find wide
application in immunoanalysis.
[0073] The use of immunoassays to detect a specific protein can
involve the separation of the proteins by electophoresis.
Electrophoresis is the migration of charged molecules in solution
in response to an electric field. Their rate of migration depends
on the strength of the field; on the net charge, size and shape of
the molecules and also on the ionic strength, viscosity and
temperature of the medium in which the molecules are moving. As an
analytical tool, electrophoresis is simple, rapid and highly
sensitive. It is used analytically to study the properties of a
single charged species, and as a separation technique.
[0074] Generally the sample is run in a support matrix such as
paper, cellulose acetate, starch gel, agarose or polyacrylamide
gel. The matrix inhibits convective mixing caused by heating and
provides a record of the electrophoretic run: at the end of the
run, the matrix can be stained and used for scanning,
autoradiography or storage. In addition, the most commonly used
support matrices--agarose and polyacrylamide--provide a means of
separating molecules by size, in that they are porous gels. A
porous gel may act as a sieve by retarding, or in some cases
completely obstructing, the movement of large macromolecules while
allowing smaller molecules to migrate freely. Because dilute
agarose gels are generally more rigid and easy to handle than
polyacrylamide of the same concentration, agarose is used to
separate larger macromolecules such as nucleic acids, large
proteins and protein complexes. Polyacrylamide, which is easy to
handle and to make at higher concentrations, is used to separate
most proteins and small oligonucleotides that require a small gel
pore size for retardation.
[0075] Proteins are amphoteric compounds; their net charge
therefore is determined by the pH of the medium in which they are
suspended. In a solution with a pH above its isoelectric point, a
protein has a net negative charge and migrates towards the anode in
an electrical field. Below its isoelectric point, the protein is
positively charged and migrates towards the cathode. The net charge
carried by a protein is in addition independent of its size--i.e.,
the charge carried per unit mass (or length, given proteins and
nucleic acids are linear macromolecules) of molecule differs from
protein to protein. At a given pH therefore, and under
non-denaturing conditions, the electrophoretic separation of
proteins is determined by both size and charge of the
molecules.
[0076] Sodium dodecyl sulphate (SDS) is an anionic detergent which
denatures proteins by "wrapping around" the polypeptide
backbone--and SDS binds to proteins fairly specifically in a mass
ratio of 1.4:1. In so doing, SDS confers a negative charge to the
polypeptide in proportion to its length. Further, it is usually
necessary to reduce disulphide bridges in proteins (denature)
before they adopt the random-coil configuration necessary for
separation by size; this is done with 2-mercaptoethanol or
dithiothreitol (DTT). In denaturing SDS-PAGE separations therefore,
migration is determined not by intrinsic electrical charge of the
polypeptide, but by molecular weight.
[0077] Determination of molecular weight is done by SDS-PAGE of
proteins of known molecular weight along with the protein to be
characterized. A linear relationship exists between the logarithm
of the molecular weight of an SDS-denatured polypeptide, or native
nucleic acid, and its Rf. The Rf is calculated as the ratio of the
distance migrated by the molecule to that migrated by a marker
dye-front. A simple way of determining relative molecular weight by
electrophoresis (Mr) is to plot a standard curve of distance
migrated vs. log10MW for known samples, and read off the logMr of
the sample after measuring distance migrated on the same gel.
[0078] In two-dimensional electrophoresis, proteins are
fractionated first on the basis of one physical property, and, in a
second step, on the basis of another. For example, isoelectric
focusing can be used for the first dimension, conveniently carried
out in a tube gel, and SDS electrophoresis in a slab gel can be
used for the second dimension. One example of a procedure is that
of O'Farrell, P. H., High Resolution Two-dimensional
Electrophoresis of Proteins, J. Biol. Chem. 250:4007-4021 (1975),
herein incorporated by reference in its entirety for its teaching
regarding two-dimensional electrophoresis methods. Other examples
include but are not limited to, those found in Anderson, L and
Anderson, N G, High resolution two-dimensional electrophoresis of
human plasma proteins, Proc. Natl. Acad. Sci. 74:5421-5425 (1977),
Ornstein, L., Disc electrophoresis, L. Ann. N.Y. Acad. Sci.
121:321349 (1964), each of which is herein incorporated by
reference in its entirety for teachings regarding electrophoresis
methods.
[0079] Laemmli, U. K., Cleavage of structural proteins during the
assembly of the head of bacteriophage T4, Nature 227:680 (1970),
which is herein incorporated by reference in its entirety for
teachings regarding electrophoresis methods, discloses a
discontinuous system for resolving proteins denatured with SDS. The
leading ion in the Laemmli buffer system is chloride, and the
trailing ion is glycine. Accordingly, the resolving gel and the
stacking gel are made up in Tris-HCl buffers (of different
concentration and pH), while the tank buffer is Tris-glycine. All
buffers contain 0.1% SDS.
[0080] One example of an immunoassay that uses electrophoresis that
is contemplated in the current methods is Western blot analysis.
Western blotting or immunoblotting allows the determination of the
molecular mass of a protein and the measurement of relative amounts
of the protein present in different samples. Detection methods
include chemiluminescence and chromagenic detection. Standard
methods for Western blot analysis can be found in, for example, D.
M. Bollag et al., Protein Methods (2d edition 1996) and E. Harlow
& D. Lane, Antibodies, a Laboratory Manual (1988), U.S. Pat.
No. 4,452,901, each of which is herein incorporated by reference in
their entirety for teachings regarding Western blot methods.
Generally, proteins are separated by gel electrophoresis, usually
SDS-PAGE. The proteins are transferred to a sheet of special
blotting paper, e.g., nitrocellulose, though other types of paper
or membranes, can be used. The proteins retain the same pattern of
separation as on the gel. The blot is incubated with a generic
protein (such as milk proteins) to bind to any remaining sticky
places on the nitrocellulose. An antibody is then added to the
solution which is able to bind to its specific protein.
[0081] The attachment of specific antibodies to specific
immobilized antigens can be readily visualized by indirect enzyme
immunoassay techniques, usually using a chromogenic substrate (e.g.
alkaline phosphatase or horseradish peroxidase) or chemiluminescent
substrates. Other possibilities for probing include the use of
fluorescent or radioisotope labels (e.g., fluorescein, .sup.125I).
Probes for the detection of antibody binding can be conjugated
anti-immunoglobulins, conjugated staphylococcal Protein A (binds
IgG), or probes to biotinylated primary antibodies (e.g.,
conjugated avidin/streptavidin).
[0082] The power of the technique lies in the simultaneous
detection of a specific protein by means of its antigenicity, and
its molecular mass. Proteins are first separated by mass in the
SDS-PAGE, then specifically detected in the immunoassay step. Thus,
protein standards (ladders) can be run simultaneously in order to
approximate molecular mass of the protein of interest in a
heterogeneous sample.
[0083] The gel shift assay or electrophoretic mobility shift assay
(EMSA) can be used to detect the interactions between DNA binding
proteins and their cognate DNA recognition sequences, in both a
qualitative and quantitative manner. Exemplary techniques are
described in Ornstein L., Disc electrophoresis-I: Background and
theory, Ann. NY Acad. Sci. 121:321-349 (1964), and Matsudiara, P T
and D R Burgess, SDS microslab linear gradient polyacrylamide gel
electrophoresis, Anal. Biochem. 87:386-396 (1987), each of which is
herein incorporated by reference in its entirety for teachings
regarding gel-shift assays.
[0084] In a general gel-shift assay, purified proteins or crude
cell extracts can be incubated with a labeled (e.g.,
.sup.32P-radiolabeled) DNA or RNA probe, followed by separation of
the complexes from the free probe through a nondenaturing
polyacrylamide gel. The complexes migrate more slowly through the
gel than unbound probe. Depending on the activity of the binding
protein, a labeled probe can be either double-stranded or
single-stranded. For the detection of DNA binding proteins such as
transcription factors, either purified or partially purified
proteins, or nuclear cell extracts can be used. For detection of
RNA binding proteins, either purified or partially purified
proteins, or nuclear or cytoplasmic cell extracts can be used. The
specificity of the DNA or RNA binding protein for the putative
binding site is established by competition experiments using DNA or
RNA fragments or oligonucleotides containing a binding site for the
protein of interest, or other unrelated sequence. The differences
in the nature and intensity of the complex formed in the presence
of specific and nonspecific competitor allows identification of
specific interactions. Refer to Promega, Gel Shift Assay FAQ,
available at <http://www.promega.com/faq/gelshfaq.html>(last
visited Mar. 25, 2005), which is herein incorporated by reference
in its entirety for teachings regarding gel shift methods.
[0085] Gel shift methods can include using, for example, colloidal
forms of COOMASSIE (Imperial Chemicals Industries, Ltd) blue stain
to detect proteins in gels such as polyacrylamide electrophoresis
gels. Such methods are described, for example, in Neuhoff et al.,
Electrophoresis 6:427-448 (1985), and Neuhoff et al.,
Electrophoresis 9:255-262 (1988), each of which is herein
incorporated by reference in its entirety for teachings regarding
gel shift methods. In addition to the conventional protein assay
methods referenced above, a combination cleaning and protein
staining composition is described in U.S. Pat. No. 5,424,000,
herein incorporated by reference in its entirety for its teaching
regarding gel shift methods. The solutions can include phosphoric,
sulfuric, and nitric acids, and Acid Violet dye.
[0086] Radioimmune Precipitation Assay (RIPA) is a sensitive assay
using radiolabeled antigens to detect specific antibodies in serum.
The antigens are allowed to react with the serum and then
precipitated using a special reagent such as, for example, protein
A sepharose beads. The bound radiolabeled immunoprecipitate is then
commonly analyzed by gel electrophoresis. Radioimmunoprecipitation
assay (RIPA) is often used as a confirmatory test for diagnosing
the presence of HIV antibodies. RIPA is also referred to in the art
as Farr Assay, Precipitin Assay, Radioimmune Precipitin Assay;
Radioimmunoprecipitation Analysis; Radioimmunoprecipitation
Analysis, and Radioimmunoprecipitation Analysis.
[0087] While the above immunoassays that utilize electrophoresis to
separate and detect the specific proteins of interest allow for
evaluation of protein size, they are not very sensitive for
evaluating protein concentration. However, also contemplated are
immunoassays wherein the protein or antibody specific for the
protein is bound to a solid support (e.g., tube, well, bead, or
cell) to capture the antibody or protein of interest, respectively,
from a sample, combined with a method of detecting the protein or
antibody specific for the protein on the support. Examples of such
immunoassays include Radioimmunoassay (RIA), Enzyme-Linked
Immunosorbent Assay (ELISA), Flow cytometry, protein array,
multiplexed bead assay, and magnetic capture.
[0088] Radioimmunoassay (RIA) is a classic quantitative assay for
detection of antigen-antibody reactions using a radioactively
labeled substance (radioligand), either directly or indirectly, to
measure the binding of the unlabeled substance to a specific
antibody or other receptor system. Radioimmunoassay is used, for
example, to test hormone levels in the blood without the need to
use a bioassay. Non-immunogenic substances (e.g., haptens) can also
be measured if coupled to larger carrier proteins (e.g., bovine
gamma-globulin or human serum albumin) capable of inducing antibody
formation. RIA involves mixing a radioactive antigen (because of
the ease with which iodine atoms can be introduced into tyrosine
residues in a protein, the radioactive isotopes .sup.125I or
.sup.131I are often used) with antibody to that antigen. The
antibody is generally linked to a solid support, such as a tube or
beads. Unlabeled or "cold" antigen is then adding in known
quantities and measuring the amount of labeled antigen displaced.
Initially, the radioactive antigen is bound to the antibodies. When
cold antigen is added, the two compete for antibody binding sites
and at higher concentrations of cold antigen, more binds to the
antibody, displacing the radioactive variant. The bound antigens
are separated from the unbound ones in solution and the
radioactivity of each used to plot a binding curve.
[0089] Enzyme-Linked Immunosorbent Assay (ELISA), or more
generically termed EIA (Enzyme ImmunoAssay), is an immunoassay that
can detect an antibody specific for a protein. In such an assay, a
detectable label bound to either an antibody-binding or
antigen-binding reagent is an enzyme. When exposed to its
substrate, this enzyme reacts in such a manner as to produce a
chemical moiety which can be detected, for example, by
spectrophotometric, fluorometric or visual means. Enzymes which can
be used to detectably label reagents useful for detection include,
but are not limited to, horseradish peroxidase, alkaline
phosphatase, glucose oxidase, .beta.-galactosidase, ribonuclease,
urease, catalase, malate dehydrogenase, staphylococcal nuclease,
asparaginase, yeast alcohol dehydrogenase, alpha.-glycerophosphate
dehydrogenase, triose phosphate isomerase, glucose-6-phosphate
dehydrogenase, glucoamylase and acetylcholinesterase. For
descriptions of ELISA procedures, see Voller, A. et al., J. Clin.
Pathol. 31:507-520 (1978); Butler, J. E., Meth. Enzymol. 73:482-523
(1981); Maggio, E. (ed.), Enzyme Immunoassay, CRC Press, Boca
Raton, 1980; Butler, J. E., In: Structure of Antigens, Vol. 1 (Van
Regenmortel, M., CRC Press, Boca Raton, 1992, pp. 209-259; Butler,
J. E., In: van Oss, C. J. et al., (eds), Immunochemistry, Marcel
Dekker, Inc., New York, 1994, pp. 759-803; Butler, J. E. (ed.),
Immunochemistry of Solid-Phase Immunoassay, CRC Press, Boca Raton,
1991); Crowther, "ELISA: Theory and Practice," In: Methods in
Molecule Biology, Vol. 42, Humana Press; New Jersey, 1995;U.S. Pat.
No. 4,376,110, each of which is incorporated herein by reference in
its entirety and specifically for teachings regarding ELISA
methods.
[0090] It is understood that any instrument that is capable of
detecting the labels used herein is appropriate for the methods
disclosed. Additional means of detection can also be used such as
reporter constructs such as the luciferase gene as well as methods
of label complementation. In short, any means known in the art for
detection of acceptable for use with the disclosed methods.
[0091] Disclosed herein are cells comprising the first protein, a
second protein, and a nucleic acid comprising the first recognition
element adjacent to a second recognition element, wherein the first
protein binds the first recognition element and the second protein
binds the second recognition element, wherein at least one of the
first and second proteins comprises a first half of a split
fluorescent protein and at least one of the first and second
proteins comprises a second half of the split fluorescent protein,
wherein binding of the first and second proteins to their
respective recognition sites results in the assembly and excitation
of the split fluorescent protein. It is understood that the
disclosed cells can be used to screen for agents that inhibits the
interaction of a first protein and a first recognition element on a
nucleic acid comprising the steps of a) administering an agent to a
cell comprising the first protein, a second protein, and a nucleic
acid comprising the first recognition element adjacent to a second
recognition element, wherein the first protein binds the first
recognition element and the second protein binds the second
recognition element; and b) detecting co-localization of the first
and second protein, wherein a decrease in co-localization of the
first and second protein relative to a control indicates an agent
that inhibits the interaction. It is understood that in the methods
disclosed herein, the first protein can comprise MBNL1, MBNL2, or
MBNL3 and the first recognition element can comprise polyCUG or
polyCCUG It is also understood that the second protein can
comprise, for example, MS2 and the second recognition element can
be an MS2 coat protein RNA recognition element. The interaction of
the disclosed proteins can be used to facilitate a detection method
not available when one or more components are not available. For
example, disclosed herein are methods of screening for an agent,
wherein at least one of the first and second proteins comprises a
donor fluorescent dye and at least one of the first and second
proteins comprises an acceptor dye, wherein excitation of the donor
fluorescent dye results in a fluorescent emission that excites the
acceptor dye if the first and second proteins are co-localized.
[0092] Thus, in one aspect, the acceptor dye fluoresces when
excited and detection of this fluorescence is an indication of
cleavage. Traditional examples of donor fluorescent dyes and
acceptor fluorescent dyes include FAM and TAMRA. In another aspect,
the acceptor dye quenches the fluorescence of the donor dye by
absorbing the energy emitted by the fluorophore, releasing it as
heat rather than fluorescence. In this aspect, the detection of
fluorescence emitted from the donor dye is an indication of
cleavage. Examples of dark quenchers are methyl red, DABCYL,
ElleQuencher.TM., and Eclipse.TM. Dark Quencher. Methyl red
quenches the lower wavelength dyes such as FAM but is not good at
quenching those that emit at a higher wavelength, e.g. Cy5.TM..
ElleQuencher.TM. was designed to quench the higher end of the
spectrum. It has been tested in Double-Dye Oligonucleotide probes
and Scorpions.TM.. It gives good results for both when tested with
dyes such as ROX and TAMRA but is also equivalent or better than
methyl red for dyes such as FAM (lower wavelength).
[0093] Also disclosed are methods, wherein at least one of the
first and second proteins comprises a first half of a split
fluorescent protein and at least one of the first and second
proteins comprises a second half of the split fluorescent protein,
wherein excitation of the split fluorescent protein results in a
fluorescent emission if the first and second proteins are
co-localized. It is understood that one example of a split
fluorescent protein that can be used in the disclosed methods is
Venus fluorescent protein (VFP). It is understood that other
methods of displaying co-localization in the cell. For example, the
disclosed herein are methods wherein at least one of the first and
second proteins comprises a first half of a split beta
galactosidase protein and at least one of the first and second
proteins comprises a second half of the split beta galactosidase
protein, wherein hydrolysis of a beta galactosidase substrate
results in fluorescence of luminescence if the first and second
proteins are co-localized.
[0094] Also disclosed are method of screening for an agent that
improves spliceopathy comprising the steps of a) introducing an
agent into a cell comprising of a splicing regulator, overexpressed
polyCUG or polyCCUG repeat RNA, and spliceopathy reporter
construct, wherein the reporter construct comprises a gene
susceptible to polyCUG or polyCCUG repeat induced spliceopathy
flanked by one or more genes encoding a labeled protein; and b)
measuring the level of the labeled protein; and c) comparing the
ratio of labeled protein, wherein an increase of labeled protein
indicates an agent that improves spliceopathy. For example,
disclosed herein, are methods wherein the reporter proteins
flanking the nucleic acid sequence of the spliceopathy reporter are
labeled proteins. It is understood that examples of splicing
regulators include but are not limited to MBNL1, MBNL2, MBNL3,
CUG-B1, and ETR-3. It is also understood that examples of
spliceopathy susceptible genes include but are not limited to TNNT3
and SERCA1. It is understood that such labeled proteins can be
labeled similarly or have different labels. Thus, for example,
disclosed are methods wherein the gene susceptible to polyCUG or
polyCCUG is flanked by a gene encoding a single labeled protein,
and wherein an increase of the labeled protein indicates an agent
that improves spliceopathy. An example of such a label can be green
fluorescence protein. Also, for example disclosed are methods
wherein the gene susceptible to polyCUG or polyCCUG is flanked by
genes encoding first and second labeled protein, wherein the first
and second proteins are differentially labeled; and wherein the
method further comprises d) comparing the ratio of the first
labeled protein to the second labeled protein, wherein a high ratio
indicates an agent that improves spliceopathy. Thus, for example,
disclosed herein are methods wherein the first and second labeled
proteins are labeled with YFP and Y.cndot.CFP respectively, and
wherein an improvement of spliceopathy is determined by comparing
the ration of YFP to Y.cndot.CFP, wherein a high ratio indicates
the agent improved spliceopathy. Also disclosed are methods of
screening for an agent that inhibits the interaction of a protein
and a ligand comprising the steps of a) introducing an agent into a
cell comprising an MBNL1 expression construct and an spliceopathy
reporter construct susceptible to poly(CUG).sup.exp induced
spliceopathy, wherein the reporter construct comprises a polyCUG
susceptible exon flanked by introns and exons encoding first and
second labeled protein, wherein the first and second proteins are
differentially labeled.
[0095] Alternatively, the disclosed methods can be achieved without
the use of labeled proteins. For example, a minigene encoding
luciferase reporter construct can be used. Thus, disclosed herein
are methods of screening for an agent that improves spliceopathy
comprising the steps of a) introducing an agent into a cell
comprising a splicing regulator protein, a poly(CUG) or poly(CCUG)
expanded RNA, and a spliceopathy reporter wherein the spliceopathy
reporter comprises a nucleic acid sequence susceptible to splicing
flanked by a reporter protein wherein the reporter protein is
luciferase; and measuring the level of lucifierase activity.
[0096] 1. Kits
[0097] Disclosed herein are kits that are drawn to reagents that
can be used in practicing the methods disclosed herein. The kits
can include any reagent or combination of reagent discussed herein
or that would be understood to be required or beneficial in the
practice of the disclosed methods. For example, the kits could
include primers to perform the amplification reactions discussed in
certain embodiments of the methods, as well as the buffers and
enzymes required to use the primers as intended. For example,
disclosed is a kit for screening for agents that inhibit the
interaction of MBNL1 with polyCUG.sup.exp mRNA. It is understood
that agents identified by the disclosed screening methods can be
used to treat DM1. Thus, disclosed herein are kits for screening
for agents that can be used to treat DM1. Thus, for example,
disclosed are kits comprising a polystyrene plate, polyCUG.sup.exp
mRNA, a capture oligodeoxynucleotide (ODN), and MBNL1, wherein the
MBNL1 is labeled. Also disclosed are kits comprising a
nitrocellulose filter plate, labeled polyCUG.sup.exp mRNA, and
MBNL1. It is understood and herein contemplated that the proteins
or polyCUG.sup.exp provided in the kits disclosed herein can be
labeled by any means known in the art. For example, the label can
be a fluorescent label such as fluoroscein isothiocyanate (FITC),
phycoerythrin (PE), TEXAS RED.RTM., Green fluorescent protein
(GFP), yellow fluorescent protein (YFP), cyan fluorescent protein
(CFP), allophycocyanin (APC), PerCP.TM., CY-CHROME.TM., or
PharRED.TM.. Alternatively, the label could be a radio label, or an
enzymatic reporter such as beta galactosidase, horseradish
peroxidase, or alkaline phosphatase.
[0098] 2. Methods of Treatment
[0099] Disclosed herein are methods treating myotonic dystrophy
(DM) in a subject in need thereof comprising administering to the
subject an agent that inhibits the interaction of muscleblind
proteins such as MBNL1, MBNL2, or MBNL3 with polyCUG.sup.exp mRNA.
It is understood and herein contemplated that many different
molecules can accomplish this task. For example, disclosed herein
are methods of treating wherein the agent comprises a morpholino
such as CAG25 (SEQ ID NO: 3) or the morpholino set forth in SEQ ID
NO: 5. It is understood that the disclosed methods of treating
myotonic dystrophy can be used to treat myotonic dystrophy type 1
(DM1) or myotonic dystrophy type 2 (DM2). Also disclosed are
methods of treating myotonic dystrophy wherein the agent is an
aminoglycosidic antibiotic compound such as, for example, neomycin
and gentamicin. Further disclosed herein are methods treating
myotonic dystrophy in a subject in need thereof comprising
administering to the subject an agent that improves
spliceopathy.
[0100] Spliceopathy refers to the abnormal regulation of
alternative splicing. It is understood that such disregulation can
result from the sequestration of splicing factors such as the
muscleblind proteins. Thus, for example, disclosed herein are
methods of treating DM1 in a subject in need thereof comprising
administering to the subject an agent that improves spliceopathy.
It is understood and herein contemplated that many different
molecules can accomplish this task. For example, disclosed herein
are methods of treating wherein the agent comprises a morpholino
such as CAG25 (SEQ ID NO: 3) or the morpholino set forth in SEQ ID
NOs: 3, 4, 5, 6. Also disclosed are methods of treating myotonic
dystrophy wherein the agent is an aminoglycosidic antibiotic
compound such as, for example, neomycin and gentamicin. Thus, for
example, specifically disclosed are methods of treating myotonic
dystrophy wherein the agent is an aminoglycosidic antibiotic
compound such as, for example, neomycin and gentamicin. Also
disclosed herein are methods of treating myotonic dystrophy type 2
(DM2) in a subject in need thereof comprising administering to the
subject an agent that improves spliceopathy. Also disclosed herein
are methods of treating myotonic dystrophy type 2 (DM2) in a
subject in need thereof comprising administering to the subject an
agent that improves spliceopathy. As with treatment for DM1, many
different molecules can accomplish this task. For example,
disclosed herein are methods of treating wherein the agent
comprises a morpholino such as CAG25 (SEQ ID NO: 3) or
aminoglycosidic antibiotic compound such as, for example, neomycin
and gentamicin can be used as treatment.
[0101] It is further understood that the methods of treating DM can
affect DM by inhibiting, improving, or reversing channelopathy.
Channelopathy is the reduction in ion channel conductance. It is
understood and herein contemplated that channelopathy can result
from spliceopathy of the ion channel such as ClC-1. Thus, disclosed
herein are methods of treating channelopathy comprising
administering to a subject in need thereof one of the antisense
nucleotides disclosed herein.
[0102] "Treatment," "treat," or "treating" mean a method of
reducing the effects of a disease or condition. Treatment can also
refer to a method of reducing the disease or condition itself
rather than just the symptoms. The treatment can be any reduction
from native levels and can be but is not limited to the complete
ablation of the disease, condition, or the symptoms of the disease
or condition. Therefore, in the disclosed methods, treatment" can
refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%
reduction in the severity of an established disease or the disease
progression. For example, a disclosed method for reducing the
effects of prostate cancer is considered to be a treatment if there
is a 10% reduction in one or more symptoms of the disease in a
subject with the disease when compared to native levels in the same
subject or control subjects. Thus, the reduction can be a 10, 20,
30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in
between as compared to native or control levels. It is understood
and herein contemplated that "treatment" does not necessarily refer
to a cure of the disease or condition, but an improvement in the
outlook of a disease or condition. Nevertheless, it is fully
contemplated herein that "treatment" can not only refer to the
ablation of the disease state, but the reversal of the condition.
It is also understood that by correcting or improving spliceopathy,
the disease state is being treated. Therefore, herein "improves
spliceopathy" or correct spliceopathy" means any change in
spliceopathy that results in a change in the degree, amount or
action of towards proper regulation of alternative splicing.
[0103] It is understood that the morpholinos used in the disclosed
methods can comprise repeating nucleotides, for example, CAG25 as
set forth in SEQ ID NO: 3. It is understood and herein contemplated
that the antisense oligonucleotides for use in the methods or
treating disclosed herein can comprise 2, 3, 4, 5, 6, 7, 8, 9 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25
repeats where a 25 repeat would be referred to as CAG75. It is
further understood that the antisense oligonucleotides do not have
to comprise complete repeats, but can comprise fractions of a
repeat. Thus, for example, CAG25 is an 8.3 repeat representing 8
full repeats plus one additional nucleotide. Thus, disclosed herein
are repeats comprising 2.3, 2.6, 3.3, 3.6, 4.3, 4.6, 5.3, 5.6, 6.3,
6.6, 7.3, 7.6, 8.3, 8.6, 9.3, 9.6, 10.3, 10.6, 11.3, 11.6,
12.312.6, 13.2, 13.6, 14.3, 14.6, 15.3, 15.6, 16.3, 16.6, 17.3,
17.6, 18.3, 18.6, 19.3, 19.6, 20.3, 20.6, 21.3, 21.6, 22.3, 22.6,
23.3, 23.6, 24.3, and 24.6.
[0104] It is further understood and herein contemplated that the
disclosed antisense oligonucleotides can be modified to be
morpholinos. Morpholino refers to synthetic oligonucleotides which
have standard nucleic acid bases, bound to morpholine rings rather
than the deoxyribose rings of DNA and the bases are linked through
phosphorodiamidate groups instead of phosphates. The morpholino
operates by binding to complementary RNA and blocks acceess to the
RNA by other molecules. Thus disclosed herein are antisense
oligonucleotides as set forth is SEQ ID NO: 3, SEQ ID NO: 4, or SEQ
ID NO:5 wherein the antisense oligonuceottide is a morpholino. It
is understood that where a particular antisense oligonucleotides is
disclosed, contemplated herein is the use of the morpholino variant
of that antisense oligonucleotides. Thus, it understood that any of
the disclosed methods of treatment can comprise a
morpholino-antisense oligonucleotides. Thus, for example,
specifically disclosed herein are methods of treatment of DM,
wherein the antisense oligonucleotides is a morpholino
oligonucleotides. Also disclosed are methods of treating DM,
wherein the PNA-antisense oligonucleotides is a the morpholino
variant of SEQ ID NO:3, SEQ ID NO:4; or SEQ ID NO:5. Further
disclosed are methods of treatment wherein the morpholino antisense
oligonucleotides is a CAG, wherein the CAG can comprise 2, 3, 4, 5,
6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, or 25 repeats. Thus, for example disclosed herein are methods
of treating DM, wherein the CAG comprises 2, 3, 4, or 5 CAG
repeats. It is further understood that the antisense
oligonucleotides do not have to comprise complete repeats, but can
comprise fractions of a repeat. Thus, for example, CAG25 is an 8.3
repeat representing 8 full repeats plus one additional nucleotide.
Thus, disclosed herein are repeats comprising 2.3, 2.6, 3.3, 3.6,
4.3, 4.6, 5.3, 5.6, 6.3, 6.6, 7.3, 7.6, 8.3, 8.6, 9.3, 9.6, 10.3,
10.6, 11.3, 11.6, 12.312.6, 13.2, 13.6, 14.3, 14.6, 15.3, 15.6,
16.3, 16.6, 17.3, 17.6, 18.3, 18.6, 19.3, 19.6, 20.3, 20.6, 21.3,
21.6, 22.3, 22.6, 23.3, 23.6, 24.3, and 24.6.
[0105] It is further understood and herein contemplated that the
disclosed antisense oligonucleotides can be modified to incorporate
peptide nucleic acids as the sugar backbone to the antisense
oligonucleotides. Peptide nucleic acids (PNA) "are synthetic
analogue of DNA and RNA, in which the naturally occurring
sugarphosphate backbone has been replaced by N-(2-aminoethyl)
glycine units. PNA can hybridize to complementary DNA or RNA strand
through Watson-Crick base-pairing to form a hybrid duplex, with
high affinity and sequence selectivity. The high binding affinity
of PNA has been attributed in part to the lack of electrostatic
repulsion. In addition to conferring hybridization stability, the
neutral polyamide backbone provides the added benefit of enzymatic
stability, making PNA resistant to both proteases and nucleases.
Together these properties make PNA an attractive reagent for
biotechnology applications. However, unlike DNA or RNA in the
unhybridized state (single strand) whose structure, to a large
degree, is extended in solution due to the negatively charged
phosphate backbone, PNA tends to fold into complex globular
structures, presumably due to the collapse of the hydrophobic
nucleobases. In fact, this conformational collapse has been
exploited in the development of stemless PNA molecular beacons,
taking advantage of the proximity between the two termini in the
unhybridized state. Several modifications have been made to the PNA
N-(2-aminoethyl) glycine backbone in attempts to increase its
rigidity." (Dragulesca-Andrasi, A et al. (2006) JACS 128:
10258-10267)
[0106] One modification, GPNA, an analogue of PNA containing
internally linked D-arginine side chains, binds to RNA with high
affinity and sequence selectivity and is readily taken up by
mammalian cells. Alternatively, a simple c-backbone modification
can transform a randomly folded peptide nucleic acid (PNA) into a
right-handed helix. These conformationally preorganized helical
PNAs bind to DNA and RNA with exceptionally high affinity and
sequence selectivity. It is understood that the antisense
oligonucleotide disclosed herein can comprise PNA. Thus, for
example, disclosed herein is PNA-CAG25. Also disclosed are the
antisense oligonucleotides set forth in SEQ ID NO:3, SEQ ID NO:4;
SEQ ID NO:5; or SEQ ID NO:6, wherein the backbone has been modified
as a PNA. It is understood that where a particular antisense
oligonucleotides is disclosed, contemplated herein is the use of
the PNA variant of that antisense oligonucleotides. Thus, it
understood that any of the disclosed methods of treatment can
comprise a PNA-antisense oligonucleotides. Thus, for example,
specifically disclosed herein are methods of treatment of DM,
wherein the antisense oligonucleotides is a peptide nucleic acid
antisense oligonucleotides. Also disclosed are methods of treating
DM, wherein the PNA-antisense oligonucleotides is a the PNA variant
of SEQ ID NO:3, SEQ ID NO:4; and SEQ ID NO:5. Further disclosed are
methods of treatment wherein the PNA antisense oligonucleotides is
a PNA-CAG, wherein the PNA-CAG can comprise 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25
repeats. Thus, for example disclosed herein are methods of treating
DM, wherein the PNA-CAG comprises 2, 3, 4, or 5 CAG repeats. It is
further understood that the antisense oligonucleotides do not have
to comprise complete repeats, but can comprise fractions of a
repeat. Thus, for example, PNA-CAG25 is an 8.3 repeat representing
8 full repeats plus one additional nucleotide. Thus, disclosed
herein are repeats comprising 2.3, 2.6, 3.3, 3.6, 4.3, 4.6, 5.3,
5.6, 6.3, 6.6, 7.3, 7.6, 8.3, 8.6, 9.3, 9.6, 10.3, 10.6, 11.3,
11.6, 12.312.6, 13.2, 13.6, 14.3, 14.6, 15.3, 15.6, 16.3, 16.6,
17.3, 17.6, 18.3, 18.6, 19.3, 19.6, 20.3, 20.6, 21.3, 21.6, 22.3,
22.6, 23.3, 23.6, 24.3, and 24.6.
[0107] Thus, for example, treating DM1 can comprise any method or
the administration of any agent that affects spliceopathy in a
manner that ameliorates a symptom or causative event associated
with DM1. For example, a morpholino that corrects spliceopathy
associated with ClC1 or displaces MBNL1 on poly(CUG).sup.exp.
[0108] Herein is disclosed that that a antisense oligonucleotide
(AON) targeting the 3' splice site of the chloride ion channel
(ClC-1) exon 7a reverses the defect of ClC-1 alternative splicing
in two mouse models of DM. By repressing the inclusion of this
exon, the AON restores the full-length reading frame in ClC-1 mRNA,
upregulates the level of ClC-1 mRNA, increases the expression of
ClC-1 protein in the surface membrane, normalizes muscle ClC-1
current density and deactivation kinetics, and eliminates myotonic
discharges. These observations indicate that the myotonia and
chloride channelopathy in DM both result from abnormal alternative
splicing of ClC-1 and that antisense-induced exon skipping offers a
powerful method for correcting alternative splicing defects in DM.
It is therefore understood and herein contemplated that the
disclosed methods can comprise methods of treating DM, wherein the
myotonia is the result of channelopathy resulting from
spliceopathy. Therefore, disclosed herein are methods or treating
myotonic dystrophy in a subject in need thereof comprising
administering to the subject an agent that corrects spliceopathy,
wherein the spliceopathy results in channelopathy.
[0109] 3. Delivery of the Compositions to Cells
[0110] There are a number of compositions and methods which can be
used to deliver nucleic acids to cells, either in vitro or in vivo.
These methods and compositions can largely be broken down into two
classes: viral based delivery systems and non-viral based delivery
systems. For example, the nucleic acids can be delivered through a
number of direct delivery systems such as, electroporation,
lipofection, calcium phosphate precipitation, plasmids, viral
vectors, viral nucleic acids, phage nucleic acids, phages, cosmids,
or via transfer of genetic material in cells or carriers such as
cationic liposomes. Appropriate means for transfection, including
viral vectors, chemical transfectants, or physico-mechanical
methods such as electroporation and direct diffusion of DNA, are
described by, for example, Wolff, J. A., et al., Science, 247,
1465-1468, (1990); and Wolff, J. A. Nature, 352, 815-818, (1991).
Such methods are well known in the art and readily adaptable for
use with the compositions and methods described herein. In certain
cases, the methods will be modified to specifically function with
large DNA molecules. Further, these methods can be used to target
certain diseases and cell populations by using the targeting
characteristics of the carrier.
[0111] a) Nucleic Acid Based Delivery Systems
[0112] Transfer vectors can be any nucleotide construction used to
deliver genes into cells (e.g., a plasmid), or as part of a general
strategy to deliver genes, e.g., as part of recombinant retrovirus
or adenovirus (Ram et al. Cancer Res. 53:83-88, (1993)).
[0113] As used herein, plasmid or viral vectors are agents that
transport the disclosed nucleic acids, such as MBNL1,
p(CUG).sup.exp, and CAG25 or other antisense oligonucleotide into
the cell without degradation and include a promoter yielding
expression of the gene in the cells into which it is delivered.
Viral vectors are, for example, Adenovirus, Adeno-associated virus,
Herpes virus, Vaccinia virus, Polio virus, AIDS virus, neuronal
trophic virus, Sindbis and other RNA viruses, including these
viruses with the HIV backbone. Also preferred are any viral
families which share the properties of these viruses which make
them suitable for use as vectors. Retroviruses include Murine
Maloney Leukemia virus, MMLV, and retroviruses that express the
desirable properties of MMLV as a vector. Retroviral vectors are
able to carry a larger genetic payload, i.e., a transgene or marker
gene, than other viral vectors, and for this reason are a commonly
used vector. However, they are not as useful in non-proliferating
cells. Adenovirus vectors are relatively stable and easy to work
with, have high titers, and can be delivered in aerosol
formulation, and can transfect non-dividing cells. Pox viral
vectors are large and have several sites for inserting genes, they
are thermostable and can be stored at room temperature. A preferred
embodiment is a viral vector which has been engineered so as to
suppress the immune response of the host organism, elicited by the
viral antigens. Preferred vectors of this type will carry coding
regions for Interleukin 8 or 10.
[0114] Viral vectors can have higher transaction (ability to
introduce genes) abilities than chemical or physical methods to
introduce genes into cells. Typically, viral vectors contain,
nonstructural early genes, structural late genes, an RNA polymerase
III transcript, inverted terminal repeats necessary for replication
and encapsidation, and promoters to control the transcription and
replication of the viral genome. When engineered as vectors,
viruses typically have one or more of the early genes removed and a
gene or gene/promotor cassette is inserted into the viral genome in
place of the removed viral DNA. Constructs of this type can carry
up to about 8 kb of foreign genetic material. The necessary
functions of the removed early genes are typically supplied by cell
lines which have been engineered to express the gene products of
the early genes in trans.
[0115] (1) Retroviral Vectors
[0116] A retrovirus is an animal virus belonging to the virus
family of Retroviridae, including any types, subfamilies, genus, or
tropisms. Retroviral vectors, in general, are described by Verma,
I. M., Retroviral vectors for gene transfer. In Microbiology-1985,
American Society for Microbiology, pp. 229-232, Washington, (1985),
which is incorporated by reference herein. Examples of methods for
using retroviral vectors for gene therapy are described in U.S.
Pat. Nos. 4,868,116 and 4,980,286; PCT applications WO 90/02806 and
WO 89/07136; and Mulligan, (Science 260:926-932 (1993)); the
teachings of which are incorporated herein by reference.
[0117] A retrovirus is essentially a package which has packed into
it nucleic acid cargo. The nucleic acid cargo carries with it a
packaging signal, which ensures that the replicated daughter
molecules will be efficiently packaged within the package coat. In
addition to the package signal, there are a number of molecules
which are needed in cis, for the replication, and packaging of the
replicated virus. Typically a retroviral genome, contains the gag,
pol, and env genes which are involved in the making of the protein
coat. It is the gag, pol, and env genes which are typically
replaced by the foreign DNA that it is to be transferred to the
target cell. Retrovirus vectors typically contain a packaging
signal for incorporation into the package coat, a sequence which
signals the start of the gag transcription unit, elements necessary
for reverse transcription, including a primer binding site to bind
the tRNA primer of reverse transcription, terminal repeat sequences
that guide the switch of RNA strands during DNA synthesis, a purine
rich sequence 5' to the 3'LTR that serve as the priming site for
the synthesis of the second strand of DNA synthesis, and specific
sequences near the ends of the LTRs that enable the insertion of
the DNA state of the retrovirus to insert into the host genome. The
removal of the gag, pol, and env genes allows for about 8 kb of
foreign sequence to be inserted into the viral genome, become
reverse transcribed, and upon replication be packaged into a new
retroviral particle. This amount of nucleic acid is sufficient for
the delivery of a one to many genes depending on the size of each
transcript. It is preferable to include either positive or negative
selectable markers along with other genes in the insert.
[0118] Since the replication machinery and packaging proteins in
most retroviral vectors have been removed (gag, pol, and env), the
vectors are typically generated by placing them into a packaging
cell line. A packaging cell line is a cell line which has been
transfected or transformed with a retrovirus that contains the
replication and packaging machinery, but lacks any packaging
signal. When the vector carrying the DNA of choice is transfected
into these cell lines, the vector containing the gene of interest
is replicated and packaged into new retroviral particles, by the
machinery provided in cis by the helper cell. The genomes for the
machinery are not packaged because they lack the necessary
signals.
[0119] (2) Adenoviral Vectors
[0120] The construction of replication-defective adenoviruses has
been described (Berkner et al., J. Virology 61:1213-1220 (1987);
Massie et al., Mol. Cell. Biol. 6:2872-2883 (1986); Haj-Ahmad et
al., J. Virology 57:267-274 (1986); Davidson et al., J. Virology
61:1226-1239 (1987); Zhang "Generation and identification of
recombinant adenovirus by liposome-mediated transfection and PCR
analysis" BioTechniques 15:868-872 (1993)). The benefit of the use
of these viruses as vectors is that they are limited in the extent
to which they can spread to other cell types, since they can
replicate within an initial infected cell, but are unable to form
new infectious viral particles. Recombinant adenoviruses have been
shown to achieve high efficiency gene transfer after direct, in
vivo delivery to airway epithelium, hepatocytes, vascular
endothelium, CNS parenchyma and a number of other tissue sites
(Morsy, J. Clin. Invest. 92:1580-1586 (1993); Kirshenbaum, J. Clin.
Invest. 92:381-387 (1993); Roessler, J. Clin. Invest. 92:1085-1092
(1993); Moullier, Nature Genetics 4:154-159 (1993); La Salle,
Science 259:988-990 (1993); Gomez-Foix, J. Biol. Chem.
267:25129-25134 (1992); Rich, Human Gene Therapy 4:461-476 (1993);
Zabner, Nature Genetics 6:75-83 (1994); Guzman, Circulation
Research 73:1201-1207 (1993); Bout, Human Gene Therapy 5:3-10
(1994); Zabner, Cell 75:207-216 (1993); Caillaud, Eur. J.
Neuroscience 5:1287-1291 (1993); and Ragot, J. Gen. Virology
74:501-507 (1993)). Recombinant adenoviruses achieve gene
transduction by binding to specific cell surface receptors, after
which the virus is internalized by receptor-mediated endocytosis,
in the same manner as wild type or replication-defective adenovirus
(Chardonnet and Dales, Virology 40:462-477 (1970); Brown and
Burlingham, J. Virology 12:386-396 (1973); Svensson and Persson, J.
Virology 55:442-449 (1985); Seth, et al., J. Virol. 51:650-655
(1984); Seth, et al., Mol. Cell. Biol. 4:1528-1533 (1984); Varga et
al., J. Virology 65:6061-6070 (1991); Wickham et al., Cell
73:309-319 (1993)).
[0121] A viral vector can be one based on an adenovirus which has
had the E1 gene removed and these virons are generated in a cell
line such as the human 293 cell line. In another preferred
embodiment both the E1 and E3 genes are removed from the adenovirus
genome.
[0122] (3) Adeno-Asscociated Viral Vectors
[0123] Another type of viral vector is based on an adeno-associated
virus (AAV). This defective parvovirus is a preferred vector
because it can infect many cell types and is nonpathogenic to
humans. AAV type vectors can transport about 4 to 5 kb and wild
type AAV is known to stably insert into chromosome 19. Vectors
which contain this site specific integration property are
preferred. An especially preferred embodiment of this type of
vector is the P4.1 C vector produced by Avigen, San Francisco,
Calif., which can contain the herpes simplex virus thymidine kinase
gene, HSV-tk, and/or a marker gene, such as the gene encoding the
green fluorescent protein, GFP.
[0124] In another type of AAV virus, the AAV contains a pair of
inverted terminal repeats (ITRs) which flank at least one cassette
containing a promoter which directs cell-specific expression
operably linked to a heterologous gene. Heterologous in this
context refers to any nucleotide sequence or gene which is not
native to the AAV or B19 parvovirus.
[0125] Typically the AAV and B19 coding regions have been deleted,
resulting in a safe, noncytotoxic vector. The AAV ITRs, or
modifications thereof, confer infectivity and site-specific
integration, but not cytotoxicity, and the promoter directs
cell-specific expression. U.S. Pat. No. 6,261,834 is herein
incorporated by reference for material related to the AAV
vector.
[0126] The disclosed vectors thus provide DNA molecules which are
capable of integration into a mammalian chromosome without
substantial toxicity.
[0127] The inserted genes in viral and retroviral usually contain
promoters, and/or enhancers to help control the expression of the
desired gene product. A promoter is generally a sequence or
sequences of DNA that function when in a relatively fixed location
in regard to the transcription start site. A promoter contains core
elements required for basic interaction of RNA polymerase and
transcription factors, and may contain upstream elements and
response elements.
[0128] (4) Large Payload Viral Vectors
[0129] Molecular genetic experiments with large human herpesviruses
have provided a means whereby large heterologous DNA fragments can
be cloned, propagated and established in cells permissive for
infection with herpesviruses (Sun et al., Nature Genetics 8: 33-41,
1994; Cotter and Robertson, Curr Opin Mol Ther 5: 633-644, 1999).
These large DNA viruses (herpes simplex virus (HSV) and
Epstein-Barr virus (EBV), have the potential to deliver fragments
of human heterologous DNA >150 kb to specific cells. EBV
recombinants can maintain large pieces of DNA in the infected
B-cells as episomal DNA. Individual clones carried human genomic
inserts up to 330 kb appeared genetically stable the maintenance of
these episomes requires a specific EBV nuclear protein, EBNA1,
constitutively expressed during infection with EBV. Additionally,
these vectors can be used for transfection, where large amounts of
protein can be generated transiently in vitro. Herpesvirus amplicon
systems are also being used to package pieces of DNA >220 kb and
to infect cells that can stably maintain DNA as episomes.
[0130] Other useful systems include, for example, replicating and
host-restricted non-replicating vaccinia virus vectors.
[0131] b) Non-Nucleic Acid Based Systems
[0132] The disclosed compositions can be delivered to the target
cells in a variety of ways. For example, the compositions can be
delivered through electroporation, or through lipofection, or
through calcium phosphate precipitation. The delivery mechanism
chosen will depend in part on the type of cell targeted and whether
the delivery is occurring for example in vivo or in vitro.
[0133] Thus, the compositions can comprise, in addition to the
disclosed CAG25 or MBNL1 vectors for example, lipids such as
liposomes, such as cationic liposomes (e.g., DOTMA, DOPE,
DC-cholesterol) or anionic liposomes, or protein transduction
domains. Liposomes can further comprise proteins to facilitate
targeting a particular cell, if desired. Administration of a
composition comprising a compound and a cationic liposome can be
administered to the blood afferent to a target organ or inhaled
into the respiratory tract to target cells of the respiratory
tract. Regarding liposomes, see, e.g., Brigham et al. Am. J. Resp.
Cell. Mol. Biol. 1:95-100 (1989); Feigner et al. Proc. Natl. Acad.
Sci. USA 84:7413-7417 (1987); U.S. Pat. No. 4,897,355. It is
understood that protein transduction domains, can comprise a domain
from a larger protein, such as HIV-1 tat protein or herpes virus
VP22, or an engineered peptide such as Endo-Porter.TM..
Furthermore, the compound can be administered as a component of a
microcapsule that can be targeted to specific cell types, such as
macrophages, or where the diffusion of the compound or delivery of
the compound from the microcapsule is designed for a specific rate
or dosage.
[0134] In the methods described above which include the
administration and uptake of exogenous DNA into the cells of a
subject (i.e., gene transduction or transfection), delivery of the
compositions to cells can be via a variety of mechanisms. As one
example, delivery can be via a liposome, using commercially
available liposome preparations such as LIPOFECTIN, LIPOFECTAMINE
(GIBCO-BRL, Inc., Gaithersburg, Md.), SUPERFECT (Qiagen, Inc.
Hilden, Germany) and TRANSFECTAM (Promega Biotec, Inc., Madison,
Wis.), as well as other liposomes developed according to procedures
standard in the art. In addition, the disclosed nucleic acid or
vector can be delivered in vivo by electroporation, the technology
for which is available from Genetronics, Inc. (San Diego, Calif.)
as well as by means of a SONOPORATION machine (ImaRx Pharmaceutical
Corp., Tucson, Ariz.).
[0135] The materials may be in solution, suspension (for example,
incorporated into microparticles, liposomes, or cells). These may
be targeted to a particular cell type via antibodies, receptors, or
receptor ligands. The following references are examples of the use
of this technology to target specific proteins to tumor tissue
(Senter, et al., Bioconjugate Chem., 2:447-451, (1991); Bagshawe,
K. D., Br. J. Cancer, 60:275-281, (1989); Bagshawe, et al., Br. J.
Cancer, 58:700-703, (1988); Senter, et al., Bioconjugate Chem.,
4:3-9, (1993); Battelli, et al., Cancer Immunol. Immunother.,
35:421-425, (1992); Pietersz and McKenzie, Immunolog. Reviews,
129:57-80, (1992); and Roffler, et al., Biochem. Pharmacol,
42:2062-2065, (1991)). These techniques can be used for a variety
of other specific cell types. Vehicles such as "stealth" and other
antibody conjugated liposomes (including lipid mediated drug
targeting to colonic carcinoma), receptor mediated targeting of DNA
through cell specific ligands, lymphocyte directed tumor targeting,
and highly specific therapeutic retroviral targeting of murine
glioma cells in vivo. The following references are examples of the
use of this technology to target specific proteins to tumor tissue
(Hughes et al., Cancer Research, 49:6214-6220, (1989); and
Litzinger and Huang, Biochimica et Biophysica Acta, 1104:179-187,
(1992)). In general, receptors are involved in pathways of
endocytosis, either constitutive or ligand induced. These receptors
cluster in clathrin-coated pits, enter the cell via clathrin-coated
vesicles, pass through an acidified endosome in which the receptors
are sorted, and then either recycle to the cell surface, become
stored intracellularly, or are degraded in lysosomes. The
internalization pathways serve a variety of functions, such as
nutrient uptake, removal of activated proteins, clearance of
macromolecules, opportunistic entry of viruses and toxins,
dissociation and degradation of ligand, and receptor-level
regulation. Many receptors follow more than one intracellular
pathway, depending on the cell type, receptor concentration, type
of ligand, ligand valency, and ligand concentration. Molecular and
cellular mechanisms of receptor-mediated endocytosis has been
reviewed (Brown and Greene, DNA and Cell Biology 10:6, 399-409
(1991)).
[0136] Nucleic acids that are delivered to cells which are to be
integrated into the host cell genome, typically contain integration
sequences. These sequences are often viral related sequences,
particularly when viral based systems are used. These viral
intergration systems can also be incorporated into nucleic acids
which are to be delivered using a non-nucleic acid based system of
deliver, such as a liposome, so that the nucleic acid contained in
the delivery system can be come integrated into the host
genome.
[0137] Other general techniques for integration into the host
genome include, for example, systems designed to promote homologous
recombination with the host genome. These systems typically rely on
sequence flanking the nucleic acid to be expressed that has enough
homology with a target sequence within the host cell genome that
recombination between the vector nucleic acid and the target
nucleic acid takes place, causing the delivered nucleic acid to be
integrated into the host genome. These systems and the methods
necessary to promote homologous recombination are known to those of
skill in the art.
[0138] c) In Vivo/Ex Vivo
[0139] As described above, the compositions can be administered in
a pharmaceutically acceptable carrier and can be delivered to the
subject's cells in vivo and/or ex vivo by a variety of mechanisms
well known in the art (e.g., uptake of naked DNA, liposome fusion,
intramuscular injection of DNA via a gene gun, endocytosis and the
like).
[0140] If ex vivo methods are employed, cells or tissues can be
removed and maintained outside the body according to standard
protocols well known in the art. The compositions can be introduced
into the cells via any gene transfer mechanism, such as, for
example, calcium phosphate mediated gene delivery, electroporation,
microinjection or proteoliposomes. The transduced cells can then be
infused (e.g., in a pharmaceutically acceptable carrier) or
homotopically transplanted back into the subject per standard
methods for the cell or tissue type. Standard methods are known for
transplantation or infusion of various cells into a subject.
[0141] 4. Expression Systems
[0142] The nucleic acids that are delivered to cells typically
contain expression controlling systems. For example, the inserted
genes in viral and retroviral systems usually contain promoters,
and/or enhancers to help control the expression of the desired gene
product. A promoter is generally a sequence or sequences of DNA
that function when in a relatively fixed location in regard to the
transcription start site. A promoter contains core elements
required for basic interaction of RNA polymerase and transcription
factors, and may contain upstream elements and response
elements.
[0143] a) Viral Promoters and Enhancers
[0144] Preferred promoters controlling transcription from vectors
in mammalian host cells may be obtained from various sources, for
example, the genomes of viruses such as: polyoma, Simian Virus 40
(SV40), adenovirus, retroviruses, hepatitis-B virus and most
preferably cytomegalovirus, or from heterologous mammalian
promoters, e.g. beta actin promoter. The early and late promoters
of the SV40 virus are conveniently obtained as an SV40 restriction
fragment which also contains the SV40 viral origin of replication
(Fiers et al., Nature, 273: 113 (1978)). The immediate early
promoter of the human cytomegalovirus is conveniently obtained as a
HindIII E restriction fragment (Greenway, P. J. et al., Gene 18:
355-360 (1982)). Of course, promoters from the host cell or related
species also are useful herein.
[0145] Enhancer generally refers to a sequence of DNA that
functions at no fixed distance from the transcription start site
and can be either 5' (Laimins, L. et al., Proc. Natl. Acad. Sci.
78: 993 (1981)) or 3' (Lusky, M. L., et al., Mol. Cell. Bio. 3:
1108 (1983)) to the transcription unit. Furthermore, enhancers can
be within an intron (Banerji, J. L. et al., Cell 33: 729 (1983)) as
well as within the coding sequence itself (Osborne, T. F., et al.,
Mol. Cell. Bio. 4: 1293 (1984)). They are usually between 10 and
300 by in length, and they function in cis. Enhancers function to
increase transcription from nearby promoters. Enhancers also often
contain response elements that mediate the regulation of
transcription. Promoters can also contain response elements that
mediate the regulation of transcription. Enhancers often determine
the regulation of expression of a gene. While many enhancer
sequences are now known from mammalian genes (globin, elastase,
albumin, -fetoprotein and insulin), typically one will use an
enhancer from a eukaryotic cell virus for general expression.
Preferred examples are the SV40 enhancer on the late side of the
replication origin (bp 100-270), the cytomegalovirus early promoter
enhancer, the polyoma enhancer on the late side of the replication
origin, and adenovirus enhancers.
[0146] The promotor and/or enhancer may be specifically activated
either by light or specific chemical events which trigger their
function. Systems can be regulated by reagents such as tetracycline
and dexamethasone. There are also ways to enhance viral vector gene
expression by exposure to irradiation, such as gamma irradiation,
or alkylating chemotherapy drugs.
[0147] In certain embodiments the promoter and/or enhancer region
can act as a constitutive promoter and/or enhancer to maximize
expression of the region of the transcription unit to be
transcribed. In certain constructs the promoter and/or enhancer
region be active in all eukaryotic cell types, even if it is only
expressed in a particular type of cell at a particular time. A
preferred promoter of this type is the CMV promoter (650 bases).
Other preferred promoters are SV40 promoters, cytomegalovirus (full
length promoter), and retroviral vector LTF.
[0148] It has been shown that all specific regulatory elements can
be cloned and used to construct expression vectors that are
selectively expressed in specific cell types such as melanoma
cells. The glial fibrillary acetic protein (GFAP) promoter has been
used to selectively express genes in cells of glial origin.
[0149] Expression vectors used in eukaryotic host cells (yeast,
fungi, insect, plant, animal, human or nucleated cells) may also
contain sequences necessary for the termination of transcription
which may affect mRNA expression. These regions are transcribed as
polyadenylated segments in the untranslated portion of the mRNA
encoding tissue factor protein. The 3' untranslated regions also
include transcription termination sites. It is preferred that the
transcription unit also contains a polyadenylation region. One
benefit of this region is that it increases the likelihood that the
transcribed unit will be processed and transported like mRNA. The
identification and use of polyadenylation signals in expression
constructs is well established. It is preferred that homologous
polyadenylation signals be used in the transgene constructs. In
certain transcription units, the polyadenylation region is derived
from the SV40 early polyadenylation signal and consists of about
400 bases. It is also preferred that the transcribed units contain
other standard sequences alone or in combination with the above
sequences improve expression from, or stability of, the
construct.
[0150] b) Markers
[0151] The viral vectors can include nucleic acid sequence encoding
a marker product. This marker product is used to determine if the
gene has been delivered to the cell and once delivered is being
expressed. Preferred marker genes are the E. Coli lacZ gene, which
encodes .beta.-galactosidase, and green fluorescent protein.
[0152] In some embodiments the marker may be a selectable marker.
Examples of suitable selectable markers for mammalian cells are
dihydrofolate reductase (DHFR), thymidine kinase, neomycin,
neomycin analog G418, hydromycin, and puromycin. When such
selectable markers are successfully transferred into a mammalian
host cell, the transformed mammalian host cell can survive if
placed under selective pressure. There are two widely used distinct
categories of selective regimes. The first category is based on a
cell's metabolism and the use of a mutant cell line which lacks the
ability to grow independent of a supplemented media. Two examples
are: CHO DHFR-cells and mouse LTK-cells. These cells lack the
ability to grow without the addition of such nutrients as thymidine
or hypoxanthine. Because these cells lack certain genes necessary
for a complete nucleotide synthesis pathway, they cannot survive
unless the missing nucleotides are provided in a supplemented
media. An alternative to supplementing the media is to introduce an
intact DHFR or TK gene into cells lacking the respective genes,
thus altering their growth requirements. Individual cells which
were not transformed with the DHFR or TK gene will not be capable
of survival in non-supplemented media.
[0153] The second category is dominant selection which refers to a
selection scheme used in any cell type and does not require the use
of a mutant cell line. These schemes typically use a drug to arrest
growth of a host cell. Those cells which have a novel gene would
express a protein conveying drug resistance and would survive the
selection. Examples of such dominant selection use the drugs
neomycin, (Southern P. and Berg, P., J. Molec. Appl. Genet. 1: 327
(1982)), mycophenolic acid, (Mulligan, R. C. and Berg, P. Science
209: 1422 (1980)) or hygromycin, (Sugden, B. et al., Mol. Cell.
Biol. 5: 410-413 (1985)). The three examples employ bacterial genes
under eukaryotic control to convey resistance to the appropriate
drug G418 or neomycin (geneticin), xgpt (mycophenolic acid) or
hygromycin, respectively. Others include the neomycin analog G418
and puramycin.
C. COMPOSITIONS
[0154] Disclosed are the components to be used to prepare the
disclosed compositions as well as the compositions themselves to be
used within the methods disclosed herein. These and other materials
are disclosed herein, and it is understood that when combinations,
subsets, interactions, groups, etc. of these materials are
disclosed that while specific reference of each various individual
and collective combinations and permutation of these compounds may
not be explicitly disclosed, each is specifically contemplated and
described herein. For example, if a particular CAG25 antisense
oligonucleotide is disclosed and discussed and a number of
modifications that can be made to a number of molecules including
the CAG25 antisense oligonucleotide are discussed, specifically
contemplated is each and every combination and permutation of CAG25
antisense oligonucleotide and the modifications that are possible
unless specifically indicated to the contrary. Thus, if a class of
molecules A, B, and C are disclosed as well as a class of molecules
D, E, and F and an example of a combination molecule, A-D is
disclosed, then even if each is not individually recited each is
individually and collectively contemplated meaning combinations,
A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are considered
disclosed. Likewise, any subset or combination of these is also
disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E
would be considered disclosed. This concept applies to all aspects
of this application including, but not limited to, steps in methods
of making and using the disclosed compositions. Thus, if there are
a variety of additional steps that can be performed it is
understood that each of these additional steps can be performed
with any specific embodiment or combination of embodiments of the
disclosed methods.
[0155] Disclosed herein are compositions that can be used to treat
DM1 or DM2. For example, disclosed herein are morpholinos such as
CAG25 and the antisense oligonucleotide as set forth in SEQ ID NO:
4, that can treat DM1. Also disclosed are small molecules such as
aminoglycoside antibiotics neomycin and gentamicin. It is
understood that other aminoglycoside family members such as those
disclosed in FIG. 18 are also disclosed for treating DM1 or
DM2.
[0156] Herein, "morpholino" refers to synthetic oligonucleotides
which have standard nucleic acid bases, bound to morpholine rings
rather than the deoxyribose rings of DNA and the bases are linked
through phosphorodiamidate groups instead of phosphates. The
morpholino operates by binding to complementary RNA and blocks
acceess to the RNA by other molecules. Disclosed herein, the
morpholino may also be used to displace a molecule that is already
bound to the complementary RNA strand.
[0157] 1. Homology/identity
[0158] It is understood that one way to define any known variants
and derivatives or those that might arise, of the disclosed genes
and proteins herein is through defining the variants and
derivatives in terms of homology to specific known sequences. For
example SEQ ID NO: 1 sets forth a particular sequence of an MBNL1
and SEQ ID NO: 2 sets forth a particular sequence of the protein
encoded by SEQ ID NO:1, an MBNL1 protein.
[0159] Specifically disclosed are variants of these and other genes
and proteins herein disclosed which have at least, 70, 71, 72, 73,
74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,
91, 92, 93, 94, 95, 96, 97, 98, 99 percent homology to the stated
sequence. Those of skill in the art readily understand how to
determine the homology of two proteins or nucleic acids, such as
genes. For example, the homology can be calculated after aligning
the two sequences so that the homology is at its highest level.
[0160] Another way of calculating homology can be performed by
published algorithms. Optimal alignment of sequences for comparison
may be conducted by the local homology algorithm of Smith and
Waterman Adv. Appl. Math. 2: 482 (1981), by the homology alignment
algorithm of Needleman and Wunsch, J. Mol. Biol. 48: 443 (1970), by
the search for similarity method of Pearson and Lipman, Proc. Natl.
Acad. Sci. U.S.A. 85: 2444 (1988), by computerized implementations
of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the
Wisconsin Genetics Software Package, Genetics Computer Group, 575
Science Dr., Madison, Wis.), or by inspection.
[0161] The same types of homology can be obtained for nucleic acids
by for example the algorithms disclosed in Zuker, M. Science
244:48-52, 1989, Jaeger et al. Proc. Natl. Acad. Sci. USA
86:7706-7710, 1989, Jaeger et al. Methods Enzymol. 183:281-306,
1989 which are herein incorporated by reference for at least
material related to nucleic acid alignment.
[0162] 2. Hybridization/Selective Hybridization
[0163] The term hybridization typically means a sequence driven
interaction between at least two nucleic acid molecules, such as a
primer or a probe and a gene. Sequence driven interaction means an
interaction that occurs between two nucleotides or nucleotide
analogs or nucleotide derivatives in a nucleotide specific manner.
For example, G interacting with C or A interacting with T are
sequence driven interactions. Typically sequence driven
interactions occur on the Watson-Crick face or Hoogsteen face of
the nucleotide. The hybridization of two nucleic acids is affected
by a number of conditions and parameters known to those of skill in
the art. For example, the salt concentrations, pH, and temperature
of the reaction all affect whether two nucleic acid molecules will
hybridize.
[0164] Parameters for selective hybridization between two nucleic
acid molecules are well known to those of skill in the art. For
example, in some embodiments selective hybridization conditions can
be defined as stringent hybridization conditions. For example,
stringency of hybridization is controlled by both temperature and
salt concentration of either or both of the hybridization and
washing steps. For example, the conditions of hybridization to
achieve selective hybridization may involve hybridization in high
ionic strength solution (6.times.SSC or 6.times.SSPE) at a
temperature that is about 12-25.degree. C. below the Tm (the
melting temperature at which half of the molecules dissociate from
their hybridization partners) followed by washing at a combination
of temperature and salt concentration chosen so that the washing
temperature is about 5.degree. C. to 20.degree. C. below the Tm.
The temperature and salt conditions are readily determined
empirically in preliminary experiments in which samples of
reference DNA immobilized on filters are hybridized to a labeled
nucleic acid of interest and then washed under conditions of
different stringencies. Hybridization temperatures are typically
higher for DNA-RNA and RNA-RNA hybridizations. The conditions can
be used as described above to achieve stringency, or as is known in
the art. (Sambrook et al., Molecular Cloning: A Laboratory Manual,
2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.,
1989; Kunkel et al. Methods Enzymol. 1987:154:367, 1987 which is
herein incorporated by reference for material at least related to
hybridization of nucleic acids). A preferable stringent
hybridization condition for a DNA:DNA hybridization can be at about
68.degree. C. (in aqueous solution) in 6.times.SSC or 6.times.SSPE
followed by washing at 68.degree. C. Stringency of hybridization
and washing, if desired, can be reduced accordingly as the degree
of complementarity desired is decreased, and further, depending
upon the G-C or A-T richness of any area wherein variability is
searched for. Likewise, stringency of hybridization and washing, if
desired, can be increased accordingly as homology desired is
increased, and further, depending upon the G-C or A-T richness of
any area wherein high homology is desired, all as known in the
art.
[0165] Another way to define selective hybridization is by looking
at the amount (percentage) of one of the nucleic acids bound to the
other nucleic acid. For example, in some embodiments selective
hybridization conditions would be when at least about, 60, 65, 70,
71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,
88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 percent of the
limiting nucleic acid is bound to the non-limiting nucleic acid.
Typically, the non-limiting primer is in for example, 10 or 100 or
1000 fold excess. This type of assay can be performed at under
conditions where both the limiting and non-limiting primer are for
example, 10 fold or 100 fold or 1000 fold below their k.sub.d, or
where only one of the nucleic acid molecules is 10 fold or 100 fold
or 1000 fold or where one or both nucleic acid molecules are above
their k.sub.d.
[0166] Another way to define selective hybridization is by looking
at the percentage of primer that gets enzymatically manipulated
under conditions where hybridization is required to promote the
desired enzymatic manipulation. For example, in some embodiments
selective hybridization conditions would be when at least about,
60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,
85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100
percent of the primer is enzymatically manipulated under conditions
which promote the enzymatic manipulation, for example if the
enzymatic manipulation is DNA extension, then selective
hybridization conditions would be when at least about 60, 65, 70,
71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,
88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 percent of the
primer molecules are extended. Preferred conditions also include
those suggested by the manufacturer or indicated in the art as
being appropriate for the enzyme performing the manipulation.
[0167] Just as with homology, it is understood that there are a
variety of methods herein disclosed for determining the level of
hybridization between two nucleic acid molecules. It is understood
that these methods and conditions may provide different percentages
of hybridization between two nucleic acid molecules, but unless
otherwise indicated meeting the parameters of any of the methods
would be sufficient. For example if 80% hybridization was required
and as long as hybridization occurs within the required parameters
in any one of these methods it is considered disclosed herein.
[0168] It is understood that those of skill in the art understand
that if a composition or method meets any one of these criteria for
determining hybridization either collectively or singly it is a
composition or method that is disclosed herein.
[0169] 3. Nucleic Acids
[0170] There are a variety of molecules disclosed herein that are
nucleic acid based, including for example the nucleic acids that
encode, for example, CAG25 as well as any other proteins disclosed
herein, as well as various functional nucleic acids. The disclosed
nucleic acids are made up of for example, nucleotides, nucleotide
analogs, or nucleotide substitutes. Non-limiting examples of these
and other molecules are discussed herein. It is understood that for
example, when a vector is expressed in a cell, that the expressed
mRNA will typically be made up of A, C, G, and U. Likewise, it is
understood that if, for example, an antisense molecule is
introduced into a cell or cell environment through for example
exogenous delivery, it is advantagous that the antisense molecule
be made up of nucleotide analogs that reduce the degradation of the
antisense molecule in the cellular environment.
[0171] a) Nucleotides and Related Molecules
[0172] A nucleotide is a molecule that contains a base moiety, a
sugar moiety and a phosphate moiety. Nucleotides can be linked
together through their phosphate moieties and sugar moieties
creating an internucleoside linkage. The base moiety of a
nucleotide can be adenin-9-yl (A), cytosin-1-yl (C), guanin-9-yl
(G), uracil-1-yl (U), and thymin-1-yl (T). The sugar moiety of a
nucleotide is a ribose or a deoxyribose. The phosphate moiety of a
nucleotide is pentavalent phosphate. An non-limiting example of a
nucleotide would be 3'-AMP (3'-adenosine monophosphate) or 5'-GMP
(5'-guanosine monophosphate).
[0173] A nucleotide analog is a nucleotide which contains some type
of modification to either the base, sugar, or phosphate moieties.
Modifications to nucleotides are well known in the art and would
include for example, 5-methylcytosine (5-me-C), 5-hydroxymethyl
cytosine, xanthine, hypoxanthine, and 2-aminoadenine as well as
modifications at the sugar or phosphate moieties.
[0174] Nucleotide substitutes are molecules having similar
functional properties to nucleotides, but which do not contain a
phosphate moiety, such as peptide nucleic acid (PNA). Nucleotide
substitutes are molecules that will recognize nucleic acids in a
Watson-Crick or Hoogsteen manner, but which are linked together
through a moiety other than a phosphate moiety. Nucleotide
substitutes are able to conform to a double helix type structure
when interacting with the appropriate target nucleic acid.
[0175] It is also possible to link other types of molecules
(conjugates) to nucleotides or nucleotide analogs to enhance for
example, cellular uptake. Conjugates can be chemically linked to
the nucleotide or nucleotide analogs. Such conjugates include but
are not limited to lipid moieties such as a cholesterol moiety.
(Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86,
6553-6556),
[0176] A Watson-Crick interaction is at least one interaction with
the Watson-Crick face of a nucleotide, nucleotide analog, or
nucleotide substitute. The Watson-Crick face of a nucleotide,
nucleotide analog, or nucleotide substitute includes the C2, N1,
and C6 positions of a purine based nucleotide, nucleotide analog,
or nucleotide substitute and the C2, N3, C4 positions of a
pyrimidine based nucleotide, nucleotide analog, or nucleotide
substitute.
[0177] A Hoogsteen interaction is the interaction that takes place
on the Hoogsteen face of a nucleotide or nucleotide analog, which
is exposed in the major groove of duplex DNA. The Hoogsteen face
includes the N7 position and reactive groups (NH2 or O) at the C6
position of purine nucleotides.
[0178] b) Sequences
[0179] There are a variety of sequences related to the protein
molecules disclosed herein, for example MBNL1, or any of the
nucleic acids disclosed herein for making CAG25, all of which are
encoded by nucleic acids or are nucleic acids. The sequences for
the human analogs of these genes, as well as other analogs, and
alleles of these genes, and splice variants and other types of
variants, are available in a variety of protein and gene databases,
including Genbank. Those sequences available at the time of filing
this application at Genbank are herein incorporated by reference in
their entireties as well as for individual subsequences contained
therein. Genbank can be accessed at
http://www.ncbi.nih.gov/entrez/query.fcgi. Those of skill in the
art understand how to resolve sequence discrepancies and
differences and to adjust the compositions and methods relating to
a particular sequence to other related sequences. Primers and/or
probes can be designed for any given sequence given the information
disclosed herein and known in the art.
[0180] c) Primers and Probes
[0181] Disclosed are compositions including primers and probes,
which are capable of interacting with the disclosed nucleic acids,
such as the poly(CUG).sup.exp as disclosed herein. In certain
embodiments the primers are used to support DNA amplification
reactions. Typically the primers will be capable of being extended
in a sequence specific manner. Extension of a primer in a sequence
specific manner includes any methods wherein the sequence and/or
composition of the nucleic acid molecule to which the primer is
hybridized or otherwise associated directs or influences the
composition or sequence of the product produced by the extension of
the primer. Extension of the primer in a sequence specific manner
therefore includes, but is not limited to, PCR, DNA sequencing, DNA
extension, DNA polymerization, RNA transcription, or reverse
transcription. Techniques and conditions that amplify the primer in
a sequence specific manner are preferred. In certain embodiments
the primers are used for the DNA amplification reactions, such as
PCR or direct sequencing. It is understood that in certain
embodiments the primers can also be extended using non-enzymatic
techniques, where for example, the nucleotides or oligonucleotides
used to extend the primer are modified such that they will
chemically react to extend the primer in a sequence specific
manner. Typically the disclosed primers hybridize with the
disclosed nucleic acids or region of the nucleic acids or they
hybridize with the complement of the nucleic acids or complement of
a region of the nucleic acids.
[0182] The size of the primers or probes for interaction with the
nucleic acids in certain embodiments can be any size that supports
the desired enzymatic manipulation of the primer, such as DNA
amplification or the simple hybridization of the probe or primer. A
typical primer or probe would be at least 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,
30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,
47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,
64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,
81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,
98, 99, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375,
400, 425, 450, 475, 500, 550, 600, 650, 700, 750, 800, 850, 900,
950, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000, 3500, or
4000 nucleotides long.
[0183] In other embodiments a primer or probe can be less than or
equal to 6, 7, 8, 9, 10, 11, 12 13, 14, 15, 16, 17, 18, 19, 20, 21,
22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,
39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,
56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,
73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,
90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 125, 150, 175, 200,
225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 550,
600, 650, 700, 750, 800, 850, 900, 950, 1000, 1250, 1500, 1750,
2000, 2250, 2500, 2750, 3000, 3500, or 4000 nucleotides long.
[0184] In certain embodiments this product is at least 20, 21, 22,
23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,
40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,
57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,
74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,
91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 125, 150, 175, 200, 225,
250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 550, 600,
650, 700, 750, 800, 850, 900, 950, 1000, 1250, 1500, 1750, 2000,
2250, 2500, 2750, 3000, 3500, or 4000 nucleotides long.
[0185] In other embodiments the product is less than or equal to
20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,
37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,
54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,
71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,
88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 125, 150, 175,
200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500,
550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1250, 1500,
1750, 2000, 2250, 2500, 2750, 3000, 3500, or 4000 nucleotides
long.
[0186] d) Functional Nucleic Acids
[0187] Functional nucleic acids are nucleic acid molecules that
have a specific function, such as binding a target molecule or
catalyzing a specific reaction. Functional nucleic acid molecules
can be divided into the following categories, which are not meant
to be limiting. For example, functional nucleic acids include
antisense molecules, aptamers, ribozymes, triplex forming
molecules, and external guide sequences. The functional nucleic
acid molecules can act as affectors, inhibitors, modulators, and
stimulators of a specific activity possessed by a target molecule,
or the functional nucleic acid molecules can possess a de novo
activity independent of any other molecules.
[0188] Functional nucleic acid molecules can interact with any
macromolecule, such as DNA, RNA, polypeptides, or carbohydrate
chains. Thus, functional nucleic acids can interact with the mRNA
of DMPK or ClC1. Often functional nucleic acids are designed to
interact with other nucleic acids based on sequence homology
between the target molecule and the functional nucleic acid
molecule. In other situations, the specific recognition between the
functional nucleic acid molecule and the target molecule is not
based on sequence homology between the functional nucleic acid
molecule and the target molecule, but rather is based on the
formation of tertiary structure that allows specific recognition to
take place.
[0189] Antisense molecules are designed to interact with a target
nucleic acid molecule through either canonical or non-canonical
base pairing. The interaction of the antisense molecule and the
target molecule is designed to promote the destruction of the
target molecule through, for example, RNAseH mediated RNA-DNA
hybrid degradation. Alternatively the antisense molecule is
designed to interrupt a processing function that normally would
take place on the target molecule, such as transcription or
replication. Antisense molecules can be designed based on the
sequence of the target molecule. Numerous methods for optimization
of antisense efficiency by finding the most accessible regions of
the target molecule exist. Exemplary methods would be in vitro
selection experiments and DNA modification studies using DMS and
DEPC. It is preferred that antisense molecules bind the target
molecule with a dissociation constant (k.sub.d) less than or equal
to 10.sup.-6, 10.sup.-8, 10.sup.-10, or 10.sup.-12. A
representative sample of methods and techniques which aid in the
design and use of antisense molecules can be found in the following
non-limiting list of U.S. Pat. Nos. 5,135,917, 5,294,533,
5,627,158, 5,641,754, 5,691,317, 5,780,607, 5,786,138, 5,849,903,
5,856,103, 5,919,772, 5,955,590, 5,990,088, 5,994,320, 5,998,602,
6,005,095, 6,007,995, 6,013,522, 6,017,898, 6,018,042, 6,025,198,
6,033,910, 6,040,296, 6,046,004, 6,046,319, and 6,057,437.
[0190] Aptamers are molecules that interact with a target molecule,
preferably in a specific way. Typically aptamers are small nucleic
acids ranging from 15-50 bases in length that fold into defined
secondary and tertiary structures, such as stem-loops or
G-quartets. Aptamers can bind small molecules, such as ATP (U.S.
Pat. No. 5,631,146) and theophiline (U.S. Pat. No. 5,580,737), as
well as large molecules, such as reverse transcriptase (U.S. Pat.
No. 5,786,462) and thrombin (U.S. Pat. No. 5,543,293). Aptamers can
bind very tightly with k.sub.ds from the target molecule of less
than 10.sup.-12 M. It is preferred that the aptamers bind the
target molecule with a k.sub.d less than 10.sup.-6, 10.sup.-8,
10.sup.-10, or 10.sup.-12. Aptamers can bind the target molecule
with a very high degree of specificity. For example, aptamers have
been isolated that have greater than a 10000 fold difference in
binding affinities between the target molecule and another molecule
that differ at only a single position on the molecule (U.S. Pat.
No. 5,543,293). It is preferred that the aptamer have a k.sub.d
with the target molecule at least 10, 100, 1000, 10,000, or 100,000
fold lower than the k.sub.d with a background binding molecule. It
is preferred when doing the comparison for a polypeptide for
example, that the background molecule be a different polypeptide.
Representative examples of how to make and use aptamers to bind a
variety of different target molecules can be found in the following
non-limiting list of U.S. Pat. Nos. 5,476,766, 5,503,978,
5,631,146, 5,731,424, 5,780,228, 5,792,613, 5,795,721, 5,846,713,
5,858,660, 5,861,254, 5,864,026, 5,869,641, 5,958,691, 6,001,988,
6,011,020, 6,013,443, 6,020,130, 6,028,186, 6,030,776, and
6,051,698.
[0191] Ribozymes are nucleic acid molecules that are capable of
catalyzing a chemical reaction, either intramolecularly or
intermolecularly. Ribozymes are thus catalytic nucleic acid. It is
preferred that the ribozymes catalyze intermolecular reactions.
There are a number of different types of ribozymes that catalyze
nuclease or nucleic acid polymerase type reactions which are based
on ribozymes found in natural systems, such as hammerhead
ribozymes, (for example, but not limited to the following U.S. Pat.
Nos. 5,334,711, 5,436,330, 5,616,466, 5,633,133, 5,646,020,
5,652,094, 5,712,384, 5,770,715, 5,856,463, 5,861,288, 5,891,683,
5,891,684, 5,985,621, 5,989,908, 5,998,193, 5,998,203, WO 9858058
by Ludwig and Sproat, WO 9858057 by Ludwig and Sproat, and WO
9718312 by Ludwig and Sproat) hairpin ribozymes (for example, but
not limited to the following U.S. Pat. Nos. 5,631,115, 5,646,031,
5,683,902, 5,712,384, 5,856,188, 5,866,701, 5,869,339, and
6,022,962), and tetrahymena ribozymes (for example, but not limited
to the following U.S. Pat. Nos. 5,595,873 and 5,652,107). There are
also a number of ribozymes that are not found in natural systems,
but which have been engineered to catalyze specific reactions de
novo (for example, but not limited to the following U.S. Pat. Nos.
5,580,967, 5,688,670, 5,807,718, and 5,910,408). Preferred
ribozymes cleave RNA or DNA substrates, and more preferably cleave
RNA substrates. Ribozymes typically cleave nucleic acid substrates
through recognition and binding of the target substrate with
subsequent cleavage. This recognition is often based mostly on
canonical or non-canonical base pair interactions. This property
makes ribozymes particularly good candidates for target specific
cleavage of nucleic acids because recognition of the target
substrate is based on the target substrates sequence.
Representative examples of how to make and use ribozymes to
catalyze a variety of different reactions can be found in the
following non-limiting list of U.S. Pat. Nos. 5,646,042, 5,693,535,
5,731,295, 5,811,300, 5,837,855, 5,869,253, 5,877,021, 5,877,022,
5,972,699, 5,972,704, 5,989,906, and 6,017,756.
[0192] Triplex forming functional nucleic acid molecules are
molecules that can interact with either double-stranded or
single-stranded nucleic acid. When triplex molecules interact with
a target region, a structure called a triplex is formed, in which
there are three strands of DNA forming a complex dependant on both
Watson-Crick and Hoogsteen base-pairing. Triplex molecules are
preferred because they can bind target regions with high affinity
and specificity. It is preferred that the triplex forming molecules
bind the target molecule with a k.sub.d less than 10.sup.-6,
10.sup.-8, 10.sup.-10, or 10.sup.-12. Representative examples of
how to make and use triplex forming molecules to bind a variety of
different target molecules can be found in the following
non-limiting list of U.S. Pat. Nos. 5,176,996, 5,645,985,
5,650,316, 5,683,874, 5,693,773, 5,834,185, 5,869,246, 5,874,566,
and 5,962,426.
[0193] External guide sequences (EGSs) are molecules that bind a
target nucleic acid molecule forming a complex, and this complex is
recognized by RNase P, which cleaves the target molecule. EGSs can
be designed to specifically target a RNA molecule of choice. RNAse
P aids in processing transfer RNA (tRNA) within a cell. Bacterial
RNAse P can be recruited to cleave virtually any RNA sequence by
using an EGS that causes the target RNA:EGS complex to mimic the
natural tRNA substrate. (WO 92/03566 by Yale, and Forster and
Altman, Science 238:407-409 (1990)).
[0194] Similarly, eukaryotic EGS/RNAse P-directed cleavage of RNA
can be utilized to cleave desired targets within eukarotic cells.
(Yuan et al., Proc. Natl. Acad. Sci. USA 89:8006-8010 (1992); WO
93/22434 by Yale; WO 95/24489 by Yale; Yuan and Altman, EMBO J.
14:159-168 (1995), and Carrara et al., Proc. Natl. Acad. Sci. (USA)
92:2627-2631 (1995)). Representative examples of how to make and
use EGS molecules to facilitate cleavage of a variety of different
target molecules be found in the following non-limiting list of
U.S. Pat. Nos. 5,168,053, 5,624,824, 5,683,873, 5,728,521,
5,869,248, and 5,877,162.
[0195] 4. Peptides
[0196] a) Protein Variants
[0197] As discussed herein there are numerous variants of the MBNL1
protein and that are known and herein contemplated. In addition, to
the known functional MBNL1 strain variants there are derivatives of
the MBNL1 proteins which also function in the disclosed methods and
compositions. Protein variants and derivatives are well understood
to those of skill in the art and in can involve amino acid sequence
modifications. For example, amino acid sequence modifications
typically fall into one or more of three classes: substitutional,
insertional or deletional variants. Insertions include amino and/or
carboxyl terminal fusions as well as intrasequence insertions of
single or multiple amino acid residues. Insertions ordinarily will
be smaller insertions than those of amino or carboxyl terminal
fusions, for example, on the order of one to four residues.
Immunogenic fusion protein derivatives, such as those described in
the examples, are made by fusing a polypeptide sufficiently large
to confer immunogenicity to the target sequence by cross-linking in
vitro or by recombinant cell culture transformed with DNA encoding
the fusion. Deletions are characterized by the removal of one or
more amino acid residues from the protein sequence. Typically, no
more than about from 2 to 6 residues are deleted at any one site
within the protein molecule. These variants ordinarily are prepared
by site specific mutagenesis of nucleotides in the DNA encoding the
protein, thereby producing DNA encoding the variant, and thereafter
expressing the DNA in recombinant cell culture. Techniques for
making substitution mutations at predetermined sites in DNA having
a known sequence are well known, for example M13 primer mutagenesis
and PCR mutagenesis. Amino acid substitutions are typically of
single residues, but can occur at a number of different locations
at once; insertions usually will be on the order of about from 1 to
10 amino acid residues; and deletions will range about from 1 to 30
residues. Deletions or insertions preferably are made in adjacent
pairs, i.e. a deletion of 2 residues or insertion of 2 residues.
Substitutions, deletions, insertions or any combination thereof may
be combined to arrive at a final construct. The mutations must not
place the sequence out of reading frame and preferably will not
create complementary regions that could produce secondary mRNA
structure. Substitutional variants are those in which at least one
residue has been removed and a different residue inserted in its
place. Such substitutions generally are made in accordance with the
following Tables 1 and 2 and are referred to as conservative
substitutions.
TABLE-US-00001 TABLE 1 Amino Acid Abbreviations Amino Acid
Abbreviations alanine AlaA allosoleucine AIle arginine ArgR
asparagine AsnN aspartic acid AspD cysteine CysC glutamic acid GluE
glutamine GlnK glycine GlyG histidine HisH isolelucine IleI leucine
LeuL lysine LysK phenylalanine PheF proline ProP pyroglutamic Glu
acidp serine SerS threonine ThrT tyrosine TyrY tryptophan TrpW
valine ValV
TABLE-US-00002 TABLE 2 Amino Acid Substitutions Original Residue
Exemplary Conservative Substitutions, others are known in the art.
Alaser Arglys, gln Asngln; his Aspglu Cysser Glnasn, lys Gluasp
Glypro Hisasn; gln Ileleu; val Leuile; val Lysarg; gln; MetLeu; ile
Phemet; leu; tyr Serthr Thrser Trptyr Tyrtrp; phe Valile; leu
[0198] Substantial changes in function or immunological identity
are made by selecting substitutions that are less conservative than
those in Table 2, i.e., selecting residues that differ more
significantly in their effect on maintaining (a) the structure of
the polypeptide backbone in the area of the substitution, for
example as a sheet or helical conformation, (b) the charge or
hydrophobicity of the molecule at the target site or (c) the bulk
of the side chain. The substitutions which in general are expected
to produce the greatest changes in the protein properties will be
those in which (a) a hydrophilic residue, e.g. seryl or threonyl,
is substituted for (or by) a hydrophobic residue, e.g. leucyl,
isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine or proline
is substituted for (or by) any other residue; (c) a residue having
an electropositive side chain, e.g., lysyl, arginyl, or histidyl,
is substituted for (or by) an electronegative residue, e.g.,
glutamyl or aspartyl; or (d) a residue having a bulky side chain,
e.g., phenylalanine, is substituted for (or by) one not having a
side chain, e.g., glycine, in this case, (e) by increasing the
number of sites for sulfation and/or glycosylation.
[0199] For example, the replacement of one amino acid residue with
another that is biologically and/or chemically similar is known to
those skilled in the art as a conservative substitution. For
example, a conservative substitution would be replacing one
hydrophobic residue for another, or one polar residue for another.
The substitutions include combinations such as, for example, Gly,
Ala; Val, Ile, Leu; Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg; and
Phe, Tyr. Such conservatively substituted variations of each
explicitly disclosed sequence are included within the mosaic
polypeptides provided herein.
[0200] Substitutional or deletional mutagenesis can be employed to
insert sites for N-glycosylation (Asn-X-Thr/Ser) or O-glycosylation
(Ser or Thr). Deletions of cysteine or other labile residues also
may be desirable. Deletions or substitutions of potential
proteolysis sites, e.g. Arg, is accomplished for example by
deleting one of the basic residues or substituting one by
glutaminyl or histidyl residues.
[0201] Certain post-translational derivatizations are the result of
the action of recombinant host cells on the expressed polypeptide.
Glutaminyl and asparaginyl residues are frequently
post-translationally deamidated to the corresponding glutamyl and
asparyl residues. Alternatively, these residues are deamidated
under mildly acidic conditions. Other post-translational
modifications include hydroxylation of proline and lysine,
phosphorylation of hydroxyl groups of seryl or threonyl residues,
methylation of the o-amino groups of lysine, arginine, and
histidine side chains (T. E. Creighton, Proteins: Structure and
Molecular Properties, W.H. Freeman & Co., San Francisco pp
79-86 [1983]), acetylation of the N-terminal amine and, in some
instances, amidation of the C-terminal carboxyl.
[0202] It is understood that one way to define the variants and
derivatives of the disclosed proteins herein is through defining
the variants and derivatives in terms of homology/identity to
specific known sequences. For example, SEQ ID NO:1 sets forth a
particular sequence of MBNL1 and SEQ ID NO:2 sets forth a
particular sequence of a MBNL1 protein. Specifically disclosed are
variants of these and other proteins herein disclosed which have at
least, 70% or 75% or 80% or 85% or 90% or 95% homology to the
stated sequence. Those of skill in the art readily understand how
to determine the homology of two proteins. For example, the
homology can be calculated after aligning the two sequences so that
the homology is at its highest level.
[0203] Another way of calculating homology can be performed by
published algorithms. Optimal alignment of sequences for comparison
may be conducted by the local homology algorithm of Smith and
Waterman Adv. Appl. Math. 2: 482 (1981), by the homology alignment
algorithm of Needleman and Wunsch, J. Mol. Biol. 48: 443 (1970), by
the search for similarity method of Pearson and Lipman, Proc. Natl.
Acad. Sci. U.S.A. 85: 2444 (1988), by computerized implementations
of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the
Wisconsin Genetics Software Package, Genetics Computer Group, 575
Science Dr., Madison, Wis.), or by inspection.
[0204] The same types of homology can be obtained for nucleic acids
by for example the algorithms disclosed in Zuker, M. Science
244:48-52, 1989, Jaeger et al. Proc. Natl. Acad. Sci. USA
86:7706-7710, 1989, Jaeger et al. Methods Enzymol. 183:281-306,
1989 which are herein incorporated by reference for at least
material related to nucleic acid alignment.
[0205] It is understood that the description of conservative
mutations and homology can be combined together in any combination,
such as embodiments that have at least 70% homology to a particular
sequence wherein the variants are conservative mutations.
[0206] As this specification discusses various proteins and protein
sequences it is understood that the nucleic acids that can encode
those protein sequences are also disclosed. This would include all
degenerate sequences related to a specific protein sequence, i.e.
all nucleic acids having a sequence that encodes one particular
protein sequence as well as all nucleic acids, including degenerate
nucleic acids, encoding the disclosed variants and derivatives of
the protein sequences. Thus, while each particular nucleic acid
sequence may not be written out herein, it is understood that each
and every sequence is in fact disclosed and described herein
through the disclosed protein sequence. For example, one of the
many nucleic acid sequences that can encode the protein sequence
set forth in SEQ ID NO:1 is set forth in SEQ ID NO:2. It is
understood that while no amino acid sequence indicates what
particular DNA sequence encodes that protein within an organism,
where particular variants of a disclosed protein are disclosed
herein, the known nucleic acid sequence that encodes that protein
from which that protein arises is also known and herein disclosed
and described.
[0207] It is understood that there are numerous amino acid and
peptide analogs which can be incorporated into the disclosed
compositions. For example, there are numerous D amino acids or
amino acids which have a different functional substituent then the
amino acids shown in Table 1 and Table 2. The opposite stereo
isomers of naturally occurring peptides are disclosed, as well as
the stereo isomers of peptide analogs. These amino acids can
readily be incorporated into polypeptide chains by charging tRNA
molecules with the amino acid of choice and engineering genetic
constructs that utilize, for example, amber codons, to insert the
analog amino acid into a peptide chain in a site specific way
(Thorson et al., Methods in Molec. Biol. 77:43-73 (1991), Zoller,
Current Opinion in Biotechnology, 3:348-354 (1992); Ibba,
Biotechnology & Genetic Engineering Reviews 13:197-216 (1995),
Cahill et al., TIBS, 14(10):400-403 (1989); Benner, TIB Tech,
12:158-163 (1994); Ibba and Hennecke, Biotechnology, 12:678-682
(1994) all of which are herein incorporated by reference at least
for material related to amino acid analogs).
[0208] Molecules can be produced that resemble peptides, but which
are not connected via a natural peptide linkage. For example,
linkages for amino acids or amino acid analogs can include
CH.sub.2NH--, --CH.sub.2S--, --CH.sub.2--CH.sub.2--CH.dbd.CH--(cis
and trans), --COCH.sub.2--CH(OH)CH.sub.2--, and --CHH.sub.2SO--
(These and others can be found in Spatola, A. F. in Chemistry and
Biochemistry of Amino Acids, Peptides, and Proteins, B. Weinstein,
eds., Marcel Dekker, New York, p. 267 (1983); Spatola, A. F., Vega
Data (March 1983), Vol. 1, Issue 3, Peptide Backbone Modifications
(general review); Morley, Trends Pharm Sci (1980) pp. 463-468;
Hudson, D. et al., Int J Pept Prot Res 14:177-185 (1979)
(--CH.sub.2NH--, CH.sub.2CH.sub.2--); Spatola et al. Life Sci
38:1243-1249 (1986) (--CH H.sub.2--S); Hann J. Chem. Soc Perkin
Trans. 1307-314 (1982) (--CH--CH--, cis and trans); Almquist et al.
J. Med. Chem. 23:1392-1398 (1980) (--COCH.sub.2--); Jennings-White
et al. Tetrahedron Lett 23:2533 (1982) (--COCH.sub.2--); Szelke et
al. European Appin, EP 45665 CA (1982): 97:39405 (1982)
(--CH(OH)CH.sub.2--); Holladay et al. Tetrahedron. Lett
24:4401-4404 (1983) (--C(OH)CH.sub.2--); and Hruby Life Sci
31:189-199 (1982) (--CH.sub.2--S--); each of which is incorporated
herein by reference. A particularly preferred non-peptide linkage
is --CH.sub.2NH--. It is understood that peptide analogs can have
more than one atom between the bond atoms, such as b-alanine,
g-aminobutyric acid, and the like.
[0209] Amino acid analogs and analogs and peptide analogs often
have enhanced or desirable properties, such as, more economical
production, greater chemical stability, enhanced pharmacological
properties (half-life, absorption, potency, efficacy, etc.),
altered specificity (e.g., a broad-spectrum of biological
activities), reduced antigenicity, and others.
[0210] D-amino acids can be used to generate more stable peptides,
because D amino acids are not recognized by peptidases and such.
Systematic substitution of one or more amino acids of a consensus
sequence with a D-amino acid of the same type (e.g., D-lysine in
place of L-lysine) can be used to generate more stable peptides.
Cysteine residues can be used to cyclize or attach two or more
peptides together. This can be beneficial to constrain peptides
into particular conformations. (Rizo and Gierasch Ann. Rev.
Biochem. 61:387 (1992), incorporated herein by reference).
[0211] 5. Pharmaceutical Carriers/Delivery of Pharmaceutical
Products
[0212] As described above, the compositions can also be
administered in vivo in a pharmaceutically acceptable carrier. By
"pharmaceutically acceptable" is meant a material that is not
biologically or otherwise undesirable, i.e., the material may be
administered to a subject, along with the nucleic acid or vector,
without causing any undesirable biological effects or interacting
in a deleterious manner with any of the other components of the
pharmaceutical composition in which it is contained. The carrier
would naturally be selected to minimize any degradation of the
active ingredient and to minimize any adverse side effects in the
subject, as would be well known to one of skill in the art.
[0213] The compositions may be administered orally, parenterally
(e.g., intravenously), by intramuscular injection, by
intraperitoneal injection, transdermally, extracorporeally,
topically or the like, including topical intranasal administration
or administration by inhalant. As used herein, "topical intranasal
administration" means delivery of the compositions into the nose
and nasal passages through one or both of the nares and can
comprise delivery by a spraying mechanism or droplet mechanism, or
through aerosolization of the nucleic acid or vector.
Administration of the compositions by inhalant can be through the
nose or mouth via delivery by a spraying or droplet mechanism.
Delivery can also be directly to any area of the respiratory system
(e.g., lungs) via intubation. The exact amount of the compositions
required will vary from subject to subject, depending on the
species, age, weight and general condition of the subject, the
severity of the allergic disorder being treated, the particular
nucleic acid or vector used, its mode of administration and the
like. Thus, it is not possible to specify an exact amount for every
composition. However, an appropriate amount can be determined by
one of ordinary skill in the art using only routine experimentation
given the teachings herein.
[0214] Parenteral administration of the composition, if used, is
generally characterized by injection. Injectables can be prepared
in conventional forms, either as liquid solutions or suspensions,
solid forms suitable for solution of suspension in liquid prior to
injection, or as emulsions. A more recently revised approach for
parenteral administration involves use of a slow release or
sustained release system such that a constant dosage is maintained.
See, e.g., U.S. Pat. No. 3,610,795, which is incorporated by
reference herein.
[0215] The materials may be in solution, suspension (for example,
incorporated into microparticles, liposomes, or cells). These may
be targeted to a particular cell type via antibodies, receptors, or
receptor ligands. The following references are examples of the use
of this technology to target specific proteins to tumor tissue
(Senter, et al., Bioconjugate Chem., 2:447-451, (1991); Bagshawe,
K. D., Br. J. Cancer, 60:275-281, (1989); Bagshawe, et al., Br. J.
Cancer, 58:700-703, (1988); Senter, et al., Bioconjugate Chem.,
4:3-9, (1993); Battelli, et al., Cancer Immunol. Immunother.,
35:421-425, (1992); Pietersz and McKenzie, Immunolog. Reviews,
129:57-80, (1992); and Roffler, et al., Biochem. Pharmacol,
42:2062-2065, (1991)). Vehicles such as "stealth" and other
antibody conjugated liposomes (including lipid mediated drug
targeting to colonic carcinoma), receptor mediated targeting of DNA
through cell specific ligands, lymphocyte directed tumor targeting,
and highly specific therapeutic retroviral targeting of murine
glioma cells in vivo. The following references are examples of the
use of this technology to target specific proteins to tumor tissue
(Hughes et al., Cancer Research, 49:6214-6220, (1989); and
Litzinger and Huang, Biochimica et Biophysica Acta, 1104:179-187,
(1992)). In general, receptors are involved in pathways of
endocytosis, either constitutive or ligand induced. These receptors
cluster in clathrin-coated pits, enter the cell via clathrin-coated
vesicles, pass through an acidified endosome in which the receptors
are sorted, and then either recycle to the cell surface, become
stored intracellularly, or are degraded in lysosomes. The
internalization pathways serve a variety of functions, such as
nutrient uptake, removal of activated proteins, clearance of
macromolecules, opportunistic entry of viruses and toxins,
dissociation and degradation of ligand, and receptor-level
regulation. Many receptors follow more than one intracellular
pathway, depending on the cell type, receptor concentration, type
of ligand, ligand valency, and ligand concentration. Molecular and
cellular mechanisms of receptor-mediated endocytosis has been
reviewed (Brown and Greene, DNA and Cell Biology 10:6, 399-409
(1991)).
[0216] a) Pharmaceutically Acceptable Carriers
[0217] The compositions, including antibodies, can be used
therapeutically in combination with a pharmaceutically acceptable
carrier.
[0218] Suitable carriers and their formulations are described in
Remington: The Science and Practice of Pharmacy (19th ed.) ed. A.
R. Gennaro, Mack Publishing Company, Easton, Pa. 1995. Typically,
an appropriate amount of a pharmaceutically-acceptable salt is used
in the formulation to render the formulation isotonic. Examples of
the pharmaceutically-acceptable carrier include, but are not
limited to, saline, Ringer's solution and dextrose solution. The pH
of the solution is preferably from about 5 to about 8, and more
preferably from about 7 to about 7.5. Further carriers include
sustained release preparations such as semipermeable matrices of
solid hydrophobic polymers containing the antibody, which matrices
are in the form of shaped articles, e.g., films, liposomes or
microparticles. It will be apparent to those persons skilled in the
art that certain carriers may be more preferable depending upon,
for instance, the route of administration and concentration of
composition being administered.
[0219] Pharmaceutical carriers are known to those skilled in the
art. These most typically would be standard carriers for
administration of drugs to humans, including solutions such as
sterile water, saline, and buffered solutions at physiological pH.
The compositions can be administered intramuscularly or
subcutaneously. Other compounds will be administered according to
standard procedures used by those skilled in the art.
[0220] Pharmaceutical compositions may include carriers,
thickeners, diluents, buffers, preservatives, surface active agents
and the like in addition to the molecule of choice. Pharmaceutical
compositions may also include one or more active ingredients such
as antimicrobial agents, antiinflammatory agents, anesthetics, and
the like.
[0221] The pharmaceutical composition may be administered in a
number of ways depending on whether local or systemic treatment is
desired, and on the area to be treated. Administration may be
topically (including ophthalmically, vaginally, rectally,
intranasally), orally, by inhalation, or parenterally, for example
by intravenous drip, subcutaneous, intraperitoneal or intramuscular
injection. The disclosed antibodies can be administered
intravenously, intraperitoneally, intramuscularly, subcutaneously,
intracavity, or transdermally.
[0222] Preparations for parenteral administration include sterile
aqueous or non-aqueous solutions, suspensions, and emulsions.
Examples of non-aqueous solvents are propylene glycol, polyethylene
glycol, vegetable oils such as olive oil, and injectable organic
esters such as ethyl oleate. Aqueous carriers include water,
alcoholic/aqueous solutions, emulsions or suspensions, including
saline and buffered media. Parenteral vehicles include sodium
chloride solution, Ringer's dextrose, dextrose and sodium chloride,
lactated Ringer's, or fixed oils. Intravenous vehicles include
fluid and nutrient replenishers, electrolyte replenishers (such as
those based on Ringer's dextrose), and the like. Preservatives and
other additives may also be present such as, for example,
antimicrobials, anti-oxidants, chelating agents, and inert gases
and the like.
[0223] Formulations for topical administration may include
ointments, lotions, creams, gels, drops, suppositories, sprays,
liquids and powders. Conventional pharmaceutical carriers, aqueous,
powder or oily bases, thickeners and the like may be necessary or
desirable.
[0224] Compositions for oral administration include powders or
granules, suspensions or solutions in water or non-aqueous media,
capsules, sachets, or tablets. Thickeners, flavorings, diluents,
emulsifiers, dispersing aids or binders may be desirable.
[0225] Some of the compositions may potentially be administered as
a pharmaceutically acceptable acid- or base-addition salt, formed
by reaction with inorganic acids such as hydrochloric acid,
hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid,
sulfuric acid, and phosphoric acid, and organic acids such as
formic acid, acetic acid, propionic acid, glycolic acid, lactic
acid, pyruvic acid, oxalic acid, malonic acid, succinic acid,
maleic acid, and fumaric acid, or by reaction with an inorganic
base such as sodium hydroxide, ammonium hydroxide, potassium
hydroxide, and organic bases such as mono-, di-, trialkyl and aryl
amines and substituted ethanolamines.
[0226] b) Therapeutic Uses
[0227] Effective dosages and schedules for administering the
compositions may be determined empirically, and making such
determinations is within the skill in the art. The dosage ranges
for the administration of the compositions are those large enough
to produce the desired effect in which the symptoms/disorder are/is
effected. The dosage should not be so large as to cause adverse
side effects, such as unwanted cross-reactions, anaphylactic
reactions, and the like. Generally, the dosage will vary with the
age, condition, sex and extent of the disease in the patient, route
of administration, or whether other drugs are included in the
regimen, and can be determined by one of skill in the art. The
dosage can be adjusted by the individual physician in the event of
any counter indications. Dosage can vary, and can be administered
in one or more dose administrations daily, for one or several days.
Guidance can be found in the literature for appropriate dosages for
given classes of pharmaceutical products. For example, guidance in
selecting appropriate doses for antibodies can be found in the
literature on therapeutic uses of antibodies, e.g., Handbook of
Monoclonal Antibodies, Ferrone et al., eds., Noges Publications,
Park Ridge, N.J., (1985) ch. 22 and pp. 303-357; Smith et al.,
Antibodies in Human Diagnosis and Therapy, Haber et al., eds.,
Raven Press, New York (1977) pp. 365-389. A typical daily dosage of
the antibody used alone might range from about 1 .mu.g/kg to up to
100 mg/kg of body weight or more per day, depending on the factors
mentioned above.
[0228] Following administration of a disclosed composition, such as
an antisense oligonucleotide morpholino or PNA, for treating,
inhibiting, or preventing an DM1, the efficacy of the therapeutic
antibody can be assessed in various ways well known to the skilled
practitioner. For instance, one of ordinary skill in the art will
understand that a composition, such as a morpholino, disclosed
herein is efficacious in treating or inhibiting a DM1 in a subject
by observing that the composition reduces symptoms associated with
the disease or reduces nuclear foci sequestration of MBNL1.
[0229] The compositions that inhibit MBNL1 interactions with
poly(CUG).sup.exp disclosed herein may be administered
prophylactically to patients or subjects who are at risk for
DM1.
[0230] 6. Compositions Identified by Screening with Disclosed
Compositions/Combinatorial Chemistry
[0231] a) Combinatorial Chemistry
[0232] The disclosed compositions can be used as targets for any
combinatorial technique to identify molecules or macromolecular
molecules that interact with the disclosed compositions in a
desired way. It is understood that when using the disclosed
compositions in combinatorial techniques or screening methods,
molecules, such as macromolecular molecules, will be identified
that have particular desired properties such as inhibition or
stimulation or the target molecule's function. The molecules
identified and isolated when using the disclosed methods, such as,
CAG25, are also disclosed.
[0233] It is understood that the disclosed methods for identifying
molecules that inhibit the interactions between, for example, MBNL1
and poly(CUG).sup.exp can be performed using high through put
means. For example, putative inhibitors can be identified using
Fluorescence Resonance Energy Transfer (FRET) to quickly identify
interactions. The underlying theory of the techniques is that when
two molecules are close in space, ie, interacting at a level beyond
background, a signal is produced or a signal can be quenched. Then,
a variety of experiments can be performed, including, for example,
adding in a putative inhibitor. If the inhibitor competes with the
interaction between the two signaling molecules, the signals will
be removed from each other in space, and this will cause a decrease
or an increase in the signal, depending on the type of signal used.
This decrease or increasing signal can be correlated to the
presence or absence of the putative inhibitor. Any signaling means
can be used. For example, disclosed are methods of identifying an
inhibitor of the interaction between any two of the disclosed
molecules comprising, contacting a first molecule and a second
molecule together in the presence of a putative inhibitor, wherein
the first molecule or second molecule comprises a fluorescence
donor, wherein the first or second molecule, typically the molecule
not comprising the donor, comprises a fluorescence acceptor; and
measuring Fluorescence Resonance Energy Transfer (FRET), in the
presence of the putative inhibitor and the in absence of the
putative inhibitor, wherein a decrease in FRET in the presence of
the putative inhibitor as compared to FRET measurement in its
absence indicates the putative inhibitor inhibits binding between
the two molecules. This type of method can be performed with a cell
system as well.
[0234] Combinatorial chemistry includes but is not limited to all
methods for isolating small molecules or macromolecules that are
capable of binding either a small molecule or another
macromolecule, typically in an iterative process. Proteins,
oligonucleotides, and sugars are examples of macromolecules. For
example, oligonucleotide molecules with a given function, catalytic
or ligand-binding, can be isolated from a complex mixture of random
oligonucleotides in what has been referred to as "in vitro
genetics" (Szostak, TIBS 19:89, 1992). One synthesizes a large pool
of molecules bearing random and defined sequences and subjects that
complex mixture, for example, approximately 10.sup.15 individual
sequences in 100 .mu.g of a 100 nucleotide RNA, to some selection
and enrichment process. Through repeated cycles of affinity
chromatography and PCR amplification of the molecules bound to the
ligand on the column, Ellington and Szostak (1990) estimated that 1
in 10.sup.10 RNA molecules folded in such a way as to bind a small
molecule dyes. DNA molecules with such ligand-binding behavior have
been isolated as well (Ellington and Szostak, 1992; Bock et al,
1992). Techniques aimed at similar goals exist for small organic
molecules, proteins, antibodies and other macromolecules known to
those of skill in the art. Screening sets of molecules for a
desired activity whether based on small organic libraries,
oligonucleotides, or antibodies is broadly referred to as
combinatorial chemistry. Combinatorial techniques are particularly
suited for defining binding interactions between molecules and for
isolating molecules that have a specific binding activity, often
called aptamers when the macromolecules are nucleic acids.
[0235] There are a number of methods for isolating proteins which
either have de novo activity or a modified activity. For example,
phage display libraries have been used to isolate numerous peptides
that interact with a specific target. (See for example, U.S. Pat.
No. 6,031,071; 5,824,520; 5,596,079; and 5,565,332 which are herein
incorporated by reference at least for their material related to
phage display and methods relate to combinatorial chemistry)
[0236] A preferred method for isolating proteins that have a given
function is described by Roberts and Szostak (Roberts R. W. and
Szostak J. W. Proc. Natl. Acad. Sci. USA, 94(23)12997-302 (1997).
This combinatorial chemistry method couples the functional power of
proteins and the genetic power of nucleic acids. An RNA molecule is
generated in which a puromycin molecule is covalently attached to
the 3'-end of the RNA molecule. An in vitro translation of this
modified RNA molecule causes the correct protein, encoded by the
RNA to be translated. In addition, because of the attachment of the
puromycin, a peptdyl acceptor which cannot be extended, the growing
peptide chain is attached to the puromycin which is attached to the
RNA. Thus, the protein molecule is attached to the genetic material
that encodes it. Normal in vitro selection procedures can now be
done to isolate functional peptides. Once the selection procedure
for peptide function is complete traditional nucleic acid
manipulation procedures are performed to amplify the nucleic acid
that codes for the selected functional peptides. After
amplification of the genetic material, new RNA is transcribed with
puromycin at the 3'-end, new peptide is translated and another
functional round of selection is performed. Thus, protein selection
can be performed in an iterative manner just like nucleic acid
selection techniques. The peptide which is translated is controlled
by the sequence of the RNA attached to the puromycin. This sequence
can be anything from a random sequence engineered for optimum
translation (i.e. no stop codons etc.) or it can be a degenerate
sequence of a known RNA molecule to look for improved or altered
function of a known peptide. The conditions for nucleic acid
amplification and in vitro translation are well known to those of
ordinary skill in the art and are preferably performed as in
Roberts and Szostak (Roberts R. W. and Szostak J. W. Proc. Natl.
Acad. Sci. USA, 94(23)12997-302 (1997)).
[0237] Another preferred method for combinatorial methods designed
to isolate peptides is described in Cohen et al. (Cohen B. A., et
al., Proc. Natl. Acad. Sci. USA 95(24):14272-7 (1998)). This method
utilizes and modifies two-hybrid technology. Yeast two-hybrid
systems are useful for the detection and analysis of
protein:protein interactions. The two-hybrid system, initially
described in the yeast Saccharomyces cerevisiae, is a powerful
molecular genetic technique for identifying new regulatory
molecules, specific to the protein of interest (Fields and Song,
Nature 340:245-6 (1989)). Cohen et al., modified this technology so
that novel interactions between synthetic or engineered peptide
sequences could be identified which bind a molecule of choice. The
benefit of this type of technology is that the selection is done in
an intracellular environment. The method utilizes a library of
peptide molecules that attached to an acidic activation domain.
[0238] Using methodology well known to those of skill in the art,
in combination with various combinatorial libraries, one can
isolate and characterize those small molecules or macromolecules,
which bind to or interact with the desired target. The relative
binding affinity of these compounds can be compared and optimum
compounds identified using competitive binding studies, which are
well known to those of skill in the art.
[0239] Techniques for making combinatorial libraries and screening
combinatorial libraries to isolate molecules which bind a desired
target are well known to those of skill in the art. Representative
techniques and methods can be found in but are not limited to U.S.
Pat. Nos. 5,084,824, 5,288,514, 5,449,754, 5,506,337, 5,539,083,
5,545,568, 5,556,762, 5,565,324, 5,565,332, 5,573,905, 5,618,825,
5,619,680, 5,627,210, 5,646,285, 5,663,046, 5,670,326, 5,677,195,
5,683,899, 5,688,696, 5,688,997, 5,698,685, 5,712,146, 5,721,099,
5,723,598, 5,741,713, 5,792,431, 5,807,683, 5,807,754, 5,821,130,
5,831,014, 5,834,195, 5,834,318, 5,834,588, 5,840,500, 5,847,150,
5,856,107, 5,856,496, 5,859,190, 5,864,010, 5,874,443, 5,877,214,
5,880,972, 5,886,126, 5,886,127, 5,891,737, 5,916,899, 5,919,955,
5,925,527, 5,939,268, 5,942,387, 5,945,070, 5,948,696, 5,958,702,
5,958,792, 5,962,337, 5,965,719, 5,972,719, 5,976,894, 5,980,704,
5,985,356, 5,999,086, 6,001,579, 6,004,617, 6,008,321, 6,017,768,
6,025,371, 6,030,917, 6,040,193, 6,045,671, 6,045,755, 6,060,596,
and 6,061,636.
[0240] Combinatorial libraries can be made from a wide array of
molecules using a number of different synthetic techniques. For
example, libraries containing fused 2,4-pyrimidinediones (U.S. Pat.
No. 6,025,371) dihydrobenzopyrans (U.S. Pat. Nos. 6,017,768 and
5,821,130), amide alcohols (U.S. Pat. No. 5,976,894), hydroxy-amino
acid amides (U.S. Pat. No. 5,972,719) carbohydrates (U.S. Pat. No.
5,965,719), 1,4-benzodiazepin-2,5-diones (U.S. Pat. No. 5,962,337),
cyclics (U.S. Pat. No. 5,958,792), biaryl amino acid amides (U.S.
Pat. No. 5,948,696), thiophenes (U.S. Pat. No. 5,942,387),
tricyclic Tetrahydroquinolines (U.S. Pat. No. 5,925,527),
benzofurans (U.S. Pat. No. 5,919,955), isoquinolines (U.S. Pat. No.
5,916,899), hydantoin and thiohydantoin (U.S. Pat. No. 5,859,190),
indoles (U.S. Pat. No. 5,856,496), imidazol-pyrido-indole and
imidazol-pyrido-benzothiophenes (U.S. Pat. No. 5,856,107)
substituted 2-methylene-2,3-dihydrothiazoles (U.S. Pat. No.
5,847,150), quinolines (U.S. Pat. No. 5,840,500), PNA (U.S. Pat.
No. 5,831,014), containing tags (U.S. Pat. No. 5,721,099),
polyketides (U.S. Pat. No. 5,712,146), morpholino-subunits (U.S.
Pat. Nos. 5,698,685 and 5,506,337), sulfamides (U.S. Pat. No.
5,618,825), and benzodiazepines (U.S. Pat. No. 5,288,514).
[0241] As used herein combinatorial methods and libraries included
traditional screening methods and libraries as well as methods and
libraries used in interative processes.
[0242] 5. Compositions with Similar Functions
[0243] It is understood that the compositions disclosed herein have
certain functions, such as displacing MBNL1 or binding
polyCUG.sup.exp mRNA. Disclosed herein are certain structural
requirements for performing the disclosed functions, and it is
understood that there are a variety of structures which can perform
the same function which are related to the disclosed structures,
and that these structures will ultimately achieve the same result,
for example inhibition of the interaction between MBNL1 and
polyCUG.sup.exp mRNA.
[0244] Throughout this application, various publications are
referenced. The disclosures of these publications in their
entireties are hereby incorporated by reference into this
application in order to more fully describe the state of the art to
which this invention pertains. The references disclosed are also
individually and specifically incorporated by reference herein for
the material contained in them that is discussed in the sentence in
which the reference is relied upon.
[0245] It will be apparent to those skilled in the art that various
modifications and variations can be made in the present invention
without departing from the scope or spirit of the invention. Other
embodiments of the invention will be apparent to those skilled in
the art from consideration of the specification and practice of the
invention disclosed herein. It is intended that the specification
and examples be considered as exemplary only, with a true scope and
spirit of the invention being indicated by the following
claims.
D. EXAMPLES
[0246] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how the compounds, compositions, articles, devices
and/or methods claimed herein are made and evaluated, and are
intended to be purely exemplary and are not intended to limit the
disclosure. Efforts have been made to ensure accuracy with respect
to numbers (e.g., amounts, temperature, etc.), but some errors and
deviations should be accounted for. Unless indicated otherwise,
parts are parts by weight, temperature is in .degree. C. or is at
ambient temperature, and pressure is at or near atmospheric.
1. Example 1
MBNL Sequestration on poly(CUG).sup.exp Leading to Spliceopathy
[0247] Transcription of the mutant allele generates DMPK mRNA
containing an expanded CUG repeat. Mutant transcripts accumulate in
discrete RNA nuclear (ribonuclear) foci. The RNA in foci is the
fully-processed DMPK mRNA (Taneja K L, et al. J Cell Biol 1995;
128(6):995-1002). Reddy and colleagues have found that
siRNA-mediated depletion of MBNL1 eliminated most of the
ribonuclear foci in DM1 myoblasts, suggesting that the
MBNL1-poly(CUG).sup.exp interaction is the key determinant of foci
formation (Dansithong W, et al. J Biol Chem 2005;
280(7):5773-5780). RNA binding proteins in the muscleblind-like
(MBNL) family, including MBNL1, MBNL2, and MBNL3, are sequestered
in ribonuclear foci. MBNL proteins show strong colocalization with
poly(CUG).sup.exp in DM1 cells and consequently are depleted from
the nucleoplasm (Lin X, et al. Hum Mol Genet. 2006; Mankodi A, et
al. Hum Mol Genet. 2001; 10:2165-2170). MBNL1 and MBNL2 are
expressed in mature skeletal muscle, heart and brain (Fardaei M, et
al. Hum Mol Genet. 2002; 11(7):805-814; Kanadia R N, et al. Gene
Expr Patterns 2003; 3(4):459-462). MBNL3 is expressed mainly in
placenta. Loss of MBNL1 function leads to abnormal regulation of
alternative splicing for a select group of pre-mRNAs, such as,
insulin receptor and chloride channel 1. When overexpressed, all
three MBNL family members can regulate splicing (Ho T H, et al.
EMBO J. 2004; 23(15):3103-3112). Other splicing factors, such as,
CUG-BP1 may contribute to spliceopathy in DM1(Savkur R S, et al.
Nat Genet. 2001; 29(1):40-47; Philips A V, et al. Science 1998;
280(5364):737-741), although they are not sequestered in nuclear
foci of poly(CUG).sup.exp. Expression of splice isoforms that are
developmentally inappropriate leads to signs and symptoms of DM1,
such as, insulin resistance and myotonia.
2. Example 2
Mouse Models of DM1
[0248] HSA.sup.LR transgenic mice express human skeletal actin mRNA
containing (CUG).sup.250 in the 3' UTR. These mice express high
levels of poly(CUG).sup.exP exclusively in skeletal muscle, and
they develop myotonic myopathy.sup.9 (see below).
Mbnl1.sup..DELTA.E3/.DELTA.E3 mice (derived in the Swanson lab) are
homozygous for a targeted allele of Mbnl1. These mice develop
myotonic myopathy but the myopathy appears less severe that in
HSA.sup.LR mice.sup.5. Mbnl1 knockout mice also develop
multisystemic features of DM1, such as, cataracts, progressive
(ultimately fatal) cardiac disease, and abnormal CNS function.
3. Example 3
Structure of poly(CUG).sup.exp and binding to MBNL1
[0249] Poly(CUG) RNA forms stable hairpin structures in vitro when
it is pathologically expanded (Napierala M, Krzyzosiak W J. J Biol
Chem 1997; 272(49):31079-31085; Tian B, et al. Rna 2000; 6:79-87).
The stem of the hairpin is an extended region of duplex RNA in
which G.cndot.C and C.cndot.G base pairs are separated by a
periodic U.cndot.U mismatch. MBNL1 binds to poly(CUG).sup.exp in
vitro in preference to poly(CUG) that is not expanded, suggesting
that it recognizes poly(CUG) in a structured (duplex) form (Miller
J W, et al. EMBO J. 2000; 19(17):4439-4448). By comparison, the
physiologic targets for splicing regulation by MBNL1 are short, 6-8
nt intronic splice enhancer/repressor elements.
4. Example 4
Progressive Myotonic Myopathy (PMM): A Composite Phenotype
[0250] The PMM in DM1 is distinct from muscle phenotypes in other
forms of dystrophy. PMM is a composite, additive phenotype
resulting from independent effects of spliceopathy on different
genes, and consequently, different pathways.
[0251] a) Myotonia
[0252] Myotonia is a delay of muscle relaxation after voluntary
contraction, caused by runs of action potentials that are generated
in the muscle fibers. In HSA.sup.LR transgenic mice, myotonia is
associated with abnormal regulation of alternative splicing for the
ClC-1 chloride channel and >70% reduction of the sarcolemmal Cl
conductance (Mankodi A, et al. Mol Cell 2002; 35-44). A parallel
abnormality of ClC-1 splicing and loss of ClC-1 protein occurs in
human DM1 and DM2. As further evidence that myotonia in DM1 is a
chloride channelopathy stemming from effects on splicing of ClC-1,
the reversal of ClC-1 spliceopathy, either by antisense
oligonucleotides that suppress splicing of misregulated exon, or by
AAV-mediated overexpression of MBNL1, lead to resolution of
myotonia in HSA.sup.LR mice (M Swanson, R Kanadia, T Wheeler, C
Thornton, unpublished). While medications provide partial relief of
myotonia, they are not very effective when myotonia is severe,
hence they are not widely prescribed. Because the myotonia is most
severe in hand muscles that also are hampered by early weakness, it
markedly interferes with manual dexterity and contributes to
disability. Furthermore, a clear correlation between severity of
myotonia and weakness in DM1 exists, indicating that calcium and
mechanical overload due to myotonic discharges can accelerate the
myopathy.
[0253] b) Insulin Resistance.
[0254] DM1 is characterized by insulin resistance in skeletal
muscle. Cooper and colleagues have shown that spliceopathy affects
alternative splicing of insulin receptor (Savkur R S, et al. Nat
Genet. 2001; 29(1):40-47) (INSR). The predominant INSR isoform
expressed in DM1 muscle is the exon 11 skipped, non-muscle isoform,
which has lower signaling capacity. Because hybrid IGF-1/insulin
receptors form in skeletal muscle, the INSR spliceopathy may also
influence IGF-1 signaling.
[0255] c) Myopathy
[0256] Several factors can contribute the pathogenesis of myopathy
in DM1: (1) simple atrophy due to reduced anabolic influence, such
as, reduced signaling through insulin and IGF-1 receptors; (2)
structural abnormalities, such as, abnormal cytoskeletal
organization that is observed in DM1 muscle fibers; (3) abnormal
nuclear function, as reflected by an increase in the number of
muscle nuclei per fiber (the earliest histologic change in DM1);
(4) ineffective regeneration/repair, as reflected by abnormal
myogenesis that characterizes the most severe, congenital form of
DM1; (5) reduced nerve-muscle trophic support, as reflected by
denervation-like changes (pyknotic nuclear clumps, angular atrophic
fibers) in muscle fibers and expanded terminal arborizations and
axonal proliferation in intramuscular nerves; and (6) calcium
overload due to myotonia.
5. Example 5
MBNL1 Sequestration: Pivotal Role in Muscle Spliceopathy
[0257] Spliceopathy in DM1 targets a select group of pre-mRNAs that
share a common temporal pattern of developmental regulation (Lin X,
et al. Hum Mol Genet. 2006). The exons affected by spliceopathy
normally undergo a synchronous splicing switch during early
postnatal development in WT mice. However, loss of MBNL1, or
expression of poly(CUG).sup.exp, results in identical failure of
these splicing transitions. The spliceopathies in HSA.sup.LR
transgenic mice, MBNL1 knockout mice, and human DM1 and DM2 are
highly concordant. Immunofluorescence examination of DM1 and DM2
muscle sections shows that MBNL1 is recruited into ribonuclear foci
to such an extent that it is markedly depleted elsewhere in the
nucleoplasm (Lin X, et al. Hum Mol Genet. 2006). Taken together,
these results indicate that MBNL1 has a pivotal role in the
pathogenesis of spliceopathy. Furthermore, phenotypic consequences
in mouse models clearly are influenced by the level of
poly(CUG).sup.exp in relation to nuclear supplies of MBNL1 protein.
For example, in lines of HSA.sup.LR transgenic mice that are
phenotypically normal and have subthreshold accumulation of
poly(CUG).sup.exp, crossing with MBNL1 null heterozygotes (lowering
MNBL1 protein by 50%) results in spliceopathy, myotonia, and
myopathy. Also, intramuscular injection of AAV-MBNL1 expression
vector leads to resolution of myotonia and correction of
spliceopathy in HSA.sup.LR mice.
6. Example 6
Cell-Based Assay for Compounds that Correct
poly(CUG).sup.exp-induced Spliceopathy
[0258] The assay has two components: (1) cells that display
poly(CUG).sup.exp-induced spliceopathy; and (2) a minigene
construct that reports on severity of spliceopathy. To develop the
reporter, .about.70 alternatively spliced exons were analyzed in
HSA.sup.LR mouse and DM1 muscle to identify exons most affected by
spliceopathy. Out of 16 exons affected by spliceopathy, exon 22 of
SERCA1 was one of the most severely affected, and the gene
structure of SERCA1 lent itself well to adaptation for the reporter
assay. The fraction of SERCA1 mRNA skipping exon 22 increased from
3.+-.0.7% in WT to 78.+-.4% in HSA.sup.LR mice, and the
proportional change in DM1 and DM2 was similar (Lin X, et al. Hum
Mol Genet 2006). SERCA1 exon 22 normally undergoes a postnatal
splicing switch in WT muscle, but this switch fails to occur in
HSA.sup.LR transgenic and MBNL1 knockout mice (FIG. 1A). Exons 22
and 23 contain alternative termination codons for SERCA1
translation (FIG. 1B). A minigene construct, pSERF, was cloned to
generate a fluorescence readout for the relative frequencies of
exon 22 skipping and inclusion. pSERF contains SERCA1 exon 22 and
its flanking introns (FIG. 1C). Splice donor and acceptor signals
from the flanking exons are fused to the coding regions for yellow
fluorescent protein (eYFP) and cyan fluorescent protein (eCFP).
Spectral separation of these proteins is more than sufficient to
resolve eYFP and eCFP components when both proteins are
co-expressed. Both fluorescent proteins function as monomers and
have rapid maturation times. The splicing outcome characteristic of
normal mature skeletal muscle is inclusion of exon 22 (FIG. 1A,
lane 4). The exon 22 inclusion (ex22+) transcript of pSERF encodes
eYFP alone. The splicing that is characteristic of DM1 muscle skips
exon 22 (ex22-, FIG. 1A, lanes 5-12). The ex22- transcript encodes
eYFP-eCFP fusion protein (Y.cndot.CFP). To test that pSERF is
properly spliced in WT muscle and misregulated in response to
poly(CUG).sup.exp, pSERF was electroporated in vivo in WT or
HSA.sup.LR mice and muscle was harvested for RNA analysis after 4
days. As expected, inclusion of exon 22 was low in HSA.sup.LR mice
and high in WT muscle (FIG. 1D). These results indicate that pSERF
can report on spliceopathy induced by poly(CUG).sup.exp.
[0259] Disclosed herein, correction of spliceopathy improves the
cardinal symptoms of DM1. A spliceopathy assay has several
advantages for identifying compounds having therapeutic potential
in DM1. Spliceopathy is a downstream consequence of the RNA
mediated disease process that is directly pertinent to symptoms of
DM1. A spliceopathy screen can capture compounds that act either on
the splicing machinery or upstream of RNA processing, having any of
the following desirable effects: (1) accelerated degradation of
poly(CUG).sup.exp RNA; (2) upregulation of MBNL1 activity
(post-transcriptional); (3) release of MBNL1 from sequestration in
nuclear foci; and (4) effects on other splicing regulators, such
as, CUG-BP1, that improve the splicing defect. In terms of
screening for compounds that inhibit recognition of
poly(CUG).sup.exp by MBNL1, the spliceopathy assay identifies
compounds that differentially inhibit the interaction of MBNL1 with
its pathological target, poly(CUG).sup.exp RNA, compared to its
physiological target, the splice enhancer element in SERCA1
pre-mRNA. Finally, the variance of assay results is low. The
readout is determined by the ratio of two splice products produced
from a single transcription unit, rather than absolute levels for
either isoform. This design minimizes variance resulting from
nonuniform delivery of cell to wells or nonspecific inhibitory
effects of compounds on cell metabolism or survival. In RNA-based
assays of alternative splicing, the coefficient of variation for
relative proportion of two alternative splice products is usually
<3%, lower than most measurements of gene expression. The
readout for the assays disclosed herein are at least this low
because direct fluorescence determination of pSERF protein products
can be reasonably precise.
[0260] a) Overview
[0261] Cell lines that overexpress MBNL1 and poly(CUG).sup.exp can
be obtained in sequential steps of stable transfection, first to
overexpress MBNL1, then to express poly(CUG).sup.exp. In both
cases, gene transfer is assisted by integrase from bacteriophage
phiC31 to obtain full-length, single-copy transgene integrations.
Stepwise introduction of MBNL1 and poly(CUG).sup.exp constructs
provides more flexibility in choosing optimal ratios of MBNL1 and
poly(CUG).sup.exp expression in cells. Spliceopathy is quantified
at each step by transient transfection with reporter construct,
pSERF. The order of procedures is:
[0262] 1. Stably transfect cells with MBNL1 expression construct,
pM1 (FIG. 6). Test splicing in these lines by transient
transfection with pSERF. Select lines that have high YFP to
Y.cndot.CFP ratio and strong inclusion of exon 22.
[0263] 2. Stably transfect with poly(CUG).sup.exp expression
construct, pLLC7 (FIG. 7). Select cell lines that show uniform
expression of the GFP-neomycin resistance cassette.
[0264] 3. Excise the GFP-neomycin resistance cassette in LLC7
transgene using cre recombinase. Verify that recombination has
activated expression of poly(CUG).sup.exp, and that cells develop
nuclear foci of poly(CUG).sup.exp.
[0265] 4. Test splicing by transient transfection with pSERF.
Select lines that have low YFP to Y.cndot.CFP ratio and strong
exclusion of SERCA1 exon 22 (i.e., the pattern of splicing that is
characteristic of DM1 muscle).
[0266] b) Stably Transfect Cell Lines to Overexpress MBNL1 and
Obtain "Muscle-Like" Splicing Outcomes.
[0267] All exons presently known to be affected by spliceopathy
require MBNL1 for normal regulation in skeletal muscle and show
DM1-like spliceopathy in the absence of MBNL1(Lin X, et al. Hum Mol
Genet. 2006). Some of these exons show antagonist regulation by
CUG-BP1 and MBNL1: the normal muscle pattern of splicing is
promoted by MBNL1, the pattern characteristic of DM1 or non-muscle
cells is promoted by CUG-BP1(Ho T H, et al. EMBO J. 2004;
23(15):3103-3112; Philips A V, et al. Science 1998;
280(5364):737-741). The levels of MBNL1 in transformed cell lines
are generally low, whereas expression of CUG-BP1 is fairly
ubiquitous. As expected, basal splicing outcomes in transformed
cell lines are similar to those observed in DM1(Ho T H, et al. EMBO
J. 2004; 23(15):3103-3112). However, overexpression of MBNL1 in
cells lines is sufficient to drive splicing outcomes to the pattern
that is characteristic of normal mature muscle (see FIG. 5 for an
example concerning fast troponin T and FIG. 13 for an example
concerning SERCA1). Thus, pM1 (FIG. 6) is used to obtain stably
transfected cell lines that overexpress MBNL1 and display splicing
phenotypes similar to WT skeletal muscle. Initial efforts focus on
293 or COS cells. These adherent, transformed cell lines have
previously been used in cell based assays in a 384 well format, and
in minigene assays to assess splicing functions of MBNL1. Stable
transfection can be assisted by incorporation of attB elements in
the plasmid and co-transfection with pCMVInt to express phiC31
integrase. This integrase mediates recombination of plasmids
containing the attB element into the mammalian genome. Integration
is irreversible and may occur at any of several hundred sites
having sequence homology to the bacteriophage attP integration
element. Thus, phiC31 can be used for stepwise introduction of
constructs into the same cell line. This integrase is effective in
cells lines of diverse origin and invariably it leads to single
copy integrations of full-length construct (Chalberg T W, et al. J
Mol Biol 2006; 357(1):28-48). Integrations tend to occur in regions
that are transcriptionally active and supportive of transgene
expression (Ishikawa Y, et al. J Gene Med 2006; 8(5):646-653).
Stable transfection assisted by phiC31 integrase is fairly
efficient.
[0268] c) Procedures
[0269] 293 cells are co-transfected with pM1 and pCMVInt (phiC31
integrase expression construct) using conditions previously defined
by Dr Calos (Chalberg T W, et al. J Mol Biol 2006; 357(1):28-48).
The ratio of pCMVInt to pM1 is 50:1. Cells are split 1:30 24 hrs
after transfection and transferred to hygromycin selection medium
the next day. Clones are isolated after 10 to 14 days of selection.
Clones are analyzed for: (1) splicing outcome by transfection with
pSERF followed by RT-PCR and fluorescence analysis; and (2) MBNL1
overexpression by immunofluorescence and immunoblot. It is herein
disclosed that endogenous levels of MBNL1 expression are low and
exon 22 inclusion is <5%. An anti-MBNL1 polyclonal antibody
A2764 was raised and was shown to be monospecific by immunoblot and
immunofluorescence (Lin X, et al. Hum Mol Genet. 2006). The optimal
cell lines are those showing a high frequency of exon 22 inclusion
(high YFP:Y.cndot.CFP ratio), increased nuclear MBNL1 that is
consistent from cell-to-cell, and an overall increase in MBNL1
protein by immunoblot, compared to untransfected controls.
[0270] (1) Stably Transfected Cell Lines that Express
poly(CUG)exp
[0271] (a) Uniformity of MBNL1 and poly(CUG)exp Expression
[0272] For a disease state that depends on the stoichiometry of a
toxic RNA, poly(CUG)exp, and its major cellular binding protein,
MBNL1, the cell system most responsive to therapeutic effects is
one in which every cell expresses poly(CUG)exp at levels just
sufficient to sequester MBNL1 and induce a strong spliceopathy
phenotype. Departure from this ideal reduces the responsiveness of
an assay to therapeutic compounds. Cell-to-cell variability of
transgene expression is likely to be a particular problem for
poly(CUG)exp. Cells with subthreshold poly(CUG)exp accumulation
never develop spliceopathy, whereas cells having a large burden of
poly(CUG)exp are "resistant" to therapeutic effects. In either
case, cells become unresponsive to test compounds and the power of
the screen is correspondingly reduced.
[0273] (b) Overall Approach Using pLLC7 Construct
[0274] Constructs for expression of expanded CUG repeats present
problems of instability and transgene silencing. To overcome these
problems, three steps are employed to make stably transfected cells
lines for optimal stability and uniformity of poly(CUG)exp
expression. First, phiC31 integrase is used to obtain full-length,
single-copy integrations.
[0275] The transgene initially expresses a GFP-neomycin resistance
selection cassette. Second, FACS is used to select cells lines that
have uniform expression of the selection cassette. Any deleterious
effects of poly(CUG)exp are avoided during initial isolation and
selection clones. Third, cre recombinase is used to excise the
neo-GFP cassette and activate expression of luciferase with
poly(CUG)exp in the 3' UTR.
[0276] (c) Instability of Expanded CTG Repeats
[0277] CTG repeat tracts >150 repeats have a strong tendency for
contraction in E. coli cloning vectors (Kang S, et al. Nat Genet.
1995; 10(2):213-218), and it is difficult to clone repeat lengths
above 250. Unavoidably, any plasmid prep containing an expanded CTG
repeat consists of a heterogeneous mixture of different expansion
lengths, and bacterial cultures seeded from the same stock show
considerable prep-to-prep variability. This has a direct bearing on
use of poly(CUG)exp-expression constructs: the genetic instability
of expanded poly(CTG) compounds the problems inherent to transient
transfection, namely, assay-to-assay variability in transfection
efficiency and non-uniform transgene expression among cells that
receive different doses of plasmid. This reduces the effectiveness
of screening and create problems with transfer of assays to
screening facilities, leading to choice of stable transfection as
the preferred method for expressing poly(CUG)exp.
[0278] (2) Transgene Silencing
[0279] Gene transfer into mammalian cells is subject to variable
transgene silencing. Silencing is enhanced by sequence repetition,
as occurs in the large, multicopy transgene arrays that are
typically produced with conventional procedures for stable
transfection. Transgene silencing is especially prominent for
transgenes that contain repetitive elements. Expanded CTG repeats
are the strongest nucleosome positioning elements (Wang Y H,
Griffith J. Genomics 1995; 25(2):570-573), and these sequences are
particularly potent inducers of transgene silencing. These
silencing effects characteristically are variable between cells,
and semi-heritable in clonal isolates. Thus, conventional methods
for obtaining stably transfected cells lines lead to complex,
multicopy (dozens to hundreds of copies) transgene arrays. These
cell lines are expected to show variable silencing, marked
cell-to-cell variability in poly(CUG).sup.exp accumulation, and
unstable expression in subclones or bulk cultures over time.
[0280] (3) DMPK 3'UTR
[0281] Pathogenicity of the mutant DMPK mRNA may be influenced by
elements that are within the DMPK 3' UTR but outside of the repeat
tract (Amack J D, et al. Hum Mol Genet. 2001; 10(18):1879-1887).
Therefore, the DMPK 3' UTR has been incorporated in the construct
to maintain cis elements that may influence the pathogenicity of
poly(CUG)exp.
[0282] d) Bizyme Selection Cassette (Neomycin Phosphotransferase
Fused to GFP)
[0283] This cassette allows selection of clones by neomycin
resistance initially and subsequently by fluorescence (Hansen S G,
et al. Biotechniques 2002; 32(5):1178, 1180, 1182-1178). The cDNA
encoding Bizyme is followed by a concatamer of three SV40
transcription terminators (Novak A, et al. Genesis 2000;
28(3-4):147-155) ("triple stop"). The triple stop element was
tested and showed that it completely prevents transcription of the
downstream luciferase and CTG repeat, until after excision of the
Bizyme selection cassette and triple stop by cre recombinase.
[0284] e) Procedures
[0285] Cells derived in methods disclosed herein are cotransfected
with LLC7 and pCMVInt, and selected for G418 resistant clones as
described above. Initially an LLC7 clone is selected containing
(CTG).sup.300-350. The threshold for nuclear retention of
transcripts containing poly(CUG).sup.exp is not sharply defined.
However, observations in C2C12 cells and transgenic mice indicate
that transcripts with 150 repeats are nuclear retained, and that
the extent of nuclear retention increases with larger expansion
lengths (Amack J D, et al. Hum Mol Genet. 1999; 8(11):1975-1984).
By starting with a repeat length that is close to the upper limit
of for cloning CTG expansions, relatively complete nuclear
retention can be obtained. This reduces background luciferase
activity and more closely approximates the near-complete nuclear
retention observed in human DM1. Next clones having uniform GFP
fluorescence are selected by FACS. These clones are transiently
transfected with cre-recombinase expression vector, then reverse
selected for clones that have lost GFP expression. The clones are
compared by fluorescence in situ hybridization (FISH), MBNL1
immunofluorescence, and splicing analysis after transient
transfection with pSERF. FISH to detect ribonuclear foci of
poly(CUG).sup.exp combined with immunofluorescence to detect MBNL1
is an established procedure. The expected outcome in cells showing
spliceopathy is sequestration of MBNL1 in ribonuclear foci. Optimal
clones show consistent sequestration of MBNL1 in ribonuclear foci,
and splicing of pSERF is expected to revert to the SERCA1 exon 22
skipped isoform (low ratio of YFP:Y.cndot.CFP fluorescence).
[0286] f) Calibration and Variance of pSERF Fluorescence Splicing
Reporter
[0287] Previous reports of emission and excitation spectra for eCFP
are somewhat variable, and there is potential for FRET interaction
between eYFP and eCFP components of Y.cndot.CFP (Pollitt S K, et
al. Neuron 2003; 40(4):685-694). Full spectral analysis is
conducted for eYFP, eCFP, and Y.cndot.CFP fusion protein in cells
and the extent of FRET in Y.cndot.CFP determined. This work is
carried out using the Varian Eclipse fluorometer. A strong FRET
interaction can provide a method to directly determine the signal
from Y.cndot.CFP (i.e., excite CFP, read at YFP emission
wavelength, adjust for cross excitation). Alternatively, measuring
FRET may not offer any advantage over simple analysis of eYFP and
eCFP emission ratios, when each is excited at wavelengths that
provide the least cross excitation. The outcome of these
experiments is to select the wavelengths that are optimal for
determining the ratio of YFP:Y.cndot.CFP when co-expressed. The
strategies disclosed herein are tested in a mixing experiment in
which known ratios of eYFP and Y.cndot.CFP are analyzed, and
calibrated against RNA splicing results determined by RT-PCR
analysis of exon 22 inclusion. It is likely that stable
transfection generates clones that display a variety of different
splicing outcomes, depending on levels of MBNL1 or
poly(CUG).sup.exp expression achieved in a particular clone. If so,
this panel of "intermediate" clones, not selected for the final
assay, but displaying varying degrees of exon 22 inclusion, are
used to correlate fluorescence readouts obtained from intact cells
using the fluorometer with subsequent RNA extraction for splicing
analysis. In this manner, the well-to-well and assay-to-assay
variance in fluorescence reading is determined across the full
spectrum of splicing outcomes. If such "intermediate" clones are
not available, the full spectrum of splicing outcomes can be
reconstitute by transfecting (unmodified) 293 cells with pSERF and
increasing amounts of pM1, to drive increasing levels of exon 22
inclusion. By either approach, the relationship between the
fraction of transcripts that include exon 22 and the ratio of
eCFP/eYFP activity is straightforward: eYFP is a component of every
splicing outcome, whereas eCFP wanes in direct proportion to the
increasing levels of exon 22 inclusion. From these results, the
coefficient of variation between wells and Z' factor is calculated
(Zhang J H, et al. J Biomol Screen 1999; 4(2):67-73), and detection
threshold for effects on spliceopathy can be estimated.
[0288] To accomplish this assay cells are transfected with pSERF,
and 24 hours later dispensed to 96 well plates using a Labsystems
Multidrop plate filler. Test compounds are added to a final
concentration of 10 .mu.M. Fluorescence analysis of pSERF splicing
are determined 24 hours later. Positive results are confirmed in a
repeat experiment using replicate wells, and subsequently assessed
using other assays.
7. Example 7
Cell-Based Assay for Compounds that Inhibit the Interaction of
MBNL1 with poly(CUG)exp In Vivo
[0289] Inhibition of MBNL1-poly(CUG)exp interaction is a logical
therapeutic objective in DM1. The spliceopathy assay can identify
compounds having this activity. However, more direct screens for
inhibitors of MBNL1-poly(CUG)exp interaction can be more sensitive,
or they can identify different sets of compounds. For example, the
spliceopathy screen can give negative results for compounds that
inhibit MBNL1 recognition of its physiologic (splice enhancer
elements) as well as its pathologic (poly(CUG)exp) targets.
Nevertheless, these compounds can preferentially inhibit the
pathologic interaction at a different concentration, or furnish
scaffolds that, with further investigation of structure activity
relationships, can be modified to preferentially target the
pathological interaction.
[0290] a) Assay for Compounds that Trigger Release of
poly(CUG)exp-Containing Transcripts to the Cytoplasm.
[0291] siRNA-mediated knockdown of MBNL1 resulted in .about.70%
reduction of foci in DM1 myoblasts. Based on these observations, it
was determined that MBNL1-poly(CUG)exp interaction is the primary
determinant of ribonuclear foci formation in DM1 myoblasts
(Dansithong W, et al. J Biol Chem 2005; 280(7):5773-5780).
Disclosed herein, poly(CUG)exp transcripts in vitro show formation
of very high molecular weight complexes in the presence of purified
recombinant MBNL1 (FIG. 4). When these high molecular weight
complexes form in cells, they result in nuclear retention of
poly(CUG)exp-containing mRNA. These findings indicate that
inhibition of MBNL1 recognition results in increased
nucleocytoplasmic transport and translation of
poly(CUG)exp-containing mRNA.
[0292] b) Effect of MBNL1 on Translation of mRNA Containing
poly(CUG)exp in the 3'Untranslated Region.
[0293] 293 cells are stably transfected with an expanded repeat
(-300 repeats, lucCUG300) or non-repeat version (lucCUGO) of pLLC7.
These cells are expected to have strong expression of luc mRNA but
modest endogenous expression of MBNL1. Using these cells, the
effect of MBNL1 knockdown (using siRNAs that target the MBNL1
coding region (Dansithong W, et al. J Biol Chem 2005;
280(7):5773-5780)) or overexpression (transient transfection of
pM1) on luciferase activity, nuclear foci, and distribution of luc
mRNA can be compared in nuclear vs cytoplasmic fractions. As
compared to MBNL1 knockdown, MBNL1 overexpression enhances nuclear
foci, reduce luciferase activity, and increase the
nuclear:cytoplasmic ratio of mRNA for lucCUG300 but not for
lucCUGO.
[0294] c) Assay for Compounds that Release poly(CUG)exp-Containing
mRNA from Nuclear Foci.
[0295] The cell lines disclosed herein, can be employed in the
cytoplasmic release assay without further modification. Clones for
luciferase activity are compared in the presence and absence of
siRNA knockout of MBNL1. Clones that are most responsive to MBNL1
knockdown, as determined by upregulation of luciferase activity,
are selected.
[0296] d) Fluorescence Complementation Assay for MBNL1-poly(CUG)exp
Interaction in Cells.
[0297] Interaction between poly(CUG)exp and MBNL1 is evaluated in
cells by combining FISH detection of poly(CUG)exp RNA with
immunofluorescence detection of MBNL1 to show colocalization.
Poly(CUG)exp and MBNL1 interaction can be demonstrated in cell
lysates by immunoprecipitation of the poly(CUG)exp transcript with
antibodies to MBNL1 (X Lin, C Thornton, unpublished). However,
neither method is highly amenable to screening assays. Therefore, a
fluorescence complementation assay is used to detect and quantify
MBNL1-poly(CUG)exp binding in cells. This approach is a
modification of the trimolecular fluorescence complementation
(TriFC) method (Rackham O, Brown CM. EMBO J. 2004;
23(16):3346-3355).
[0298] e) Experimental System
[0299] The system involves 3 components: (1) chimeric transcripts
that contain poly(CUG)exp adjacent to the RNA element recognized by
bacteriophage MS2 coat protein; (2) MBNL1 protein fused to the C
terminal portion of Venus fluorescent protein (VFPC); and (3) MS2
coat protein (MS2CP) fused to the N terminal portion of VFP (VFPN)
(FIG. 8). When all 3 components are co-expressed, the expected
result in the basal state is strong VFP fluorescence because
MBNL1.cndot.VFPC and MS2CP.cndot.VFPN assemble in close proximity
on the chimeric poly(CUG)exp transcript, whereas compounds that
inhibit the poly(CUG)exp-MBNL1 interaction reduce VFP fluorescence.
The specificity and sensitivity of TriFC as a method to detect
binding of two proteins to adjacent regions of mRNA was initially
demonstrated for the zipcode binding protein, IMP1, and MS2CP
(Rackham O, Brown C M. EMBO J. 2004; 23(16):3346-3355). TriFC
generated strong fluorescence signals when MS2CP.cndot.VFPN and
IMP1.cndot.VFPC were co-expressed with a transcript containing
adjacent MS2 and IMP1 recognition elements in the 3' UTR. Mutations
in the RNA target which eliminated MS2CP or IMP1 binding caused
loss of fluorescence signal (Rackham O, Brown C M. EMBO J. 2004;
23(16):3346-3355). Results were similar with other RNA binding
proteins and their respective RNA recognition elements. The
background was low and VFP had the advantages of bright
fluorescence, monomeric structure, and rapid maturation (Nagai T,
et al. Nat Biotechnol 2002; 20(1):87-90).
[0300] (1) Procedures to Develop TriFC Assay
[0301] To develop cell lines that express an mRNA target for
assembly of the trimolecular complex, a modified "CTG donor
plasmid" containing a chimeric poly(CTG)exp-MS2RE insert is cloned.
This fragment is subcloned directly into pLLC7 (FIG. 7). The
chimeric sequence contains two tracts of expanded CTG repeats, each
consisting of 130 repeats, interspersed with two MS2 binding sites,
each consisting of a dimer of the MS2RE (see FIG. 8). After
selecting cell lines with uniform expression of the GFP-neomycin
resistance cassette, the cassette is removed by cre
recombination.
[0302] Two fusion proteins are constructed, MS2CP and MBNL1, tagged
with the N- and C-portions of split Venus protein (VFPN and VFPC).
MS2CP.cndot.VFPN has been shown to retain high affinity for MS2
binding sites (Rackham O, Brown C M. EMBO J. 2004;
23(16):3346-3355). MBNL1-VFPC fusion protein is expected to retain
high affinity for poly(CUG)exp, because MBNL1 tagged with eGFP
retained its poly(CUG)exp binding and splicing regulatory activity
(Ho T H, et al. EMBO J. 2004; 23(15):3103-3112; Fardaei M, et al.
Nucleic Acids Res 2001; 29(13):2766-2771).
[0303] The specificity and localization of the trimolecular
interaction is examined. Cells stably expressing chimeric
poly(CUG)exp.cndot.MS2RE and control cells expressing poly(CUG)exp
with no MS2 binding sites are transiently transfected with
constructs encoding MS2CP.cndot.VFPN, MBNL1-VFPC, or both.
Fluorescence detection of Venus protein is combined with FISH
detection of poly(CUG)exp.
[0304] When the MBNL1-VFPC binding to chimeric
poly(CUG)exp.cndot.MS2RE transcripts results in strong VFP
fluorescence, then stable transfection of the MBNL1-VFPC construct
is conducted. Analysis of well-to-well and assay-to-assay variance
is carried out using the Varian fluorometer and cells stably
transfected to express the chimeric transcript and MBNL1-VFPC, and
transiently transfected to express MS2CP.cndot.VFPN.
8. Example 8
Biochemical Assay for Compounds that Inhibit MBNL1-poly(CUG)exp
Interaction In Vitro
[0305] A biochemical screen with simple components is probably the
most sensitive means to detect inhibitors of MBNL1 binding to
poly(CUG).sup.exp. A biochemical screen can identify compounds not
captured by cell-based screens due to reasons of cell toxicity,
compound instability, impermeability, or protein binding. Such
compounds nevertheless can provide scaffolds that can be modified
to improve activity in vivo. In addition, it is possible to
identify simple compounds that bind poly(CUG).sup.exp and show
modest displacement of MBNL1, and then enhance the binding affinity
by presenting the compound in dimer or oligomeric structures that
are spaced according to the periodicity of the poly(CUG).sup.exp
duplex. Finally, a biochemical screen provides an important tool
for secondary evaluation of hits from the cell-based screens.
[0306] MBNL1 protein is hydrophobic and basic (pH 8.9) so the
majority of protein synthesized in bacteria is denatured or in
inclusions. By tagging human MBNL1 at the C-term with
poly-histidine (His.sub.6) and at the N-term with glutathione S
transferase (GST) and a Prescission protease cleavage site,
GST-MBNL1-His.sub.6 was expressed at high levels in BL21(DE3)
cells. A purification was developed (FIG. 2A) that produces
.about.95% full-length MBNL1: (1) affinity chromatography on
Ni-column; (2) affinity chromatography on GST-sepharose followed by
protease cleavage to release MBNL1-His.sub.6; and (3) G25-sephadex
with exchange buffer. Initially precipitation of MBNL1-His.sub.6
was found in several storage buffers. However, solubility in 100 mM
Tris pH 8.0, 50 mM NaCl, 10% glycerol, 0.1% Triton X-100 was good.
The yields are 1 to 10 mg of MBNL1 protein per liter of bacterial
culture, depending on the MBNL1 isoform. The purification scheme
was optimized using MBNL1-41 and MBNL1-42 kD isoforms, which seem
to have similar binding and splicing factor activities (see FIG. 5
below), as well as a truncated form which removes hydrophobic
sequence at the C-terminal and improves the yield of soluble
protein from E. coli. The truncated protein includes all four
zinc-fingers and other regions that are conserved among MBNL family
members, and its poly(CUG) binding activity remains intact.
Importantly, the purified recombinant MBNL1 does not display
ribonuclease activity when incubated with test transcripts.
[0307] Constructs for in vitro transcription of poly(CUG).sup.109
have been prepared and conditions for enzymatic synthesis and
purification of non-radioactively labeled transcripts have been
optimized (FIG. 2B). The poly(CUG).sup.109 transcript contains an
A-rich sequence at the 3' end. In addition, the triplet repeat is
fused directly to a modified T7 promoter to eliminate A nucleotides
5' to the poly(CUG) tract. This allows biosynthetic labeling to
high specific activity using fluorescently labeled ATP, while
avoiding premature terminations or internal labeling within the
poly(CUG) tract that might impact RNA folding or MBNL1 binding. In
addition, the A-rich 3' tail is devoid of secondary structure and
also functions as anchoring sequence for capture of
poly(CUG).sup.109 by oligonucleotides attached to the surface of
microtiter plates. Before binding assays, transcripts are denatured
at 80.degree. C. and renatured under conditions that favor folding
of poly(CUG).sup.109 as a single hairpin structure.
[0308] Next, optimal conditions were defined (ionic-strength,
monovalent cations, Mg.sup.++and other additions, temperature) for
MBNL1-poly(CUG).sup.109 binding in solution. All forms of
recombinant MBNL1 that were tested (GST fused or cleaved,
full-length or truncation of C terminal hydrophobic region, 41 or
42 kD isoform) bind to poly(CUG).sup.109 at low protein
concentration (FIG. 3, Kd values .about.10 nM), as determined by
nitrocellulose filter binding assays. In this assay, nonspecific
binding of labeled RNA to nitrocellulose was very low (<0.2% of
maximal positive signal). As described below, disclosed herein are
methods comprising a non-radioactive microtiter plate filter
binding assay similar to the one used to examine binding activities
of different forms of recombinant MBNL1 in FIG. 3.
[0309] Also disclosed herein are methods involving the interaction
of soluble, fluorescently-labeled MBNL1 protein with
poly(CUG).sup.109 tethered at the surface of a microtiter plate.
Different commercial microtiter plates for capacity to bind in
vitro transcribed poly(CUG).sup.109 and background activity
(non-specific binding of labeled MBNL1). For tethering
poly(CUG).sup.109 to plates, a "capture" oligodeoxynucleotide (ODN)
complementary to the A-rich 3' terminus of the poly(CUG).sup.109
transcript, labeled either with biotin or a reactive group at the
5' end for attachment to the plate (shown in FIG. 3C in the case of
biotin) was used. Surprisingly, the streptavidin-coated polystyrene
plates (Nunc) had greater capacity to bind poly(CUG).sup.109 and
displayed lower background binding of MBNL1 protein than plates
with reactive surface chemistries that allowed direct covalent
attachment of the capture oligonucleotide. Next, the concentration
of capture ODN that saturates binding sites (12.5 pmoles/100
.mu.l/well) and the concentration of poly(CUG).sup.109 that
saturates the capture ODN (of 5 pmole added per well, 0.5 pmole was
bound to capture ODN) were determined. Additionally, conditions for
fluorescence labeling of MBNL1 protein via conjugation between
primary amines on protein and fluorescein-EX dyes (Molecular
Probes) were determined. The detection threshold and range of
linearity was determined for labeled protein in gels and
microplates (FIG. 2B). The interaction of recombinant MBNL1 with
poly(CUG).sup.109 was examined by gel shift assay, confirming the
prediction that increasing the ratio of MBNL1 to transcript results
in formation of high molecular complexes due to binding of many
protein molecules per transcript (FIG. 4, top panel). Finally, the
efficiency of fluorescence-labeled MBNL1 binding to
poly(CUG).sup.109 transcripts tethered on plates was determined
(FIG. 4, bottom panel), which was sensitive to MBNL1 concentration
as low as 6.25 nM and showed a 50-fold dynamic range.
[0310] a) Filter Retention Assay
[0311] The two components of this assay, fluorescence-labeled
poly(CUG).sup.109 (FIG. 2B) and recombinant MBNL1 (FIG. 2A), have
been developed and optimal conditions for MBNL1 binding to
poly(CUG).sup.exp have been defined (50 mM Tris pH 8.0, 50 mM NaCl,
50 mM KCl, 1 mM Mg.sup.++, 0.1 mM DTT) (see Preliminary Studies).
Pilot binding assays have been carried out using nitrocellulose
filters (FIG. 3A). Next these same reagents and conditions are
employed to test commercially available filter plates. The
Multiscreen.sub.HTS nitrocellulose filter plate (Millipore) has
been previously used for a similar purpose (Bittker J A, et al. Nat
Biotechnol 2002; 20(10):1024-1029). This plate is designed for
automated handling in high throughput screens and is likely to have
protein binding characteristics similar to the filter binding assay
in FIG. 3A.
[0312] b) RNA Attachment Assay.
[0313] The alternative assay for inhibitors of
MBNL1-poly(CUG).sup.exp interaction involves tethering of unlabeled
poly(CUG).sup.exp to microtiter plates, and measuring the amount of
fluorescently-labeled MBNL1 retained on the plate due to RNA
binding (FIG. 3C). Methods for synthesis and tethering of
poly(CUG).sup.109 RNA are described in Preliminary Studies and
diagrammed in FIG. 3C. Results of fluorescence labeling of purified
recombinant MBNL1 are shown in FIG. 2B. Coupling efficiency for
labeling was .about.2-6 fluorochromes per molecule of GST-MBNL1,
using the FluoReporter protein labeling kit (Molecular Probes).
GST-MBNL1 is used for these assays, because the presence of GST
fusion partner does not appear to influence affinity of MBNL1 for
poly(CUG).sup.exp (FIG. 3A), and increasing the mass of MBNL1
permits higher fluorescence activity with less risk of fluorochrome
attachment at the RNA binding site. As described in herein,
streptavidin-coated polystyrene microtiter plates (Nunc) were noted
as providing the highest capacity for poly(CUG).sup.109 binding and
lowest background of MBNL1 binding in the absence of
poly(CUG).sup.exp. The material requirements for these assays are
feasible. 10-100 nM concentration of MBNL1 is suitable (lanes 4-6,
FIG. 4, bottom panel). At these concentrations with 100 .mu.l per
well, 1 mg protein is sufficient for at least 2000 wells. The
potential advantage of the RNA attachment assay, over the filter
binding assay, is greater sensitivity to inhibitors because it
detects varying degrees of displacement of MBNL1 from
poly(CUG).sup.109, whereas even a single residual MBNL1 molecule
bound to poly(CUG).sup.109 is sufficient to induce retention on the
filter. The potential disadvantage is the need for more liquid
handling steps including aspiration of wells, which may dictate
that this assay remains a work station procedure that cannot be
scaled up to high throughput.
[0314] Disclosed herein, poly(CUG).sup.exp displays considerable
resistance to ribonuclease cleavage, owing to its highly stable
secondary structure (Tian B, et al. Rna 2000; 6:79-87). However,
the A-rich 3' end of the poly(CUG).sup.109 transcript, which
contains the fluorescence label in the filter binding assay and
mediates tethering in the plate attachment assay, is not protected
in this manner. The sequence of this tail has been specifically
designed to avoid dinucleotide combinations that confer RNase
sensitivity. Further protection is achieved by addition of a
complementary morpholino to the filter binding assay, or use of a
morpholino as "capture" oligonucleotide in the plate attachment
assay. Addition of irrelevant, unstructured RNA to test buffer and
use of short incubation times further reduce RNase activity.
9. Example 9
Correction of ClC-1 Splicing Eliminates Chloride Channelopathy and
Myotonia in Mouse Models of Myotonic Dystrophy
[0315] DM type 1 (DM1), the most common muscular dystrophy
affecting adults, is caused by expansion of a CTG repeat in the 3'
untranslated region of the gene encoding the DM protein kinase
(DMPK) (Brook, J. D., et al. (1992) Cell 68:799-808). Evidence
suggests that DM1 is not caused by abnormal expression of DMPK
protein, but rather that it involves a toxic gain-of-function by
mutant DMPK transcripts that contain an expanded CUG repeat
(CUG.sup.exp) (Osborne, R. J., and Thornton, C. A. (2006) Hum Mol
Genet. 15 Spec No 2:R162-169). The transcripts containing a
CUG.sup.exp tract elicit abnormal regulation of alternative
splicing, or spliceopathy (Philips, A. V., et al. (1998) Science
280:737-741). The splicing defect, which selectively affects a
specific group of pre-mRNAs, is thought to result from reduced
activity of splicing factors in the muscleblind (MBNL) family
(Kanadia, R.N., et al. (2003) Science 302:1978-1980), increased
levels of CUG binding protein 1 (Philips, A. V., et al. (1998)
Science 280:737-741; Charlet, B. N., et al. (2002) Mol Cell
10:45-53), or both. Decreased activity of MBNL proteins can be
attributed to sequestration of these proteins in nuclear foci of
CUG.sup.exp RNA (Miller, J. W., et al. (2000) Embo J 19:4439-4448;
Lin, X., et al. (2006) Hum Mol Genet. 15:2087-2097).
[0316] Disclosed herein, transgenic mice expressing CUG.sup.exp RNA
(HSA.sup.LR mice) displayed myotonia and chloride channel 1 (ClC-1)
splicing defects similar to those observed in DM1 (Mankodi, A., et
al. (2002) Mol Cell 10:35-44). Myotonia in the HSA.sup.LR model
results from abnormal inclusion of exon 7a in the ClC-1 mRNA, owing
to sequestration of MBNL1, a factor required for repression of exon
7a splicing in muscle fibers (Kanadia, R. N., et al. (2003) Science
302:1978-1980). This mechanism is supported by several lines of
evidence: (1) inclusion of exon 7a causes frame shift and
introduction of a premature termination codon in the ClC-1 mRNA
(Mankodi, A., et al. (2002) Mol Cell 10:35-44; Charlet, B. N., et
al. (2002) Mol Cell 10:45-53); (2) truncated ClC-1 protein encoded
by the exon 7a+isoform is devoid of channel activity (Berg, J., et
al. (2004) Neurology 63:2371-2375); and (3) disruption of Mbnl1 in
mice leads to increased inclusion of ClC-1 exon 7a and myotonia
(Kanadia, R. N., et al. (2003) Science 302:1978-1980). The
postulate that myotonia in DM1 results from deficiency of ClC-1 is
based on observations that mouse models of DM1 display a 70-80%
reduction of muscle chloride conductance (Mankodi, A., et al.
(2002) Mol Cell 10:35-44; Lueck, J. D., et al. (2007) J Gen Physiol
129:79-94), coupled with previous estimates that a 75% reduction of
ClC-1 conductance is sufficient to cause myotonic discharges in
muscle fibers (Furman, R. E., and Barchi, R. L. (1978) Ann Neurol
4:357-365). However, the mechanism of ClC-1 downregulation and its
requirement for myotonia in DM1 is controversial. Effects on sodium
or potassium channels have also been implicated in DM1-associated
myotonia (Franke, C., et al. (1990) J Physiol 425:391-405; Renaud,
J. F., et al. (1986) Nature 319:678-680; Behrens, M. I., et al.
(1994) Muscle Nerve 17:1264-1270). In addition, evidence that
chloride channelopathy in DM1 results from downregulation of ClC-1
transcription, rather than abnormal splicing, has been reported
(Ebralidze, A., et al. (2004) Science 303:383-387). To provide a
causal link between ClC-1 alternative splicing, chloride
channelopathy, and myotonia in DM1, a morpholino AON was used to
selectively repress the inclusion of exon 7a.
[0317] a) Results
[0318] The strategy for suppressing the inclusion of exon 7a is
diagrammed in FIG. 23A. The morpholino AONs were complementary to
the 3' or 5' splice sites of exon 7a in the ClC-1 pre-mRNA (FIG.
23B). To examine tissue uptake, a carboxyfluorescein-labeled
morpholino was injected into tibialis anterior (TA) muscle of
HSA.sup.LR mice. Examination of tissue sections indicated that
uptake of antisense morpholino was limited to the needle track. To
improve uptake and distribution of AON, voltage pulses were used to
electroporate muscle fibers after the AON injection. This led to
uptake of antisense morpholino throughout the TA muscle (FIG.
24A-C). Of note, the AON was present in both nucleus and cytoplasm,
but appeared to accumulate preferentially in the nucleus (FIG.
24A,E).
[0319] To determine the effect of morpholino on splicing in
HSA.sup.LR mice, total RNA was extracted from TA muscle after AON
injection. Analysis of ClC-1 splicing by RT-PCR showed that
antisense morpholino had the intended effect of suppressing the
inclusion of exon 7a, whereas control morpholino with inverted
sequence had no effect on ClC-1 splicing in the contralateral TA
(FIG. 25A,D). AON targeting the 3' splice site, or co-injection of
AONs targeting the 3' and 5' splice sites, was more effective than
targeting the 5' splice site alone (FIG. 28). Effective and
sustained skipping of exon 7a was achieved after a single injection
of morpholino AON. Inclusion of exon 7a was suppressed to wild type
(WT) levels for at least 3 weeks after a single injection (FIG.
25A,D), and a partial exclusion of exon 7a was still evident after
8 weeks (FIG. 25B,E). Notably, the antisense morpholino did not
affect the formation of nuclear foci containing CUG.sup.exp RNA and
MBNL1 protein (FIG. 29), nor did it correct the alternative
splicing of other genes that are misregulated in DM1, such as,
Titin (FIG. 25C), ZASP, or Sercal (Lin, X., et al. (2006) Hum Mol
Genet. 15:2087-2097). These data indicate that morpholino AON
specifically corrects the ClC-1 splicing defect rather than
producing a general reversal of DM-associated spliceopathy or Mbnl1
sequestration.
[0320] Morpholino AONs influence splicing outcomes without inducing
degradation of their target RNAs (Mercatante, D. R., et al. (2001)
Curr Cancer Drug Targets 1:211-230). Therefore, the predicted
effect of repressing exon 7a inclusion was to eliminate the
premature termination codon in ClC-1 mRNA and thereby reduce its
degradation through the nonsense-mediated decay pathway (Lueck, J.
D., et al. (2007) Am J Physiol Cell Physiol 292:C1291-1297).
Consistent with this prediction, treatment with antisense
morpholino, but not control morpholino with inverted sequence, led
to increased levels of ClC-1 mRNA, as determined by quantitative
real time RT-PCR (FIG. 25F). These results indicate that effects of
CUG.sup.exp RNA on ClC-1 expression are mainly at the
post-transcriptional level. Furthermore, treatment with morpholino
AON increased the level of ClC-1 protein in the sarcolemma, as
indicated by immunofluorescence using antibodies directed against
the C-terminus (FIG. 24F,G).
[0321] Herein whole cell patch clamp analysis of single flexor
digitorum brevis (FDB) muscle fibers was used to show that ClC-1
current density is reduced and channel deactivation accelerated in
FDB fibers of untreated HSA.sup.LR mice (Lueck, J. D., et al.
(2007) J Gen Physiol 129:79-94). Therefore, the effect of AON
treatment on ClC-1 channel function was determined. Hindlimb foot
pads of 10-12-day-old WT and HSA.sup.LR mice were
injected/electroporated with carboxyfluorescein-labeled antisense
or invert morpholino. Patch clamp analysis was performed
three-to-five days after injection, at a time when fibers were
still small enough to maintain an effective voltage clamp (Lueck,
J. D., et al. (2007) J Gen Physiol 129:79-94). Individual FDB
fibers were isolated and macroscopic ClC-1 channel activity was
measured in fibers exhibiting green fluorescence (FIG. 24D,E).
ClC-1 current density (FIG. 26A,B) and deactivation kinetics (FIG.
26D) were rescued to WT values as early as 3 days after morpholino
AON injection, while current density and deactivation kinetics in
fibers treated with invert morpholino were not different from those
of untreated HSA.sup.LR fibers. The slower rate of channel
deactivation observed for WT and AON-treated fibers is most likely
not due to a current-dependent effect on channel gating, because
reducing WT ClC-1 current magnitude in half with a prepulse does
not significantly alter the kinetics of channel deactivation
(Lueck, J. D., et al. (2007) J Gen Physiol 129:79-94). Rescue of
ClC-1 activity was not due to a shift in channel activation since
the voltage dependence of relative channel open probability (Po)
was not different between antisense and invert-injected HSA.sup.LR
and WT fibers (FIG. 26C). These results demonstrate that morpholino
AON rescue of ClC-1 spliceopathy is sufficient to completely
restore normal ClC-1 current density and channel deactivation
kinetics.
[0322] The effects of repressing exon 7a inclusion on muscle
physiology in vivo was determined. Electromyography (EMG) analysis
by a blinded examiner revealed that myotonia was markedly reduced
or absent in TA muscles of HSA.sup.LR mice after injection of
antisense morpholino, whereas myotonia in the invert-injected
contralateral TA was not different from uninjected muscle (FIG.
26E,F). Myotonia reduction correlated with the degree of exon 7a
skipping at 3 and 8 week time points, indicating that a single
injection of antisense morpholino provided a sustained reduction in
myotonia.
[0323] Homozygous deletion of Mbnl1 exon 3 in mice
(Mbnl1.sup..DELTA.E3/.DELTA.E3) resulted in loss of Mbnl1 protein
from muscle, spliceopathy that is similar to DM1 patients and
HSA.sup.LR mice, reduction of ClC-1 expression and activity, and
myotonia (Kanadia, R. N., et al. (2003) Science 302:1978-1980; Lin,
X., et al. (2006) Hum Mol Genet. 15:2087-2097; Lueck, J. D., et al.
(2007) J Gen Physiol 129:79-94). To examine the effects of AON in
this model, antisense morpholino or invert control was injected
into TA muscle of Mbnl1.sup..DELTA.E3/.DELTA.E3 mice. As in the
HSA.sup.LR transgenic model, antisense morpholino repressed the
inclusion of exon 7a (FIG. 27A,B), increased the expression of
ClC-1 protein at the sarcolemma (FIG. 27C,D), and reduced the
myotonia in Mbnl1.sup..DELTA.E3/.DELTA.E3 mice (FIG. 27E). Thus,
while the pathogenesis of spliceopathy in DM is a subject of
debate, rescue of the myotonia by antisense morpholino does not
depend on the exact manner in which the ClC-1 splicing defect is
generated.
[0324] b) Discussion
[0325] Current models of DM1 pathogenesis postulate that DMPK mRNA
containing an expanded CUG repeat alters the function of splicing
factors, leading to misregulated alternative splicing for a
specific group of pre-mRNAs (Philips, A. V., et al. (1998) Science
280:737-741). In operational terms, one difficulty with this model
is that functional differences between alternative splice isoforms,
or biological consequences of altering the ratio of two alternative
splice products, are not easy to determine. Furthermore, in the
context of dozens to hundreds of transcripts whose splicing is so
affected, the phenotypic consequences of any particular splicing
change may be difficult to ascertain. A key finding of the present
study is that misregulated alternative splicing of ClC-1 is
required for the development of myotonia, a cardinal symptom of
DM1, in both HSA.sup.LR and Mbnl1.sup..DELTA.E3/.DELTA.E3 mice.
These results provide the clearest indication to date that
CUG.sup.exp-induced spliceopathy is directly involved in producing
a clinical feature of DM1. Furthermore, the results indicate an
approach for dissecting the functional significance of any
particular splicing change. Antisense oligonucleotides can be used
to correct a specific splicing defect in DM1 cells, or to induce a
DM1-like effect in WT cells.
[0326] Myotonia is the symptom by which DM1 is most often
recognized, and, due to preferential involvement of hand and
forearm muscles, it compromises the manual dexterity and
contributes to disability. In light of the abnormal calcium
homeostasis observed in DM1 cells and model systems (Benders, A.
A., et al. (1997) J Clin Invest 100:1440-1447; Benders, A. A., et
al. (1996) Acta Physiol Scand 156:355-367), and the effects of DM1
on alternative splicing of the SERCA1 calcium reuptake pump of the
sarcoplasmic reticulum (Kimura, T., et al. (2005) Hum Mol Genet.
14:2189-2200; Lin, X., et al. (2006) Hum Mol Genet. 15:2087-2097),
it also seems that excessive calcium release due to myotonic
discharges aggravate the degeneration of DM1 muscle fibers.
Although previous studies have implicated sodium channels or
calcium-activated potassium channels in DM1 (Franke, C., et al.
(1990) J Physiol 425:391-405; Renaud, J. F., et al. (1986) Nature
319:678-680; Behrens, M. I., et al. (1994) Muscle Nerve
17:1264-1270), a second finding of the present study is
confirmation that DM1-associated myotonia results primarily from a
chloride channelopathy.
[0327] The number of functional ClC-1 channels in the sarcolemma
was markedly decreased, the rate of channel deactivation was
increased, and the maximum ClC-1 channel open probability was
reduced in both HSA.sup.LR and Mbnl1.sup..DELTA.E3/.DELTA.E3 mice
(Lueck, J. D., et al. (2007) J Gen Physiol 129:79-94). The observed
acceleration in channel deactivation and reduction in maximal
channel open probability are consistent with previously reported
dominant negative effects imparted by exon 7a encoded protein
products (Berg, J., et al. (2004) Neurology 63:2371-2375). The
observations that electroporation of AON in HSA.sup.LR muscle
reduced levels of exon 7a-containing transcript (FIG. 25D,E),
increased full length ClC-1 transcript (FIG. 25F), and completely
normalized ClC-1 current density (FIG. 26B) and deactivation gating
(FIG. 26D) supports the assertion that chloride channelopathy in
DM1 involves a complex combination of transdominant RNA- and
protein-based mechanisms.
[0328] AONs influence RNA processing by annealing to pre-mRNA and
blocking the access of splicing factors to splice sites or
cis-acting regulatory elements (Dominski, Z., and Kole, R. (1993)
Proc Natl Acad Sci USA 90:8673-8677). AONs that induce skipping of
constitutively spliced exons have been used to bypass stop codons
or restore the proper reading frame in the dystrophin mRNA
(Dunckley, M. G., et al. (1998) Hum Mol Genet. 7:1083-1090; Wilton,
S. D., et al. (1999) Neuromuscul Disord 9:330-338; Alter, J., et
al. (2006) Nat Med 12:175-177). Exon 7a can show heightened
susceptibility to AONs because splicing signals in alternative
exons tend to be intrinsically weak and this exon is normally
skipped in a fraction of ClC-1 transcripts (Lueck, J. D., et al.
(2007) Am J Physiol Cell Physiol 292:C1291-1297). However, ClC-1
mRNAs that include exon 7a contain premature termination codons and
undergo rapid degradation (Lueck, J. D., et al. (2007) Am J Physiol
Cell Physiol 292:C1291-1297). Therefore, these splice products are
underrepresented at steady state, and the exact efficiency of
AON-induced exon skipping was not determined. Despite this
limitation, the decrease of exon 7a+isoforms to WT levels and the
normalized activity of ClC-1 channels in treated muscle fibers
indicates that this intervention is highly effective and
surprisingly prolonged.
[0329] These results are the first to show that symptoms of DM1 are
reversible using a targeted, non-gene therapeutic approach to
restore a normal pattern of alternative splicing. While several
drugs with anti-myotonia properties are currently available, they
provide only partial relief of symptoms, and their use in DM1 is
limited by the lack of controlled trials supporting their efficacy
and safety (Trip, J., et al. (2006) Cochrane Database Syst
Rev:CD004762). Results here indicate that targeting the ClC-1
splicing defect is highly effective for treating the myotonia in
DM1.
[0330] c) Methods
[0331] (1) Design of Oligonucleotides.
[0332] Morpholino oligonucleotides (Gene Tools LLC) were
5'-CCAGGCACGGTctgcaacagagaag-3' (SEQ ID NO: 4) targeting the ClC-1
3' splice site, 5'-gaagagacaacgtctggcacggacc-3' (SEQ ID NO: 5)
inverted control, and 5'-ggaagtgaaacttgcCTCCATCAGG-3' (SEQ ID NO:
6) targeting the ClC-1 5' splice site.
[0333] (2) Morpholino Injections.
[0334] HSA.sup.LR (Mankodi, A., et al. (2000) Science
289:1769-1773) or Mbnl1.sup..DELTA.E3/.DELTA.E3 mice (Kanadia, R.
N., et al. (2003) Science 302:1978-1980) were anesthetized by
intraperitoneal injection of 100 mg/kg ketamine, 10 mg/kg xylazine,
and 3 mg/kg acepromazine. TA muscle was pretreated by intramuscular
injection of bovine hyaluronidase (15 .mu.l, 0.4 U/.mu.l) (Sigma)
(McMahon, J. M., et al. (2001) Gene Ther 8:1264-1270). Two hours
later, 10 or 20 mg of morpholino in a total volume of 20 .mu.l
phosphate buffered saline (PBS) was injected using a 30-gauge
needle. TA muscle was then electroporated using electrodes placed
parallel to the long axis of the muscle. Electroporation parameters
were 100 V/cm, 10 pulses at 1 Hz, and 20 ms duration per pulse.
Antisense or control morpholino with inverted sequence was injected
into TA muscles of opposite limbs. The determination of which TA
received antisense morpholino was randomized, and investigators
remained blinded to this assignment until EMG analyses were
completed. Other analyses were performed without blinding. For
experiments to determine the distribution of injected oligos, the
antisense morpholino was labeled with carboxyfluorescein and
cryosections of muscle (10 .mu.M) were examined by fluorescence
microscopy, with or without fixation in 4% paraformaldehyde. Some
sections were co-labeled with TRITC-wheat germ agglutinin (Parsons,
S. A., et al. (2003) Mol Cell Biol 23:4331-4343) (50 .mu.g/ml in
PBS; Sigma) and 4',6-diamidino-2-phenylindole (DAPI) to highlight
the surface membranes and nuclei of muscle fibers.
[0335] (3) RNA Analysis.
[0336] Mice were sacrificed three or eight weeks after morpholino
injection. TA muscles were removed and frozen in liquid nitrogen.
Total RNA was isolated with TriReagent (Molecular Research Center).
cDNA synthesis was primed with oligo dT as described previously
(Mankodi, A., et al. (2002) Mol Cell 10:35-44). Assays for
alternative splicing of ClC-1 and Titin were described previously
(Mankodi, A., et al. (2002) Mol Cell 10:35-44; Lin, X., et al.
(2006) Hum Mol Genet. 15:2087-2097). Primer sequences were 277.
ClC-1 forward: ClCm-7 5'-TGAAGGAATACCTCACACTCAAGG-3' (SEQ ID NO: 7)
and reverse: ClCm-30 5'-CACGGAACACAAAGGCACTG-3' (SEQ ID NO: 8);
mTitin forward: mTTN1 5'-GTGTGAGTCGCTCCAGAAACG-3' (SEQ ID NO: 9)
and reverse: mTTN2 5'-CCACCACAGGACCATGTTATTTC-3' (SEQ ID NO:
10).
[0337] RT-PCR products (22 cycles) were separated on agarose gels,
stained with SYBR green II, and scanned on a laser fluorimager
(Molecular Dynamics). Band intensity was quantified using
ImageQuant software. Total levels of ClC-1 mRNA were determined by
quantitative real-time RT-PCR (Taqman, Applied Biosystems) relative
to housekeeping gene RNA polymerase II transcription factor
IIB.
[0338] (4) Immunofluorescence.
[0339] Frozen transverse sections of TA muscle (10 .mu.M) were
stained with affinity-purified rabbit polyclonal anti-ClC-1
antibody (1:50; Alpha Diagnostic International) as previously
described (Kanadia, R. N., et al. (2006) Proc Natl Acad Sci USA
103:11748-11753). Muscle sections from ClC-1 null mice and WT FVB
mice served as negative and positive controls on each slide.
Z-plane stacks consisting of 8 images separated by 0.25 .mu.M were
captured and deconvolved using Autoquant v9.3 software (Autoquant
Imaging). Maximum-projection images were obtained using Metavue
software (Universal Imaging Corporation). Exposure time and
thresholding were identical for all comparisons of antisense vs.
invert controls.
[0340] (5) Macroscopic Recordings of ClC-1 Current.
[0341] Delivery of antisense and invert morpholinos into FDB fibers
was achieved by injection and electroporation of hindlimb footpads.
Briefly, 12-14-day-old HSA.sup.LR mice were anesthetized by
intraperitoneal injection of 100 mg/kg ketamine, 10 mg/kg xylazine,
and 3 mg/kg acepromazine. Hindlimb foot pads then were injected
with bovine hyaluronidase followed 1 hour later with 20 .mu.g (10
.mu.l, 2 .mu.g/.mu.l in PBS) of antisense or invert
carboxyfluorescein-labeled morpholino. Uptake of morpholinos was
enhanced by electroporation (100 V/cm, 20 pulses at 1 Hz, and 20 ms
per pulse) of the foot pad immediately after injection (DiFranco,
M., et al. (2006) Protein Expr Purif 47:281-288). Three to five
days after injection/electroporation, individual FDB muscle fibers
were isolated as previously described (Lueck, J. D., et al. (2007)
J Gen Physiol 129:79-94). Brightfield and fluorescence (488 nm
excitation) images of single FDB fibers were acquired using a
40.times. (1.4 NA) objective and a TILL IMAGO QE cooled-CCD camera.
Only fibers exhibiting clear striations, clean surfaces and green
fluorescence were chosen for electrophysiological recordings. ClC-1
currents were measured and analyzed in whole cell patch clamp
experiments (Hamill, O. P., et al. (1981) Pflugers Arch 391:85-100)
using an approach identical to that described in detail elsewhere
(Lueck, J. D., et al. (2007) J Gen Physiol 129:79-94). ClC-1
current density (pA/pF) was calculated in order to compare data
across fibers of different sizes.
[0342] (6) Electromyography (EMG).
[0343] EMG was performed under general anesthesia as described
previously (Kanadia, R. N., et al. (2003) Science 302:1978-1980).
Images and video recordings of electromyographic myotonia in
HSA.sup.LR and Mbnl1.sup..DELTA.E3/.DELTA.E3 mice are shown in
previous reports (Kanadia, R. N., et al. (2003) Science
302:1978-1980; Mankodi, A., et al. (2000) Science 289:1769-1773). A
minimum of 15 needle insertions were performed for each muscle
examined. Myotonic discharges were graded on a 4 point scale: 0, no
myotonia; 1, occasional myotonic discharge in <50% of needle
insertions; 2, myotonic discharge in >50% of needle insertions;
3: myotonic discharge with nearly every insertion.
[0344] (7) Statistical Analysis.
[0345] Group data are expressed as mean.+-.s.d., except for patch
clamp data in FIG. 26 which are expressed as mean.+-.s.e.m. Between
group comparison was performed by two-tailed t-test or two way
ANOVA as indicated.
10. Example 10
Protein Displacement Therapy with Peptide Nucleic Acid PNA)
Oligomers Composed of CAG Repeats
[0346] Previously it was shown that expanded CUG repeat RNA forms
stable hairpin structures (Tian B, et al. RNA 2000; 6:79-87). FIG.
30A shows that PNA-CAG repeat oligos of lengths ranging from 2 to 5
CAG repeats can invade (CUG)109 hairpins and effectively interact
with expanded CUG repeat hairpin structures in vitro. FIG. 30B
shows that these PNA-CAG oligos can also inhibit the interaction of
(CUG)109 RNA with MBNL1 protein in vitro. FIG. 30A was performed by
non-denaturing polyacrylamide gel scanned with laser fluorimager
shows migration of fluorescein-labeled (CUG)109 transcript (10 nM).
This transcript was incubated 30 min with different concentrations
(8, 4, 2, 1, 0.5, 0.25 .mu.M) of PNAs containing different length
of CAG repeat sequence (PNA-CAG-6, Nterm-CAGCAG; PNA-CAG-9,
Nterm-CAGCAGCAG; PNA-CAG-12, Nterm-CAGCAGCAGCAG (SEQ ID NO: 14); or
PNA-CAG-15, Nterm-CAGCAGCAGCAGCAG) (SEQ ID NO: 15). FIG. 30B
provides a variation including 5 PNA concentrations (4, 2, 1, 0.5,
0.25 .mu.M) and after additional 30 min incubation step with
recombinant MBNL1 (200 nM). Lane "C" in each panel is a control
showing migration of fluorescein-labeled (CUG)109 transcript
without PNA or MBNL1. Rapid migration of this transcript is caused
by hairpin formation. Panel "A" shows that CAG-repeat PNA is able
to interact with (CUG)109 transcript, retarding its migration on
gel. Lanes "C+" in panel "B" show controls that contain (CUG)109
transcript and MBNL1 protein without PNA. These lanes show diffuse
smear of (CUG)109 transcript, due to formation of heterogenous high
molecular weight RNA-protein complexes. Addition of PNA displaces
the MBNL1 protein from expanded CUG repeat RNA, disrupting these
complexes and reconstituting a sharp band of (CUG)109 transcript.
Thus, disclosed herein is the use of antisense oligonucleotides as
protein displacement therapy in myotonic dystrophy. It is disclosed
herein that myotonic dystrophy has a unique disease process that
makes it quite susceptible to treatment: function of a group of
proteins, the muscleblind (MBNL) proteins, is compromised because
they are stuck onto a mutant RNA that contains CUG repeats, i.e.,
the proteins are sequestered. Earlier examples disclosed herein
were concerned with the use of antisense oligonucleotides that have
the morpholino chemistry: CAG25. Herein, is evidence that antisense
oligonucleotides having the same sequence (CAG repeats, antisense
to CUG repeats) but a different chemistry, peptide nucleic acids
(PNAs), are also effective.
11. Example 11
In Vivo Treatment of a Mouse Model for DM Using PNA-CAG
[0347] FIG. 35 shows that injection of peptide nucleic acid (PNA)
comprised of CAG repeats caused reduction of electromyographic
myotonia in HSA.sup.LR transgenic mouse model of myotonic
dystrophy. PNA-(CAG)6mer or PNA-(CAG)9mer (i.e., 2 or 3 CAG
repeats) was injected into tibialis anterior muscle on a single
occasion. Myotonia was assessed by electromyography 3 weeks
following the intramuscular injection. As control, vehicle alone
(phosphate buffered saline) was injected in the tibialis anterior
muscle of the contralateral limb. All mice had robust action
myotonia prior to treatment. Assignment as to which limb received
PNA vs control was randomized, and EMG analysis was performed
blinded to this assignment.
12. Example 12
Screening for Compounds that Inhibit Interaction of MBNL1 Protein
and CUG Expansion RNA: Fluorescence Anisotropy Assay Shows
Interaction of CUG Expansion RNA with Recombinant MBNL1 Protein In
Vitro
[0348] Fluorescein-labeled (CUG).sub.36 RNA (2 nM) was incubated
with MBNL1 protein (100 nM) and anisotropy was measured at time
points ranging from 1 to 90 minutes. Increasing values for
fluorescence anisotropy indicate interaction of fluorescein-labeled
(CUG).sub.36 transcript with MBNL1 protein. Values are averages
from 4 experiments and error bars shows SD.
13. Example 13
Fluorescence Anisotropy Assay to Screen for Compounds that Inhibit
Interaction of CUG Repeat RNA with Recombinant MBNL1 Protein
[0349] MBNL1 protein is known to bind CUG repeat RNAs that form a
stable secondary structure (hairpin structure). Aminoglycoside
antibiotics were examined to determine whether these compounds can
inhibit the interaction of MBNL1 protein with CUG repeat RNA.
Aminoglycosides were selected because they are known to bind
structured RNA. Fluorescein-labeled (CUG).sub.36 transcript (2 nM)
was incubated first with aminoglycoside compound (10 or 50 .mu.M)
and then with excess amount of recombinant MBNL1 protein (100 nM).
To calculate the fraction of CUG repeat RNA that remains bound to
MBNL1 protein ("% bound CUG.sup.exp", vertical axis), results are
expressed as the percentage of maximal fluorescence anisotropy in
assays from which aminoglycosides were omitted. Among the compounds
tested, neomycin showed the strongest inhibition of MBNL1 binding
to CUG repeat RNA. Values are the average+/-SD from three
measurements.
14. Example 14
Diagram of Enzymatic Complementation Assay to Screen for Compounds
that Inhibit Interaction of CUG Repeat RNA with Recombinant MBNL1
Protein
[0350] (CUG).sub.109 transcripts are tethered to the surface of a
streptavidin-coated microtiter plate using a capture
oligonucleotide that is biotinylated. The capture oligo anneals to
complementary sequence at the 3' end of the CUG repeat RNA.
Recombinant human MBNL1 is expressed as a fusion with the PL
fragment of beta-galactosidase. PL is a 55 amino acid fragment of
beta-galactosidase. Preliminary experiments determined that fusion
of MBNL1 with the PL fragment did not inhibit the binding of MBNL1
protein to CUG repeat RNA. After incubation with test compound,
unbound MBNL1-PL is washed away (panel B). Next, the complementing
fragment of beta-galactosidase is added to determine the amount of
MBNL1-PL that continues to interact with (CUG).sub.109 RNA and
thereby is retained on the microtiter plate. The binding of
complementing fragment of beta-galactosidase to PL reconstitutes
its enzymatic activity. This activity is then determined by adding
substrate to provide a fluorescence or chemiluminescence signal
from active beta-galactosidase.
15. Example 15
Enzymatic Complementation Assay to Screen for Compounds that
Inhibit Interaction of CUG Repeat RNA with Recombinant MBNL1
Protein
[0351] Operation of the beta-galactosidase enzymatic
complementation assay was demonstrated using two kinds of
inhibitors. On the left panel, excess soluble (CUG).sub.109 RNA was
added to the assay reaction. The soluble (CUG).sub.109 RNA binds to
MBNL1-PL protein and prevents its retention on the microtiter
plate, reflected by reduced beta-galactosidase activity (expressed
on the vertical axis in terms of relative luminescence activity).
On the right panel, compounds having the ability to intercalate
into CUG-repeat-RNA-hairpins (EtBr, ethidium bromide; or SybrGreen
stain) were added at the indicated concentrations. Both compounds
reduce the amount of MBNL1-PL retained on plate, reflected by
reduced beta-galactosidase activity. These results show that the
enzymatic complementation assay can identify compounds that inhibit
MBNL1-CUG interaction either by binding to MBNL1 protein or binding
to CUG repeat RNA.
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Sequence CWU 1
1
1514207DNAArtificial SequenceDescription of Aritificial Sequence
note = synthetic construct 1gaatgagttg tggcgcccac aatgctccca
tgacaaggag ctgacaagtt ccattttccg 60tcgcgggcat cttggaatca tgactcccac
aatgccttgg gcacttggtc gacagtgggg 120ccgcctctga aaaaaaaatg
tgagagcagt cactcaggaa atgttgttta aggggaacct 180tctggatcct
tttcatggca ccatggcaag aagaagctgt atcttatcta tggaagataa
240agcatggagt tggctaatgg atgctgatag gaccatctag ttgcaggaaa
acaagctcag 300ggctcccact gattctacat tatgggccgt tgctccaggg
agaactgcaa atatcttcat 360ccacccccac atttaaaaac gcagttggag
ataaatggac gcaataactt gattcagcag 420aagaacatgg ccatgttggc
ccagcaaatg caactagcca atgccatgat gcctggtgcc 480ccattacaac
ccgtgccaat gttttcagtt gcaccaagct tagccaccaa tgcatcagca
540gccgccttta atccctatct gggacctgtt tctccaagcc tggtcccggc
agagatcttg 600ccgactgcac caatgttggt tacagggaat ccgggtgtcc
ctgtacctgc agctgctgca 660gctgctgcac agaaattaat gcgaacagac
agacttgagg tatgtcgaga gtaccaacgt 720ggcaattgca accgaggaga
aaatgattgt cggtttgctc atcctgctga cagcacaatg 780attgacacca
atgacaacac agtcactgtg tgtatggatt acatcaaagg gagatgctct
840cgggaaaagt gcaaatactt tcatccccct gcacatttgc aagccaagat
caaggctgcc 900caataccagg tcaaccaggc tgcagctgca caggctgcag
ccaccgcagc tgccatgact 960cagtcggctg tcaaatcact gaagcgaccc
ctcgaggcaa cctttgacct gggaattcct 1020caagctgtac ttcccccatt
accaaagagg cctgctcttg aaaaaaccaa cggtgccacc 1080gcagtcttta
acactggtat tttccaatac caacaggctc tagccaacat gcagttacaa
1140cagcatacag catttctccc accaggctca atattgtgca tgacacccgc
tacaagtgtt 1200gttcccatgg tgcacggtgc tacgccagcc actgtgtccg
cagcaacaac atctgccaca 1260agtgttccct tcgctgcaac agccacagcc
aaccagatac ccataatatc tgccgaacat 1320ctgactagcc acaagtatgt
tacccagatg tagaattttc atcactaaac aatcatgcta 1380aagaggaaag
gacagtgtgc ttggttagag taaaggacga ggtcattagc catattgtat
1440atatcgtcaa gcaacacaca caaaagttcc tcagccacaa gacatccaca
tattgcatgt 1500taaccagaag aaaagacaac attttccgga aatccactgc
acactgttgc ctatacactt 1560tgtacattta attgatattt gtgctgaggt
gatattcctg tctaaaagaa caacattgtc 1620tttcttttct agcacagagt
tatgcattca aagatgcata cctagttagt ttcctatata 1680ttcatgccat
cttgaaaaga cagactatgg tgtaaccatg attctattat gtattggtac
1740gtctgtagac caagatataa ttttttaaaa ataagtttat ttctttcaag
gtttacaaat 1800aacaaaggtg caccttgtat ttaaaattgc cattatagat
gagagcgtgc atgcacagtc 1860atttttgttt aagagtaata tttttaatgt
aatagattgt aagacgtggt gagggaggga 1920tctgacagag atgaatgtgc
caagcaaaac cacaactgtg tatattttaa agcacatcat 1980ggctttaagt
accatgttgt taaggattct catgaagtgc catagactgt acatcaaatt
2040agagtattat ttcttcagtg ttattgtttt cagagccaca ttttgttgca
tatttgctag 2100tactaatcag tcaaagggca ccattctttt tttttttttt
gaaaccaaag ctgtctcaga 2160aatggccaat ttaactttac agtaacaata
gacagcacaa cacaaactct ctcaatacag 2220ataaactcac acatactgga
gatatatata taatagatat atataaaatt attttaatgc 2280attgtagtgt
aatatttatg catactatac tgtataacat gttattcaaa agggattgcc
2340atttctgaga cacagtaaca aaaaaatgag gaaattattt tgcttctatt
tatagcctct 2400gtcaaaagtc aaaagactat aaatgctttg caaaaatggt
ttcacgtttg cttaaatgct 2460tcatcacagt cacattcaaa atagtgactc
taaacaaaga agaaagcagc actgtcatca 2520gatgcatgat aaaccaaaat
atgaaaatgg gaaatgttta attaacctag taattgggtg 2580ggttaagtac
atgggtgaat tttatatgtg atttttgttt tgttttgttt tgttcagatt
2640aactgcttat agccttagaa agccttttac aaaattaaaa aaaaaaaaat
agatgtgcat 2700tcagttttta agaatggaat catccaaagg aattcctttt
tttgaggttt ggatgttgca 2760gctagtaaag gatatttttg ctctgttcag
cagttctaaa aattgctgaa gtaggggcca 2820ggtcactggt agttatagta
tggaatggga gaagtgaaag ttcagttata gaactttcca 2880tacttccaag
tttactgcaa gtttttatgc ttgagagaga tgctttctaa tataagactg
2940atgtgttgat tttactgatt gtactgtaca tctattaaag ccttagatta
ttacattacg 3000ggttggaacc cataccaatg taatttcaat cgtgttaaga
aagtaatggt gacttcacat 3060gttattgtag ttagttacat tatagaatat
tacttatttt tcttgttaaa atgtagtttt 3120tcatttccta catttattag
attttcattt tctattaaca attgaatacc atttcagttt 3180atagacttgt
tttattagat tttaccaatg aatttttcaa aatacaaaaa aaagtagttt
3240ttccttcata acatactcag ttttgaatta catgtagtgt cacatgaata
ttcgtattgt 3300taactaaatg atttatattt tactgattta atattacagt
gtaagaatgt cagtcattgt 3360tagttcttgt ctagttttca ttaaaagaac
aaagatcttt tatatggata tcttataaat 3420atataatcat tgctaagtaa
gaagttaagt tgttgctatc gcaacaatcc tggcagacaa 3480ttgagtaata
ttttgatgat ttattttgtt tgtaattagt tattataaga agatctagat
3540cctagatatt agaataaaat ttattttcta ctgtatccat ttcaaatgtt
aaaatattgt 3600ttaatatttt tgaaatccct gagtatcagg ccttgttata
aataagctgc ataatcaata 3660aatagaacaa gggacttttt gttgataatc
caaatactca aagtttacgt aatgaaaatt 3720atagcgtgtg tgcaaactct
tgagggttga ttatgctgca atttagcatg ttggaacgtc 3780tagggagaag
gttgactttt tgcacttctg tatatagtca aaagagagaa acctgtataa
3840tagtaagatc ttattttgaa taaaaacgtc tataattaca aggagttttg
ttaaggctaa 3900tacaatgaca gactgagcaa aattgcttgc aaaagtggca
cagagttagc actccatacc 3960ccttcaaaca tgttgctttg ctttcttgtg
gacagcttgt agtttgccag gattttttca 4020gctggaaaga tacgccatcc
tttcaaaccc tcatgactga caaaaactcc atggggccaa 4080atctgcctga
agatcattac caaaaatagc aggtacttct accattaagg tgaaatcatg
4140gatcagatat tccttacatt tttcaaaact actgcatgtt taaaacttca
acaaaaaaaa 4200aaaaaaa 42072343PRTArtificial SequenceDescription of
Aritificial Sequence note = synthetic construct 2Met Gly Arg Cys
Ser Arg Glu Asn Cys Lys Tyr Leu His Pro Pro Pro1 5 10 15His Leu Lys
Thr Gln Leu Glu Ile Asn Gly Arg Asn Asn Leu Ile Gln 20 25 30Gln Lys
Asn Met Ala Met Leu Ala Gln Gln Met Gln Leu Ala Asn Ala 35 40 45Met
Met Pro Gly Ala Pro Leu Gln Pro Val Pro Met Phe Ser Val Ala 50 55
60Pro Ser Leu Ala Thr Asn Ala Ser Ala Ala Ala Phe Asn Pro Tyr Leu65
70 75 80Gly Pro Val Ser Pro Ser Leu Val Pro Ala Glu Ile Leu Pro Thr
Ala 85 90 95Pro Met Leu Val Thr Gly Asn Pro Gly Val Pro Val Pro Ala
Ala Ala 100 105 110Ala Ala Ala Ala Gln Lys Leu Met Arg Thr Asp Arg
Leu Glu Val Cys 115 120 125Arg Glu Tyr Gln Arg Gly Asn Cys Asn Arg
Gly Glu Asn Asp Cys Arg 130 135 140Phe Ala His Pro Ala Asp Ser Thr
Met Ile Asp Thr Asn Asp Asn Thr145 150 155 160Val Thr Val Cys Met
Asp Tyr Ile Lys Gly Arg Cys Ser Arg Glu Lys 165 170 175Cys Lys Tyr
Phe His Pro Pro Ala His Leu Gln Ala Lys Ile Lys Ala 180 185 190Ala
Gln Tyr Gln Val Asn Gln Ala Ala Ala Ala Gln Ala Ala Ala Thr 195 200
205Ala Ala Ala Met Thr Gln Ser Ala Val Lys Ser Leu Lys Arg Pro Leu
210 215 220Glu Ala Thr Phe Asp Leu Gly Ile Pro Gln Ala Val Leu Pro
Pro Leu225 230 235 240Pro Lys Arg Pro Ala Leu Glu Lys Thr Asn Gly
Ala Thr Ala Val Phe 245 250 255Asn Thr Gly Ile Phe Gln Tyr Gln Gln
Ala Leu Ala Asn Met Gln Leu 260 265 270Gln Gln His Thr Ala Phe Leu
Pro Pro Gly Ser Ile Leu Cys Met Thr 275 280 285Pro Ala Thr Ser Val
Val Pro Met Val His Gly Ala Thr Pro Ala Thr 290 295 300Val Ser Ala
Ala Thr Thr Ser Ala Thr Ser Val Pro Phe Ala Ala Thr305 310 315
320Ala Thr Ala Asn Gln Ile Pro Ile Ile Ser Ala Glu His Leu Thr Ser
325 330 335His Lys Tyr Val Thr Gln Met 340325DNAArtificial
SequenceDescription of Aritificial Sequence note = synthetic
construct 3agcagcagca gcagcagcag cagca 25425DNAArtificial
SequenceDescription of Aritificial Sequence note = synthetic
construct 4ccaggcacgg tctgcaacag agaag 25525DNAArtificial
SequenceDescription of Aritificial Sequence note = synthetic
construct 5gaagagacaa cgtctggcac ggacc 25625DNAArtificial
SequenceDescription of Aritificial Sequence note = synthetic
construct 6ggaagtgaaa cttgcctcca tcagg 25724DNAArtificial
SequenceDescription of Aritificial Sequence note = synthetic
construct 7tgaaggaata cctcacactc aagg 24820DNAArtificial
SequenceDescription of Aritificial Sequence note = synthetic
construct 8cacggaacac aaaggcactg 20921DNAArtificial
SequenceDescription of Aritificial Sequence note = synthetic
construct 9gtgtgagtcg ctccagaaac g 211023DNAArtificial
SequenceDescription of Aritificial Sequence note = synthetic
construct 10ccaccacagg accatgttat ttc 231131RNAArtificial
SequenceDescription of Aritificial Sequence note = synthetic
construct 11gugcuucucu guugcagacc gugccugggc a 311231RNAArtificial
SequenceDescription of Aritificial Sequence note = synthetic
construct 12gccccugaug gaggcaaguu ucacuuccuc c 311325DNAArtificial
SequenceDescription of Aritificial Sequence note = synthetic
construct 13ggactacctc cgttcaaagt gaagg 251412DNAArtificial
SequenceDescription of Aritificial Sequence note = synthetic
construct 14cagcagcagc ag 121515DNAArtificial SequenceDescription
of Aritificial Sequence note = synthetic construct 15cagcagcagc
agcag 15
* * * * *
References