U.S. patent application number 12/773137 was filed with the patent office on 2011-06-09 for methods for altering mrna splicing and treating familial dysautonomia and other mechanistically related disorders.
This patent application is currently assigned to The General Hospital Corporation. Invention is credited to James F. Gusella, Susan A. Slaugenhaupt.
Application Number | 20110136836 12/773137 |
Document ID | / |
Family ID | 34426055 |
Filed Date | 2011-06-09 |
United States Patent
Application |
20110136836 |
Kind Code |
A1 |
Slaugenhaupt; Susan A. ; et
al. |
June 9, 2011 |
Methods for Altering MRNA Splicing and Treating Familial
Dysautonomia and Other Mechanistically Related Disorders
Abstract
This invention relates to methods for altering the splicing of
mRNA in cells. In particular, this invention also relates to
methods for increasing the ratio of wild type to misspliced forms
of mRNA and corresponding encoded proteins in cells possessing a
mutant gene encoding either the i) misspliced mRNA corresponding to
the mutant protein or ii) a component in the splicing machinery
responsible for processing the misspliced mRNA. In addition, this
invention relates to treating individuals having a disorder
associated with a misspliced mRNA, such as Familial Dysautonomia or
Neurofibromatosis 1, by administering to such an individual a
cytokinin such as kinetin.
Inventors: |
Slaugenhaupt; Susan A.;
(Hingham, MA) ; Gusella; James F.; (Framingham,
MA) |
Assignee: |
The General Hospital
Corporation
Boston
MA
|
Family ID: |
34426055 |
Appl. No.: |
12/773137 |
Filed: |
May 4, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10956601 |
Oct 1, 2004 |
7737110 |
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12773137 |
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60536287 |
Jan 13, 2004 |
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60508465 |
Oct 3, 2003 |
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Current U.S.
Class: |
514/263.1 |
Current CPC
Class: |
C12Q 1/6883 20130101;
A61K 31/355 20130101; A61P 3/04 20180101; A61K 31/353 20130101;
A61P 25/28 20180101; A61P 25/00 20180101; C12Q 2600/158 20130101;
A61K 31/52 20130101; C12Q 2600/156 20130101; C12Q 2600/136
20130101; A61K 31/353 20130101; A61K 2300/00 20130101; A61K 31/355
20130101; A61K 2300/00 20130101; A61K 31/52 20130101; A61K 2300/00
20130101 |
Class at
Publication: |
514/263.1 |
International
Class: |
A61K 31/52 20060101
A61K031/52; A61P 25/28 20060101 A61P025/28; A61P 25/00 20060101
A61P025/00 |
Goverment Interests
[0002] This invention was made with United States Government
support under grants from the National Institutes of Neurological
Disorders and Stroke. The United States Government has certain
rights in this invention.
Claims
1-45. (canceled)
46. A method for treating neuronal degeneration in a subject in
need thereof comprising administering to said subject a composition
that comprises an effective concentration, of one or more
6-(substituted amino)purine cytokinins selected from the group
consisting of the compounds of kinetin, benzyl adenine, isopentenyl
adenine, (6-(3-hydroxymethyl-3-methylallyl)-aminopyrine),
6-(3,3-dimethylallyl)aminopyrine, 6-(benzyl)aminopyrine,
6-(phenyl)aminopyrine, 6-(n-alkyl)aminopyrine, in which the n-alkyl
group has 4, 5, or 6 carbons, 6-(cyclohexyl)methylaminopurine, and
those compounds of Formula I, ##STR00003## in which R.sub.1 is
furfuryl, phenyl, benzyl, n-alkyl of 4, 5, or 6 carbons, branched
alkyl of 4, 5, or 6 carbons, (cyclohexyl)methyl, 3,3-dimethylallyl,
and 3-hydroxymethyl -3-methylallyl.
47. (canceled)
48. A method for treating neuronal degeneration or familial
dysautonomia in a subject in need thereof, comprising administering
to said subject an effective amount of a composition that comprises
one or more cytokinins, one or more cytokinins and one or more
tocotrienols, or one or more cytokinins and (-)-epigallocatechin
gallate.
49. The method according to claim 48, wherein at least one of the
one or more tocotrienols is tocotrienols selected from the group
consisting of .alpha.-tocotrienol, .beta.-tocotrienol,
.gamma.-tocotrienol, and .delta.-tocotrienol.
50. The method according to claim 48, wherein at least one of the
one or more tocotrienols is .delta.-tocotrienol.
51-54. (canceled)
55. The method according to claims 46 or 48 wherein the subject is
a mammal.
56. The method according to claims 46 or 48 wherein the subject is
a human.
57. The method according to claim 48, wherein at least one of the
one or more cytokinins selected from the group consisting of
benzyladenine and kinetin.
58. The method according to claim 48, wherein at least one of the
one or more cytokinins is kinetin.
59. A method of treating familial dysautonomia in a subject in need
thereof, comprising administering to said subject a composition
that comprises an effective concentration of one or more
6-(substituted amino)purine cytokinins selected from the group
consisting of the compounds of kinetin, benzyladenine, isopentenyl
adenine, (6-(3-hydroxymethyl-3-methylallyl)-aminopyrine),
6-(3,3-dimethylally)aminopyrine, 6-(benzyl)aminopyrine,
6-(phenyl)aminopyrine, 6-(n-alkyl)aminopyrine, in which the n-alkyl
group has 4, 5, or 6 carbons, 6-(cyclohexyl)methylaminopurine, and
those compounds of Formula I, ##STR00004## in which R.sub.1 is
furfuryl, phenyl, benzyl, n-alkyl of 4, 5, or 6 carbons, branched
alkyl of 4, 5, or 6 carbons, (cyclohexyl)methyl, 3,3-dimethylallyl,
and 3-hydroxymethyl-3-methylallyl.
60. (canceled)
61. The method according to claim 59, wherein at least one of the
one or more tocotrienols selected from the group consisting of
.alpha.-tocotrienol, .beta.-tocotrienol, .gamma.-tocotrienol, and
.delta.-tocotrienol.
62. The method according to claim 59, wherein at least one of the
one or more tocotrienols is .delta.-tocotrienol.
63-135. (canceled)
136. The method according to claim 59, wherein the subject is a
mammal.
137. The method according to claim 59, wherein the subject is a
human.
Description
[0001] This application is a divisional of application Ser. No.
10/956,601, filed Oct. 1, 2004, which claims priority to U.S.
provisional application Ser. No. 60/508,465, filed Oct. 3, 2003 and
U.S. provisional application Ser. No. 60/536,287, filed Jan. 13,
2004, the contents of each of which are incorporated in their
entirety.
FIELD OF THE INVENTION
[0003] This invention relates to methods for altering the splicing
of mRNA in cells. In addition, this invention also relates to
methods for correcting the ratio of wild type to mutant spliced
forms of mRNA and corresponding encoded proteins in cells
possessing a mutant gene encoding either i) the misspliced mRNA
corresponding to the mutant protein or ii) a component of the
splicing machinery. In addition, this invention relates to treating
individuals having a disorder associated with a misspliced mRNA,
such as familial dysautonomia, by administering to such an
individual a cytokinin such as kinetin.
[0004] In particular, the invention relates to enhancing correct
mRNA splicing in order to increase cellular levels of normal or
wild type IKAP mRNA or protein encoded by a IKBKAP gene in various
cell types. The defective splicing of pre-mRNA is a major cause of
human disease.
BACKGROUND OF THE INVENTION
[0005] Exon skipping is a common result of splice mutations and has
been reported in a wide variety of genetic disorders.sup.1, yet the
underlying mechanism is poorly understood. Often, such mutations
are incompletely penetrant, and low levels of normal transcript and
protein are maintained.sup.1. Familial dysautonomia (FD)
(MIM#2239001), also known as Riley Day syndrome or hereditary
sensory and autonomic neuropathy III (HSAN-III), is the best-known
and most common member of a group of congenital sensory and
autonomic neuropathies (HSAN) characterized by widespread sensory
and variable autonomic dysfunction (Axelrod F B: (1996) Autonomic
and Sensory Disorders. In: Principles and Practice of Medical
Genetics, 3rd edition, A E H Emory and D L Rimoin eds. Churchill
Livingstone, Edinburgh. pp 397-411; Axelrod F B (2002) Hereditary
Sensory and Autonomic Neuropathies: Familial Dysautonomia and other
HSANs. Clin Auton Res 12 Supplement 1, 2-14). FD affects neuronal
development and is associated with progressive neuronal
degeneration. Multiple systems are impacted resulting in a markedly
reduced quality of life and premature death (Axelrod F B: (1996)
Autonomic and Sensory Disorders. In: Principles and Practice of
Medical Genetics, 3rd edition, A E H Emory and D L Rimoin eds.
Churchill Livingstone, Edinburgh. pp 397-411; Axelrod F B (2002)
Hereditary Sensory and Autonomic Neuropathies: Familial
Dysautonomia and other HSANs. Clin Auton Res 12 Supplement 1,
2-14).
[0006] FD is a recessive disorder that has a remarkably high
carrier frequency of 1 in 30 in the Ashkenazi Jewish
population.sup.5. FD is caused by mutations in the IKBKAP
gene.sup.2,3 (Genbank Accession No. NM.sub.--003640.), and all
cases described to date involve an intron 20 mutation that results
in a unique pattern of tissue-specific exon skipping. Accurate
splicing of the mutant IKBKAP allele is particularly inefficient in
the nervous system. Three FD mutations have been identified in the
I-k-B kinase (IKK) complex-associated protein (IKBKAP):
IVS20.sup.+6T.fwdarw.C, which leads to variable, tissue-specific
skipping of exon 20 (FIG. 1a), R696P, and P914L.sup.2,3,6. All FD
patients tested to date carry at least one IVS20.sup.+6T.fwdarw.C
mutation, with more than 99.5% being homozygous, and the remainder
being heterozygous with either R696P or P914L on the alternate
allele.
[0007] The IVS20.sup.+6T.fwdarw.C mutation does not cause complete
loss of function. Instead, it results in a tissue-specific decrease
in splicing efficiency of the IKBKAP transcript; cells from
patients retain some capacity to produce normal mRNA and IKAP
protein (Slaugenhaupt et al. (2001) Tissue-specific expression of a
splicing mutation in the IKBKAP gene causes familial dysautonomia.
Am J Hum Genetics 68:598-605). The mRNA is widely distributed.
Highest levels are in the nervous system, but substantial amounts
are also present in peripheral organs (Mezey et al. (2003) Of
splice and men: what does the distribution of IKAP mRNA in the rat
tell us about the pathogenesis of familial dysautonomia? Brain
Research 983:209). It has been reported previously that all FD
tissues tested express both wild-type (WT) and mutant (MU) IKBKAP
mRNA.sup.2,4. The effect of the most common (splicing) mutation
varies from tissue to tissue--neuronal tissues seem primarily to
express mutant mRNA; somatic tissues express roughly equal levels
of normal and mutant mRNA. Accurate measurement of the ratio of the
two mRNA species using both densitometry and real-time quantitative
PCR has revealed that the levels of WT IKBKAP mRNA vary between
tissues and are lowest in central and peripheral nervous
systems.sup.4. This leads to a drastic reduction in the amount of
IKAP protein in these tissues.
[0008] There are other disorders that are caused, at least in part,
by missplicing including Neurofibromatosis 1 (NF1), also known as
von Recklinghausen NF or Peripheral NF. NF1 occurs in 1:4,000
births and is characterized by multiple cafe-au-lait spots and
neurofibromas on or under the skin. Enlargement and deformation of
bones and curvature of the spine may also occur (Riccardi, 1992,
Neurofibromatosis: phenotype, natural history, and pathogenesis.
2nd ed. Baltimore: Johns Hopkins University Press). Occasionally,
tumors may develop in the brain, on cranial nerves, or on the
spinal cord. About 50% of people with NF also have learning
disabilities (Chapter 6 in Rubenstein and Korf, 1990,
Neurofibromatosis: a handbook for patients, families, and
health-care professionals. New York: Thieme Medical
Publishers).
[0009] The NF1 gene was identified and the protein product
characterized in 1990 (Cawthon et al., 1990, Cell 62: 193-201;
Wallace et al., 1990, Science 249:181-6). The entire sequence of
the expressed NF1 gene has been reported (Viskochil et al., 1993,
Annu Rev Neurosci 16: 183-205; Gutmann and Collins, 1993, Neuron
10: 335-43; Genbank Accession No. NM.sub.--000267). The gene is has
at least 59 exons and codes for a 2818 amino acid protein called
neurofibromin. To date, 180 different NF1 mutations have been
identified. The NF1 Genetic Analysis Consortium maintains a
database of mutations identified in more than 45 collaborating
laboratories throughout the world. According to data from the
Consortium, the NF1 mutations described to date include 4
chromosomal rearrangements, 89 deletions (14 deletions involving
the entire gene, 35 deletions involving multiple exons, and 37
small deletions), 23 insertions (3 large and 20 small), 45 point
mutations (29 stop mutations and 16 amino acid substitutions), and
18 intronic mutations affecting splicing, and 4 mutations in 3'
untranslated region of the gene. About 30% of NF1 patients carry a
splice mutation resulting in the production of one or several
shortened transcripts (Vandenbroucke et al., 2002, BMC Genomics
3:13 and Serra et al., 2001, Hum Genet. 108:416-29).
[0010] Cytokinins are a class of plant hormones defined by their
ability to promote cell division in plant tissue explants in the
presence of an auxin, such as indoleacetic acid, and nutrients,
including vitamins, mineral salts, and sugar. In promoting cell
division of plant cells, cytokinins are active at low
concentrations (as low 0.01 parts per million (ppm)), but exhibit
activity only in the presence of an auxin. Certain cytokinins,
including zeatin and 6-(3,3-dimethylallyl)-aminopurine, also occur
as the base moiety components of transfer RNA in yeast, bacterial,
animal cells and plant cells. The cytokinin kinetin
(6-furfuryl-aminopurine) forms complexes with certain RNA-binding
proteins of wheat embryo extracts and appears to promote protein
synthesis in plants (see, e.g., Spirin and Ajtkhozhin (1985) Trends
in Biochem. Sci., p. 162). Kinetin and other cytokinins are used in
conjunction with auxin used in horticulture and in plant tissue
culture, such as in the production of plantlets from plant callus
tissue. Cytokinins are also used in the production of protein-rich
yeast (see e.g., East German Patent No. 148,889 (1981) (Derwent
World Patent Index Abstract)) and to augment the growth of
microbial cultures (Merck Index, 10th Ed. (1983) Entry 5148, Merck
and Co., Rahway, N.J., U.S.A.).
[0011] Kinetin belongs to the family of N.sup.6-substituted adenine
derivatives known as cytokinins, or plant growth factors, that also
includes zeatin, benzyladenine and 2iP (FIG. 1b). Kinetin is also
known as 6-furfurylaminpurine (C.sub.10H.sub.9N.sub.5) and has a
molecular weight of 215.21 (Soriano-Garcia and Parthsarathy,1975,
Biochem Biophys Res Commun 64:1062-8). Kinetin is currently
marketed as an anti-aging ingredient in skin treatments due to its
ability to ameliorate aging characteristics in cultured human
fibroblasts.sup.8, possibly through anti-oxidant
activity.sup.9.
[0012] Certain cytokinins have been shown to inhibit the growth of
tumor cells in vitro (see, e.g., Katsaros et al. (1987) FEBS Lttrs.
223:97-103). It appears that this effect is mediated via the
cytotoxic affects of adenosine analogs, such as the 6-(substituted
amino) purine cytokinins, that interfere with tRNA methylating
enzymes (Wainfan et al, (1973) Biochem. Pharmacol. 22:493-500).
When immortalized fibroblast cells are contacted with adenosine
analogs the cultured cells exhibit decreased growth rate and a
change in morphology from the normal flattened elongated morphology
typical of cultured fibroblasts to a very elongated spindle-shape
characteristic of a cytotoxic response. The very elongated shape of
immortalized cells exhibiting this response is not shape
characteristic of young, healthy, primary cultures of normal
diploid fibroblasts.
[0013] Kinetin has been shown to be capable of delaying or
preventing a host of age-related changes of human skin fibroblasts
grown in laboratory culture which has led to its incorporation into
topical skin products. West MID (1994) The cellular and molecular
biology of skin aging. Arch Dermatol 130:87-95. Fibroblasts, which
produce collagen and elastin, have been shown to decrease in number
and vitality as skin ages not only in vitro, but also in vivo. The
number of fibroblasts decreases at least 50% between birth and the
age of 80 years. West MID (1994) The cellular and molecular biology
of skin aging. Arch Dermatol 130:87-95. Rattan S I, Clark B F.
(1994) Kinetin delays the onset of aging characteristics in human
fibroblasts. Biochem Biophys Res Commun 201:665-72. Kinetin has
been shown to delay or prevent a range of cellular changes
associated with in vitro aging of human skin cells, including
alterations in cell morphology, growth rate, size, cytoskeletal
organization, macromolecular synthetic activity and accumulation of
lipofuscin aging pigments but kinetin did not alter the maximum in
vitro life span of human skin cells or their ability to multiply in
culture. Rattan S I, Clark B F. (1994) Kinetin delays the onset of
aging characteristics in human fibroblasts. Biochem Biophys Res
Commun 201:665-72. Thus, kinetin was devoid of activities
associated with cellular immortalization, malignant transformation
and carcinogenesis. Rattan S I, Clark B F. (1994) Kinetin delays
the onset of aging characteristics in human fibroblasts. Biochem
Biophys Res Commun 201:665-72.
[0014] Using rats as a mammalian model, it has been shown that
plant cytokinins can affect lipid peroxidation in erythrocyte,
muscle, liver, heart and kidney tissue, Celik I, Tuluce Y, Ozok
N.(2002) Effects of indoleacetic acid and kinetin on lipid
peroxidation levels in various rat tissues. Turk J Biol 26;193-196.
Celik et al.(2002) showed that kinetin had much less toxicity as
compared to indoleacetic acid (IAA), when administered orally to
rats. Celik I, Tuluce Y, Ozok N.(2002) Effects of indoleacetic acid
and kinetin on lipid peroxidation levels in various rat tissues.
Turk J Biol 26;193-196. It was shown that IAA interacts primarily
with the liver and kidney tissue cells, resulting in lipid
peroxidation synthesis, whereas kinetin had no such effect in the
liver or kidney.
SUMMARY OF THE INVENTION
[0015] This invention relates to methods and compositions for
increasing the amount of wild type protein encoded by cells
possessing a misspliced mRNA due to either a mutation in i) the
misspliced mRNA corresponding mutant protein or ii) a component of
the splicing machinery. In preferred embodiments, the misspliced
mRNA is a mutant mRNA. The increased wild type protein results from
contacting cells with or administering to individuals one or more
compounds which increases the amount of properly spliced mRNA. In
preferred embodiments, the compound is a cytokinin. In a more
preferred embodiment, the cytokinin is kinetin,
(6-furfurylaminopurine).
[0016] In one embodiment of this invention, cells possessing a
mutant gene resulting in misspliced mRNA are contacted with a
cytokinin which results in an enhanced ratio of correctly spliced
mRNA compared to misspliced mRNA.
[0017] In another embodiment of this invention, cells or
individuals are contacted with a cytokinin (preferably kinetin) to
treat neuronal degeneration.
[0018] In another embodiment of this invention a cytokinin
(preferably kinetin) is administered to an individual with a
disorder or disease associated with a mutation resulting in
misspliced mRNA.
[0019] In a preferred embodiment, individuals with familial
dysautonomia (FD) are treated with a therapeutically effective
amount of a cytokinin (preferably kinetin) to decrease missplicing
of the IKBKAP transcript and increase the ratio of wild type IKAP
protein versus mutant IKAP protein. In a specific embodiment, a
pharmaceutical composition comprises the therapeutically effective
amount of the cytokinin and is administered to a subject suffering
or likely to suffer from FD.
[0020] In another preferred embodiment, individuals with
Neurofibromatosis 1 (NF1) caused by missplicing are treated with a
therapeutically effective amount of a cytokinin (preferably
kinetin) to decrease missplicing of the NF1 transcript and increase
the ratio of wild type neurofibromin protein versus mutant
neurofibromin protein. In a specific embodiment, a pharmaceutical
composition comprises the therapeutically effective amount of the
cytokinin and is administered to a subject suffering or likely to
suffer from NF1.
[0021] The methods of the invention encompass in vivo and in vitro
screening assays to identify compounds that alter the splicing of
misspliced mRNA transcripts. In one embodiment, the misspliced mRNA
transcript is a mutant IKBKAP mRNA transcript. In a more specific
embodiment, the mutant IKBKAP mRNA transcript carries a mutation
that is present in a mutant IKBKAP mRNA in a subject with FD. In an
even more specific embodiment, the mutant IKBKAP mRNA transcript
has the IVS20.sup.+6T.fwdarw.C mutation. In another another
embodiment, the misspliced mRNA transcript is a mutant NF1 mRNA
transcript. In a more specific embodiment, the mutant NF1 mRNA
transcript carries a mutation that is present in a mutant NF1 mRNA
in a subject with NF1. Candidate compounds are screened for the
ability to alter the splicing of misspliced mRNA transcripts
comprising contacting a mammalian cell comprising DNA which
comprises the gene (or a fragment or variant thereof) which is
misspliced with a candidate compound and determining the amount of
misspliced and/or wild type mRNA transctipt. In a specific
embodiment, candidate compounds are screened for the ability to
alter the inclusion of exon 20 of a wild type or mutated IKBKAP
gene. In more specific embodiments, the ratio of exon 20 inclusion
to exon 20 skipping is determined for spliced IKBKAP mRNA
transcripts in the presence and absence of the candidate compound.
An alteration in the ratio of exon 20 skipping to exon 20 inclusion
indicates that the candidate compound did alter the splicing of the
misspliced mRNA.
[0022] It is an object of the present invention to provide a
compound or compounds which are suitable as therapeutic agent(s)
for the treatment of disorders involving missplicing of mRNAs,
especially FD and NF1. A further object of the present invention is
to provide a process and compositions which are suitable for
altering the splicing of mRNA in a mammalian cell.
[0023] The present invention provides a composition which is
capable of affecting mRNA splicing. In a preferred embodiment, the
composition is capable of altering mRNA splicing of the IKBKAP
gene. In a specific preferred embodiment, the composition is
capable of altering IKBKAP gene splicing by increasing the
inclusion of exon 20. In another preferred embodiment, the
composition is capable of altering mRNA splicing of the NF1 gene.
As such the composition is also useful as a pharmaceutical
composition to prevent, manage, and/or treat FD and/or NF1 caused
by missplicing according to the methods of the invention.
[0024] In some embodiments, the one or more cytokinins used in the
methods of the invention are administered to a subject in need
thereof in combination with at least one other compound that
provides a therapeutic effect. Examples of such other compounds
include, but are not limited to, antioxidants (such as
(-)-epigallocatechin gallate) and tocotrienols (such as
.alpha.-tocotrienol, .beta.-tocotrienol, .gamma.-tocotrienol, and
.delta.-tocotrienol).
BRIEF DESCRIPTION OF THE DRAWINGS
[0025] FIG. 1 Kinetin significantly increases production of
wild-type IKBKAP transcript in FD cells. (a) Schematic diagram
illustrating the location of the IVS20.sup.+6.fwdarw.C mutation in
IKBKAP and the two IKBKAP isoforms produced from this allele in FD
patients. (b) Chemical structures of the cytokinins tested in this
study: kinetin (6-furfurylaminopurine), benzyladenine
(6-benzylaminopurine), 2i P
(6-(.gamma.,.gamma.-dimethyallylamino)purine), and zeatin
(6-(4-hydroxy-3-methylbut-2-enylamino)purine). (c) RT-PCR analysis
of 2 independent FD lymphoblast cell lines, tested in triplicate,
following treatment with 10 .mu.M kinetin in 0.1% DMSO or DMSO
alone. Previous experiments demonstrated that DMSO has no effect on
IKBKAP splicing. WT:MU ratios were determined using the integrated
density value (IDV) obtained for each band and are shown beneath
each lane. The sizes of the WT (including exon 20) and MU
(excluding exon 20) PCR products are indicated on the right. Primer
sequences and PCR conditions have been previously
described.sup.4.
[0026] FIG. 2 The action of kinetin on IKBKAP splicing is dose and
time dependent and results in an increase of IKAP protein in FD
lymphoblast cell lines. (a) RT-PCR demonstrating increasing WT:MU
IKBKAP ratios as a result of increased kinetin concentration. Sizes
of the WT and MU bands are shown on the right, and the IDV ratios
are shown beneath each lane. (b) Western blot probed with an IKAP
monoclonal antibody showing that increasing concentrations of
kinetin result in an increase in IKAP protein production in FD
cells. The lower panel shows the same blot probed with an IDE
antibody as a protein loading control. (c) Graph of WT:MU IKBKAP
ratios at increasing kinetin concentrations determined by QPCR of
FD cells (treated with kinetin in water in duplicate and each
amplified in triplicate) showing that higher doses of kinetin
continue to increase WT:MU IKBKAP. Examination of panel (a) shows
that at kinetin concentrations of 100 .mu.M the MU band is barely
discernable using densitometry, therefore QPCR was used for this
study. (d) Graph of WT:MU ratios generated using IDV values
following culturing of FD cells in 50 .mu.M kinetin for increasing
lengths of time. All treatments were performed in duplicate and
average data points are plotted.
[0027] FIG. 3 Kinetin has no effect on incorporation of exon 31 in
the alternatively spliced MYO5A gene (a) schematic diagram
illustrating two of the alternative transcripts produced by MYO5A.
The primers used for amplification are illustrated by arrows
indicated on each isoform. (b) representative example of RT-PCR
from FD lymphoblast cells showing no change in the ratio of MYO5A
isoforms by kinetin treatment. Nine independent cell lines were
tested.
[0028] FIG. 4 Kinetin increases WT:MU IKBKAP transcript ratio in
the presence or absence of nonsense mediated decay (NMD) of the
mutant transcript. Two FD lymphoblast cell lines, FD1 and FD2, were
untreated (U) or treated with 50 pg/ml cycloheximide (C) to inhibit
NMD, 100 .mu.M kinetin (K), or cycloheximide+kinetin (C+K). RT-PCR
amplification and fractionation of the amplified product on a 1.5%
agarose gel was performed on cell extracts. WT:MU IKBKAP transcript
ratios were determined using the integrated density value (IDV)
obtained for each band and are shown beneath each lane. Primer
sequences and PCR conditions have been previously described
.sup.4
[0029] FIG. 5 Kinetin enhances inclusion of exon 20 in both the MU
and WT IKBKAP minigene. (a) schematic diagram illustrating the
minigene constructs and the location of the IVS20.sup.+6T.fwdarw.C
mutation. Vector specific primers used for RT-PCR analysis are
shown. (b) RT-PCR of MU and WT minigene RNA isolated from HEK293
cells following transfection and treatment with kinetin. PCR was
performed using the primers T7 and BGH-R. Trace amounts of MU
IKBKAP can be seen in the untreated WT lanes. PCR fragment sizes of
the two spliced products are shown on the left. (c) RT-PCR of the
same RNA in panel (b) using T7 and a primer that spans the 19-21
exon junction known to specifically amplify the MU band.sup.4,
showing absence of MU transcript in the WT lane following kinetin
treatment. The size of this fragment is shown on the left.
DETAILED DESCRIPTION OF THE INVENTION
[0030] Novel methods are provided for increasing the amount of mRNA
spliced in a wild type fashion and/or wild type protein in cells or
individuals using one or more compounds that alters the splicing of
mRNA transcripts. In preferred embodiments, the ratio of wild-type
to misspliced mRNA or wild type to mutant protein in a cell or
individual is increased by administration of the one or more
compounds. Methods of the invention can be used to prevent, manage,
or treat disorders associated with missplicing. In preferred
embodiments, the compound is a cytokinin, preferably a
6-(substituted amino) purine cytokinin, more preferably
benzyladenine, most preferably kinetin. In other embodiments, the
compound is one that increases production of properly spliced mRNA
to a degree that is substantially similar to or greater than
kinetin.
Disorders Treated with Methods of the Invention
[0031] The invention further relates to methods for altering the
splicing of IKBKAP by contacting cells with a cytokinin,
(preferably benzyladenine and more preferably kinetin). In
particular, the invention relates to enhancing correct mRNA
splicing in order to increase cellular levels of normal or wild
type IKAP mRNA or protein encoded by a mutant IKBKAP gene in
lymphoblast, fibroblast and neuronal cells. In specific
embodiments, the invention relates to the use of kinetin for
increasing the inclusion of exon 20 from the IKBKAP gene in spliced
mRNA transcripts. In more specific embodiments, kinetin provides a
treatment for individuals with FD by increasing the level of normal
IKAP mRNA and protein.
[0032] The methods of the invention can be used to prevent, manage,
or treat other disorders, in addition to FD, characterized by
missplicing (see Table 1 for non-FD disorders). The missplicing in
the other disorders may result from a mutation in i) the misspliced
transcript, or ii) a component of the splicing machinery
responsible for processing the misspliced transcript.
[0033] In a specific embodiment, the non-FD disorder prevented,
managed, or treated by the methods of the invention is NF1 caused
by missplicing. NF1 gene splicing can be altered (e.g., to increase
levels of wild type splicing) by contacting cells with a cytokinin,
(preferably benzyladenine and more preferably kinetin).
TABLE-US-00001 TABLE 1 Misspliced mRNA Disorder transcript Mutant
gene Neurofibromatosis 1 (NF1) NF1 NF1 Neurofibromatosis 2 (NF2)
NF2 NF2 Familial isolated growth growth hormone growth hormone
hormone deficiency type (GH-1) (GH-1) II (IGHD II) Frasier syndrome
Wilms tumor Wilms tumor suppressor suppressor gene (WT1) gene (WT1)
Frontotemporal demetia and tau (MAPT) tau (MAPT) Parkinsonism lined
to Chromosome 17 (FTDP-17) Atypical cystic fibrosis cystic fibrosis
cystic fibrosis transmembrane transmembrane conductance conductance
regulator (CFTR) regulator (CFTR) Menkes Disease (MD) ATP7A ATP7A
Occipital Horn Syndrome ATP7A ATP7A Myotonic dystrophy DM protein
kinase type 1 (DM1) (DMPK) Myotonic dystrophy ZNF9 type 2 (DM2)
Retinitis pigmentosa (RP) opsin PRPF31, HRRP3, or PRPC8 Spinal
muscular Survivor of atrophy (SMA) Motor Neuron gene 2 (SMN2)
[0034] This invention further provides methods for treating
individuals at risk for developing a disorder associated with a
misspliced mRNA transcript. In a preferred embodiments, the
individual is at risk for developing familial dysautonomia or NF-1.
Persons at risk for developing familial dysautonomia or NF1 may be
identified by genetic screening for the presence of a mutation
associated with familial dysautonomia (see e.g., International
Patent Application No. PCT/US02/00473, filed Jan. 7, 2002,
entitled: GENE FOR IDENTIFYING INDIVIDUALS WITH FAMILIAL
DYSAUTONOMIA and which is incorporated herein by reference in its
entirety) or NF-1 caused by missplicing (see e.g., U.S. Pat. Nos.
5,227,292, 5,605,799, 5,859,195, and 6,238,861). Accordingly, the
compositions for use with the methods of this invention may be
administered to an individual at various times during the course of
the disease and during different degrees of expression of clinical
symptoms. In a preferred embodiment, this invention provides
methods and compositions for treating individuals with and at risk
for developing the degenerative symptoms associated with familial
dysautonomia, such as neuronal degeneration.
Compounds for use in the Methods of the Invention
[0035] Compositions administered for the treatment of a disorder
associated with a misspliced mRNA transcript can comprise one or
more compounds that increase the amount of mRNA transcript spliced
in a wild type manner and/or alter the ratio of wild type to
misspliced mRNA transcripts. In preferred embodiments, at least one
of the compounds in the composition is a cytokinin. In more
preferred embodiment, the cytokinins are 6-(substituted
amino)purine cytokinins. 6-(substituted amino)purine cytokinins
include, but are not limited to, benzyladenine, kinetin, and
6-amino analogs thereof of Formula I:
##STR00001##
[0036] in which R.sub.1 is furfuryl, phenyl, benzyl, n-alkyl of 4,
5, or 6 carbons, branched alkyl of 4, 5, or 6 carbons, (cyclohexyl)
methyl, 3,3-dimethylallyl, and 3-hydroxymethyl-3-methylallyl. Among
the 6-(substituted amino)purine cytokinins that are intended to be
used, singly or in combination, as a compound in the methods herein
are kinetin, benzyladenine, isopentenyl adenine,
(6-(3-hydroxymethyl-3-methylallyl)-aminopurine),
6-(3,3-dimethylallyl)aminopurine, 6-(benzyl)aminopurine,
6-(phenyl)aminopurine, 6-(n-alkyl)aminopurine, in which the n-alkyl
group has 4, 5 or 6 carbons, and 6-(cyclohexyl)methylaminopurine.
Most preferred is kinetin (6-(furfuryl)aminopurine). Other such
6-(substituted amine) purine cytokinins may be tested for the
ability to improve proper mRNA splicing in cells in vitro.
[0037] In other preferred embodiments, the one or more cytokinins
are administered to a subject in need thereof in combination with
at least one other compound that provides a therapeutic effect.
Examples of such other compounds include, but are not limited to,
antioxidants (such as (-)-epigallocatechin gallate) and
tocotrienols (such as .alpha.-tocotrienol, .beta.-tocotrienol,
.gamma.-tocotrienol, and .delta.-tocotrienol). Such other compounds
to be administered in combination with the one or more cytokinins
may or may not be part of the same composition that comprises the
one or more cytokinins.
[0038] The term "in combination" is not limited to the
administration of the compounds at exactly the same time, but
rather it is meant that the compounds are administered to a subject
in a sequence and within a time interval such that they can act
together to provide an increased benefit than if they were
administered otherwise. For example, each compound may be
administered at the same time or sequentially in any order at
different points in time; however, if not administered at the same
time, they should be administered sufficiently close in time so as
to provide the desired therapeutic effect. Each compound can be
administered separately, in any appropriate form and by any
suitable route.
[0039] In various embodiments, the compounds are administered less
than 1 hour apart, at about 1 hour apart, at about 1 hour to about
2 hours apart, at about 2 hours to about 3 hours apart, at about 3
hours to about 4 hours apart, at about 4 hours to about 5 hours
apart, at about 5 hours to about 6 hours apart, at about 6 hours to
about 7 hours apart, at about 7 hours to about 8 hours apart, at
about 8 hours to about 9 hours apart, at about 9 hours to about 10
hours apart, at about 10 hours to about 11 hours apart, at about 11
hours to about 12 hours apart, no more than 24 hours apart or no
more than 48 hours apart. In preferred embodiments, two or more
compounds are administered within the same patient visit.
[0040] Those compounds that improve proper mRNA splicing either by
increasing the amount of wild type mRNA transcript and/or by
increasing the ratio of wild-type to mutant mRNA or protein may be
formulated as pharmaceuticals for administration to individuals or
added to tissue culture medium at effective concentrations. Such
concentrations are preferably about 0.1 ppm to about 500 ppm,
preferably 10 ppm to about 100 ppm, in tissue culture medium and
about 10 ppm to about 5000 ppm, preferably about 100 to about 1000
ppm, in pharmaceutical compositions. The precise concentrations,
particularly for in vivo use, may be determined empirically and may
be higher, particularly for in vivo use, depending upon the ability
of the carrier or vehicle to deliver the compound or compounds to
the treated cells or tissue and the manner in which compositions is
contacted with the treated cells or tissue.
Identification of Compounds for use in Methods of the Invention
[0041] The invention provides methods of screening for compounds
that can alter splicing of a misspliced mRNA transcript, especially
a mutant mRNA transcript that is misspliced. In a specific
embodiment, the mutant mRNA transcript is a IKBKAP mRNA transcript.
In a more specific embodiment, the a mutant IKBKAP mRNA transcript
is present in a subject with FD. In an even more specific
embodiment, the mutant IKBKAP mRNA transcript has the IVS20
.sup.+6T.fwdarw.C mutation. In another specific embodiment, the
mutant mRNA transcript is a NF1 mRNA transcript. In a more specific
embodiment, the a mutant NF1 mRNA transcript is present in a
subject with NF1.
[0042] Although not intending to be bound by a particular mechanism
of action, a compound for use in the methods of the invention can
alter the splicing of a misspliced mRNA by i) increasing wild type
splicing of mutant mRNA transcripts, ii) decreasing mutant splicing
of mutant mRNA transcripts, iii) increasing the amount of wild type
splicing of wild type transcripts by mutant splicing machinery,
and/or iv) decreasing the amount of mutant splicing of wild type
mRNA transcripts by mutant splicing machinery. In other
embodiments, compounds for use in the methods of the invention can
alter the amount of mutant protein produced from either a i) mutant
mRNA transcript or ii) wild type mRNA transcript spliced by mutant
splicing machinery. Although not intending to be bound by a
particular mechanism of action, a compound for use in the methods
of the invention can alter the amount of mutant protein produced
from a misspliced mRNA transcript by i) increasing the translation
of mRNA transcripts spliced in a manner consistent with wild type
transcripts, ii) decreasing the translation of mRNA transcripts
spliced in a manner inconsistent with wild type transcripts.
[0043] The methods of screening generally involve incubating a
candidate compound with animals or cells that express a misspliced
mRNA transcript and then assaying for an alteration in the splicing
of the misspliced mRNA transcript thereby identifying a compound
for use in the methods of the invention. The DNA comprising the
gene which is misspliced may be endogenous or it may be
heterologous, e.g., contained on a vector which has been inserted
into the cell used in the assay such as by transfection or
transduction or contained in a transgene which has been used to
make a transgeneic. In embodiments where the DNA comprising the
gene which is misspliced is heterologous, fragments of the full
length gene may be used comprising at least the portion of the gene
that is misspliced. In more specific embodiments, the fragment
comprises exon 20 of IKBKAP.
[0044] In some embodiments, the amount of wild type spliced mRNA,
mutant spliced mRNA, and/or both is determined. Any method known in
the art can be used to assay for levels of mRNA transcripts,
including, but not limited to, those assays to detect i) mRNA
levels (e.g., by northern blots, RT-PCR, Q-PCR, etc.) or ii)
protein levels (e.g., ELISA, western blots, etc.). In specific
embodiments, an increase in the ratio of wild type mRNA transcripts
to misspliced mRNA transcripts indicates that the compound
decreases missplicing. In another specific embodiment, an increase
in the ratio of wild type spliced mRNA transcripts to a control
gene transcripts indicates that the compound decreases missplicing.
In another specific embodiment, a decrease in the ratio of
misspliced mRNA transcripts to a control gene transcripts indicates
that the compound decreases missplicing. As used herein, the term
"control gene" refers to a gene whose splicing or expression are
not altered by any mutation that the animal or cell used in the
assay may have or by contact of the candidate compound. Control
genes may be endogenous (e.g. actin, etc.) or heterologous (e.g., a
reporter gene such as luciferase, GFP, CAT, or
.beta.-galactosidase,ect.).
[0045] In other embodiments, the fragment of the misspliced gene
comprising the portion that is misspliced may be part of a fusion
protein with a reporter gene (e.g., luciferase, GFP, CAT, or
.beta.-galactosidase). Such a fusion protein will allow a signal
from the reporter gene if the portion of the misspliced gene has
been removed due to splicing. No signal or a reduced signal will be
present from the reporter gene if the portion of the misspliced
gene has not been removed due to splicing. If a candidate compound
decreases missplicing, then the exon that is normally excluded due
to missplicing will be included in the fusion protein and thus
decrease the signal from the reporter gene. In a specific
embodiment, the reporter gene comprises exon 20 of IKBKAP. In
another specific embodiment, the reporter gene comprises exon 36 of
NF1.
[0046] In some embodiments, expression of a misspliced mRNA
transcript confers a phenotype to the animal or cell expressing the
transcript that can be assayed (e.g., altered growth rate,
longevity, behavior, etc.). Candidate compounds that can be used in
the methods of the invention will cause a change in at least one of
the misspliced mRNA transcript-associated phenotypes. In a
preferred embodiment, the change in the misspliced mRNA
transcript-associated phenotype is such that it approximates (or is
substantially similar) to that of an organism or cell expressing a
corresponding wild type mRNA transcript. In other embodiments,
candidate compounds are assayed for their ability to alter the
splicing of misspliced mRNA transcripts and/or alter misspliced
mRNA transcript-associated phenotypes in a manner that is
substantially similar to or better than compounds known to alter
the splicing of misspliced mRNA transcripts in a therapeutically
desirable way (e.g., cytokinins including kinetin). As used herein
"substantially similar to" refers to a ratio of wild type to
misspliced mRNAs and/or a misspliced mRNA transcript-associated
phenotype that is more similar to that of a cell or organism i)
expressing a wild type counterpart of the misspliced mRNA
transcript or ii) expressing the misspliced mRNA transcript treated
with a cytokinin (especially kinetin) than a cell or organism
expressing the misspliced mRNA transcript and not treated with a
cytokinin. Any animal model known in the art can be used to assay
candidate compounds including, but not limited to, those described
in Costa et al., 2002, Nature 415: 526-30 and Costa et al., 2001,
Nat Genet. 27:399-405.
[0047] The screening methods of the invention also encompass the
use of biochemical assays (e.g., in vitro transcription and/or
translation assays) to identify compounds. Candidate compounds
found to alter the splicing of misspliced mRNA in biochemical
assays can then be assayed in animal or cell-based assays to
determine any phenotype-altering properties.
[0048] The screening methods of the invention may be adapted for
use in high throughput screen for compositions that can be
effective for the treatment of disorders associated with
missplicing of mRNA transcript(s), especially familial dysautonomia
and NF1.
[0049] As used herein, the term "compound" refers to a molecule
that has a desired biological effect. Compounds include, but are
not limited to, proteinaceous molecules, including, but not limited
to, peptide, polypeptide, protein, post-translationally modified
protein, antibodies etc.; or a large molecule, including, but not
limited to, inorganic or organic compounds; or a small molecule
(less than 500 daltons), including, but not limited to, inorganic
or organic compounds; or a nucleic acid molecule, including, but
not limited to, double-stranded DNA, single-stranded DNA,
double-stranded RNA, single-stranded RNA, or triple helix nucleic
acid molecules. Compounds can be natural products derived from any
known organism (including, but not limited to, animals, plants,
bacteria, fungi, protista, or viruses) or from a library of
synthetic molecules.
[0050] In one embodiment, a compound that decreases the amount of a
misspliced mRNA transcript is identified by: [0051] a) contacting a
cell or organism with a compound, wherein said cell or organism
expresses said misspliced mRNA transcript; and [0052] b)
determining the ratio of wild type to misspliced mRNA transcripts
in said contacted cell or organism, wherein an increase in the
ratio of wild type to misspliced mRNA transcripts of said contacted
cell or organism as compared to the ratio of wild type to
misspliced mRNA transcripts of a cell or organism expressing said
misspliced mRNA transcript not contacted with the compound (i.e., a
control cell or organism) indicates that the compound decreases the
amount of said misspliced mRNA transcript.
[0053] In another embodiment, a compound that decreases the amount
of a misspliced mRNA transcript is identified by: [0054] a)
contacting a cell or organism with a compound, wherein said cell or
organism expresses said misspliced mRNA transcript; and [0055] b)
determining the ratio of wild type to misspliced mRNA transcripts
in said contacted cell or organism, wherein the ratio of wild type
to misspliced mRNA transcripts of said contacted cell or organism
is substantially similar to the ratio of wild type to misspliced
mRNA transcripts of a cell or organism expressing said misspliced
mRNA transcript contacted with kinetin indicates that the compound
decreases the amount of misspliced mRNA transcript.
[0056] In another embodiment, a compound that alters the amount of
a misspliced mRNA transcript is identified by: [0057] a) contacting
a cell or organism with a compound, wherein said cell or organism
exhibits at least one phenotype that is altered as a result of its
expression of said misspliced mRNA transcript when compared to a
wild type cell or organism; and [0058] b) determining the phenotype
of said contacted cell or organism, wherein a difference in the
phenotype of said contacted cell or organism as compared to the
phenotype of a cell or organism expressing said misspliced mRNA
transcript not contacted with the compound (i.e., a control cell or
organism) indicates that the compound alters the amount of said
misspliced mRNA transcript.
[0059] In another embodiment, a compound that decreases the amount
of a misspliced mRNA transcript is identified by: [0060] a)
contacting a cell or organism with a compound, wherein said cell or
organism exhibits at least one phenotype that is altered as a
result of its expression of said misspliced mRNA transcript when
compared to a wild type cell or organism; and [0061] b) determining
the phenotype of said contacted cell or organism, wherein the
phenotype of said contacted cell or organism is substantially
similar to the phenotype of a cell or organism expressing said
misspliced mRNA transcript contacted with kinetin indicates that
the compound decreases the amount of misspliced mRNA
transcript.
Treatment of Disorders Using Methods of the Invention
[0062] Disorders associated with misspliced mRNA can be prevented,
managed, or treated by the methods of the invention. Individuals
suffering from or likely to suffer from such a disorder are
administered compositions comprising compounds that have a desired
therapeutic effect (e.g., increasing the amount mRNA spliced in the
wild type fashion). Disorders that can be treated by the methods of
the invention include, but are not limited to, FD, NF1 cased by
missplicing, and those disorders listed in Table 1. Compositions
for the treatment of such disorders comprise one or more cytokinins
at concentrations effective to produce a therapeutic effect. In
preferred embodiments, at least one of the one or more cytokinins
is a 6-(substituted amino)purine cytokinins. In more preferred
embodiments, the 6-(substituted amino)purine cytokinin is kinetin
or benzyladenine. Compositions administered to individuals in need
thereof can further comprise other compounds that have a desired
therapeutic effect including, but not limited to tocotrienols (e.g.
.delta.-tocotrienol) and/or antioxidents (e.g.,
(-)-epigallocatechin gallate) in order to ameliorate or correct the
adverse effects in an individual resulting from improperly spliced
mRNA. In other embodiments, compositions for the treatment of
disorders associated with missplicing comprise a therapeutically
effect amount of a compound that increases production of properly
spliced mRNA to a degree that is substantially similar to or
greater than kinetin.
[0063] Compositions comprising compounds that have a desired
therapeutic effect can also be administered to cells in vitro. Such
cells in tissue culture provide a useful means for assessing the
effectiveness of candidate compounds for increasing the ratio of
wild type to mutant protein. Such cells may be either from tissue
explants, or immortalized cell cultures. Examples of specific cell
types include but are not limited to, lymphoblast, fibroblast and
neuronal. The cells in culture can be bathed, suspended or grown in
a culture medium used for mammalian cells. The medium contains an
effective concentration of the composition comprising the compounds
with therapeutic effect (e.g., one or more 6-(substituted
amino)purine cytokinins selected from the group consisting of
kinetin, benzyladenine, isopentenyl adenine,
(6-(3-hydroxymethyl-3-methylallyl)-aminopyrine),
6-(3,3-dimethylallyl)aminopyrine, 6-(benzyl)aminopyrine,
6-(phenyl)aminopyrine, 6-(n-alkyl)aminopyrine, in which the n-alkyl
group has 4, 5, or 6 carbons, 6-(cyclohexyl)methylaminopurine, and
those compounds of Formula I,
##STR00002##
[0064] in which R.sub.1 is furfuryl, phenyl, benzyl, n-alkyl of 4,
5, or 6 carbons, branched alkyl of 4, 5, or 6 carbons, (cyclohexyl)
methyl, 3,3-dimethylallyl, and 3-hydroxymethyl-3-methylallyl).
Preferred compounds, include, but are not limited to, kinetin and
benzyladenine, most preferred is kinetin. A preferred concentration
of the compounds of formula I in the medium is about 0.1 ppm to
about 500 ppm, more preferably about 0.1 to 100 ppm, or a
concentration of equivalent activity to a kinetin concentration of
between about 10.sup.-6 M (1 .mu.M or about 5 ppm) to about
5.times.10.sup.-4 M (50 .mu.M or about 250 ppm). For the most
preferred cytokinin, kinetin, the more preferred concentration
range is about 25 .mu.M to about 250 .mu.M or about 5 ppm to about
50 ppm in the culture medium. It is understood that the precise
concentration for each 6-(substituted amino) purine cytokinin or
mixture thereof may be empirically determined by testing a range of
concentration and selecting those in which the ratio of wild-type
to misspliced mRNA or protein is increased.
[0065] Additionally, such treated cells may be administered to an
individual as ex vivo therapy either in addition to or instead of
administration of a composition comprising the compounds with a
desired therapeutic effect.
Administration
[0066] The compositions may be formulated in numerous forms,
depending on the various factors specific for each patient (e.g.,
the severity and type of disorder, age, body weight, response, and
the past medical history of the patient), the one or more compounds
in the composition, the form of the compounds (e.g., in liquid,
semi-liquid or solid form), and/or the route of administration
(e.g., oral, intravenous, intramuscular, intra-arterial,
intramedullary, intrathecal, intraventricular, transdermal,
subcutaneous, intraperitoneal, intranasal, enteral, topical,
sublingual, vaginal, or rectal means). Pharmaceutical carriers,
vehicles, exipients, or diluents may be included in the
compositions of the invention including, but not limited to, water,
saline solutions, buffered saline solutions, oils (e.g., petroleum,
animal, vegetable or synthetic oils), starch, glucose, lactose,
sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium
stearate, glycerol monostearate, talc, sodium chloride, dried skim
milk, glycerol, propylene, glycol, ethanol, dextrose and the like.
The composition, if desired, can also contain minor amounts of
wetting or emulsifying agents, or pH buffering agents. These
compositions can take the form of solutions, suspensions, emulsion,
tablets, pills, capsules, powders, sustained-release formulations
and the like.
[0067] Oral compositions will generally include an inert diluent or
an edible carrier and may be provided as a liquid suspension or
solution or compressed into tablets or enclosed in gelatin
capsules. For the purpose of oral therapeutic administration, the
active compound or compounds can be incorporated with excipients
and used in the form of solutions or suspensions, tablets, capsules
or troches. The tablets, pills, capsules, troches and the like can
contain any of the following ingredients, or compounds of a similar
nature: a binder, such as microcrystalline cellulose, gum
tragacanth and gelatin; an excipient such as starch and lactose, a
disintegrating agent such as, but not limited to, alginic acid and
corn starch; a lubricant such as, but not limited to, magnesium
stearate; a glidant, such as, but not limited to, colloidal silicon
dioxide; a sweetening agent such as sucrose or saccharin; and a
flavoring agent such as peppermint, methyl salicylate, and fruit
flavoring. Further details on techniques for formulation and
administration are provided in the latest edition of Remington's
Pharmaceutical Sciences (Mack Publishing Co., Easton, Pa.).
[0068] When the dosage unit form is a capsule, it can contain, in
addition to material of the above type, a liquid carrier such as a
fatty oil. In addition, dosage unit forms can contain various other
materials which modify the physical form of the dosage unit, for
example, coatings of sugar and other enteric agents. The compounds
can also be administered as a component of an elixir, suspension,
syrup, wafer, chewing gum or the like. A syrup may contain, in
addition to the active compounds, sucrose as a sweetening agent and
certain preservatives, dyes and colorings and flavors. The active
materials can also be mixed with other active materials which do
not impair the desired action, or with materials that supplement
the desired action.
[0069] Solutions or suspensions used for oral administration can
include any of the following components: a sterile diluent, such as
water for injection, saline solution, fixed oil, polyethylene
glycol, glycerine, propylene glycol or other synthetic solvent;
antimicrobial agents, such as benzyl alcohol and methyl parabens;
antioxidants, such as ascorbic acid and sodium bisulfite; chelating
agents, such as ethylenediaminetetraacetic acid (EDTA); buffers,
such as acetates, citrates and phosphates; and agents for the
adjustment of tonicity such as sodium chloride or dextrose. Liquid
preparations can be enclosed in ampules, disposable syringes or
multiple dose vials made of glass, plastic or other suitable
material. Suitable carriers may include physiological saline or
phosphate buffered saline (PBS), and the suspensions and solutions
may contain thickening and solubilizing agents, such as glucose,
polyethylene glycol, and polypropylene glycol and mixtures thereof.
Liposomal suspensions, including tissue-targeted liposomes, may
also be suitable as pharmaceutically acceptable carriers. These may
be prepared according to methods known to those skilled in the
art.
[0070] Pharmaceutical formulations suitable for parenteral
administration may be formulated in aqueous solutions, preferably
in physiologically compatible buffers such as Hanks' solution,
Ringer's solution, or physiologically buffered saline. Aqueous
injection suspensions may contain substances, which increase the
viscosity of the suspension, such as sodium carboxymethyl
cellulose, sorbitol, or dextran. In addition, suspensions of the
active compounds may be prepared as appropriate oily injection
suspensions. Suitable lipophilic solvents or vehicles include fatty
oils such as sesame oil, or synthetic fatty acid esters, such as
ethyloleate or triglycerides, or liposomes. Optionally, the
suspension may also contain suitable stabilizers or agents which
increase the solubility of the compounds to allow for the
preparation of highly concentrated solutions.
[0071] For topical or nasal administration, penetrants or
permeation agents that are appropriate to the particular barrier to
be permeated are used in the formulation. Such penetrants are
generally known in the art.
[0072] The pharmaceutical compositions of the present invention may
be manufactured in a manner that is known in the art, e.g., by
means of conventional mixing, dissolving, granulating,
dragee-making, levigating, emulsifying, encapsulating, entrapping,
or lyophilizing processes.
[0073] The pharmaceutical composition may be provided as a salt and
can be formed with many acids, including but not limited to,
hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic,
and the like. Salts tend to be more soluble in aqueous solvents, or
other protonic solvents, than are the corresponding free base
forms.
Dosages
[0074] The compounds for use in the methods of the invention are
present in compositions in an amount sufficient to have a
therapeutic effect on the treated individual without serious toxic
effects. The determination of an effective concentration or dose is
well within the capability of those skilled in the art. The
effective concentration or dose may be determined empirically by
testing the compounds in individuals who would benefit from
treatment, or using in vitro and in vivo systems, including tissue
culture (e.g., using lymphoblast, fibroblast, or neuronal cells) or
suitable animal models.
[0075] A therapeutically effective dose refers to that amount of a
compound (e.g., cytokinin such as kinetin or other 6-(substituted
amino) purine cytokinin) which prevents, ameliorates, reduces, or
eliminates the symptoms of a disorder associated with a misspliced
mRNA. Therapeutic efficacy and toxicity may be determined by
standard pharmaceutical procedures in cell cultures or experimental
animals, e.g., ED50 (the dose therapeutically effective in 50% of
the population) and LD50 (the dose lethal to 50% of the
population). The dose ratio of toxic to therapeutic effects is the
therapeutic index, which can be expressed as the ratio, ED50/LD50.
Pharmaceutical compositions, which exhibit large therapeutic
indices, are preferred. The data obtained from cell culture assays
and animal studies are used in determining a range of dosages for
human use. Preferred dosage contained in a pharmaceutical
composition is within a range of circulating concentrations that
include the ED50 with little or no toxicity. The dosage varies
within this range depending upon the dosage form employed,
sensitivity of the patient, and the route of administration.
[0076] The exact dosage will be determined by the practitioner, who
will consider the factors related to the individual requiring
treatment. Dosage and administration are adjusted to provide
sufficient levels of the active compound or to maintain the desired
effect of the active compound. Factors, which may be taken into
account, include the severity of the individual's disease state,
general health of the patient, age, weight, and gender of the
patient, diet, time and frequency of administration, drug
combination(s), reaction sensitivities, and tolerance/response to
therapy. Guidance as to particular dosages and methods of delivery
is provided in the literature and is generally available to
practitioners in the art.
[0077] The concentration of active compound in the compositions
will depend on absorption, inactivation, excretion rates of the
active compound, the dosage schedule, and amount administered as
well as other factors known to those of skill in the art. Typically
a therapeutically effective dosage should deliver a concentration
of at least about 10 ppm up to about 5000 ppm, preferably 50 ppm to
about 1000 ppm, of the active compound to the treated individual.
The active ingredient may be administered at once, or may be
divided into a number of smaller doses to be administered at
intervals of time. It is understood that the precise dosage of
treatment is a function of individual being treated and may be
determined empirically using known testing protocols or by
extrapolation from in vivo or in vitro test data. It is to be
further understood that for any particular individual, specific
dosage regimens should be adjusted over time according to the
individual need and the professional judgment of the medical
personal administering or supervising the administration of the
compositions, and that the concentration ranges set forth herein
are exemplary only and are not intended to limit the scope or
practice of the described compositions.
[0078] In certain embodiments, compositions are effective at
concentrations of the 6-(substituted amino)purine cytokinin
typically in the range of between about 0.1 ppm to about 100 ppm.
When kinetin is used, the preferred concentration range is between
about 10.sup.-6 M and 5.times.10.sup.-4 M in the blood. At these
concentrations, cytokinins, apparently have no or minimal toxic
effect on mammalian cells in tissue culture. In embodiments where
.delta.-tocotrienol is administered in combination with the one or
more cytokinins, the preferred concentration range is between 0.25
.mu.g/ml and 50 .mu.g/ml in the blood. In embodiments where
(-)-epigallocatechin gallate is administered in combination with
the one or more cytokinins, the preferred concentration range is
between 5 .mu.g/ml and 60 .mu.g/ml in the blood.
EXAMPLES
Materials and Methods
[0079] Patient cell lines. Patient lymphoblast cell lines
previously established by EBV transformation were utilized. The
cells have been previously described. .sup.2,14. Institutional
Review Board approval had been obtained for the establishment and
use of these lines through New York University Medical Center,
Massachusetts General Hospital, and Harvard Medical School. Two
fibroblast cell lines were also used. GUS12829 was established and
has been previously described. GM02343 was purchased from the
Coriell Cell Repository.
[0080] Drug Screen. The panel of 1040 independent compounds used
was specifically designed for the NINDS sponsored Neurodegeneration
Drug Screening Consortium (MicroSource Discovery Systems). A single
FD lymphoblast cell line was cultured in 24 well format and treated
with 10 .mu.M compound dissolved in DMSO for 72 hours. RNA was
extracted using Tri-Reagent.TM.(Molecular Research Center) and
reverse transcription and IKBKAP PCR was performed as previously
described.sup.4.
[0081] Cell Culture and Treatment. FD lymphoblast lines were grown
in RPMI-1640 and primary fibroblast lines in Dulbecco's modified
Eagle's media (DMEM) with Earle's balanced salts. Both media were
supplemented with 2 mM L-glutamine and 1% penicillin/streptomycin
and either 10% (lymphoblast) or 20% (fibroblast) fetal bovine serum
(Invitrogen). Kinetin was obtained from both MicroSource Discovery
Systems and Sigma and dissolved at 10 mM in either DMSO or water.
FD cells were cultured in the presence of kinetin for 72 hours
except where noted. Benzyladenine, zeatin, and 2iP were obtained
from Sigma and dissolved in DMSO at 10 mM. These compounds were
tested at concentrations of 1 .mu.M, 10 .mu.M, 50 .mu.M, and 100
.mu.M. Cycloheximide was obtained from Sigma and dissolved at 10
mg/ml in DMSO. Cells were treated with 100 .mu.M kinetin for eight
hours, then 50 .mu.g/ml cycloheximide was added to the cultures for
four hours.sup.16.
[0082] Determination of IKBKAP transcript ratios. The ratio of
WT:MU IKBKAP transcripts was determined following amplification by
fractionation on 1.5% agarose gels stained with ethidium
bromide.sup.4. Each gel band was assessed using an Alpha 2000.TM.
Image Analyzer and software coupled with automatic background
subtraction (Alpha Innotech Corporation). WT:MU ratios were
obtained using the integrated density value (IDV) for each
band.sup.4. Real-time quantitative PCR (QPCR) was performed as
described using primers specific to WT IKBKAP, MU IKBKAP, and 18S
ribosomal RNA.sup.4. In order to assess total IKBKAP expression the
following primers were used: EX3F-5'-TCAGGACTTGCTGGATCAGGA (SEQ ID
NO:1) and EX4R -5'-CCACTGGCTACACTCCCTTCT (SEQ ID NO:2) located in
IKBKAP exons 3 and 4 and an exon 3 TaqMan probe:
TCTGGAGACGTCATACTCTGCAGTCTCAGC (SEQ ID NO:3). For the Myosin VA
assay, primers were designed that flank the alternatively spliced
exon 31 in order to assess transcript levels of the two isoforms.
Primer sequences were as follows: MYO-F: GAA TAC AAT GAC AGA TTC
CAC (SEQ ID NO:4); MYO-R: CAG GCT GGC CTC AAT TGC (SEQ ID NO:5).
Following reverse-transcription and PCR, products were separated on
a 1.5% agarose gel and IDV ratios determined as described
above.
[0083] Western Blot Analysis. Patient lymphoblast cell lines were
treated with increasing concentrations of kinetin for 72 hours.
Extracted protein was run on a 6% polyacrylamide gel. The samples
were transferred to nitrocellulose and stained with Ponceau-S to
visualize protein loading. IKAP was detected using a monoclonal
antibody raised against amino acids 796-1008 of IKAP (BD
Bioscience) that detects the 150 kD full-length IKAP protein. The
same blot was probed with an antibody to insulin-degrading enzyme
(IDE) as a protein loading control. Bands were quantitated using an
Alpha 2000.TM. Image Analyzer.
[0084] In vivo IKBKAP minigene splicing assay. IKBKAP genomic DNA
was amplified from an unaffected and an FD individual using primers
in exon 19 and 21: EX19F -5'-CATTACAGGCCGGCCTGAG (SEQ ID NO:6) and
EX21R -5'-CAGCTTAGAAAGTTACCTTAG (SEQ ID NO:7). The amplified
products were cloned into pcDNA3.1/V5-His Topo (Invitrogen) and
sequenced for verification. HEK293 cells were plated and kinetin
was added to the tissue culture media 4 hours later. Minigene
constructs were transiently transfected 12 hours later using
Genejuice (Novagen) as directed by the manufacturer. After 48 hours
RNA was isolated using the RNAeasy kit (Qiagen) and reverse
transcribed using Superscript.TM. II reverse transcriptase
(Invitrogen) as described.sup.4. PCR was performed using vector
specific primers: T7 (TAATACGACTCACTATAGG) (SEQ ID NO:8) and BGHR
(TAGAAGGCACAGTCGAGG) (SEQ ID NO:9), which amplify both WT and MU
transcripts, or the MU specific primer EX19/21 F
(CAAAGCTTGTATTACAGACT) (SEQ ID NO:10) and BGH-R to specifically
amplify only MU transcripts. PCR was performed as follows: 26
cycles of [94.degree. C., 30 s; 56.degree. C., 30 s; 72.degree. C.,
30 s] and products resolved on a 1.5% agarose gel and visualized by
ethidium bromide staining.
Example 1
IKBKAP mRNA Ratio Analysis
[0085] An assay that provides stable and consistent measurement of
the ratio of WT:MU FD IKBKAP mRNA splice products in lymphoblast
cell lines was developed.sup.4. This assay was employed as part of
the Neurodegeneration Drug Screening Consortium.sup.7 to identify
compounds that increase the relative production of WT mRNA. A
single, standard FD lymphoblast cell line was assayed after 72
hours in the presence of 10 .mu.M test drug, using compounds
dissolved in DMSO. A panel of 1040 bioactive compounds (NINDS
Custom Collection, MicroSource Discovery Systems) were screened,
most of which have been approved for use by the Food and Drug
Administration (FDA). Prior to initiating the screen, it was
confirmed that DMSO had no effect on IKBKAP splicing and was not
toxic to the cells at the test concentration of 0.1%. One compound,
kinetin (6-furfurylaminopurine) (FIG. 1b), dramatically enhanced
correct splicing in FD cells at the test concentration of 10 .mu.M
(FIG. 1c).
[0086] Concentrations of kinetin up to 100 .mu.M were tested and
significant enhancement of WT IKBKAP production with increasing
concentration in two independent FD lymphoblast lines was observed
(FIG. 2a). The increase in WT:MU mRNA was mirrored by an increase
in production of IKAP protein (FIG. 2b), confirming the increased
production of functional WT mRNA. To avoid DMSO toxicity at higher
concentrations, kinetin was purchased from an independent source
(Sigma) and dissolved in water. Real-time quantitative PCR analysis
showed that the WT:MU IKBKAP mRNA ratio increased steadily from
approximately 1:1 in untreated cells to 12:1 in cells treated with
400 .mu.M kinetin, the maximum dose tested due to the appearance of
some cellular toxicity (FIG. 2c). By contrast, total IKBKAP mRNA
was relatively unaffected, with a slight increase (.about.1.5 fold)
being detected only at doses higher than 50 .mu.M (data not shown).
In an FD lymphoblast assay, neither zeatin nor 2iP (dissolved in
DMSO at 10 mM) had any effect on WT:MU IKBKAP ratio. Benzyladenine
did enhance inclusion of exon 20 in the IKBKAP transcripts
approximately 2-fold by 100 .mu.M, a less dramatic effect than
kinetin. The fact that only kinetin and benzyladenine alter IKBKAP
splicing suggests that the nature of the adenine N.sup.6-linked
modification plays a role in this function.
[0087] To define how quickly kinetin has its effect on IKBKAP
splicing, a time-course at 50 .mu.M kinetin was performed (FIG.
2d). The increase in the WT:MU ratio was seen at one hour, was
maximal by 8 hours, and was maintained for at least 72 hours in
culture without kinetin replenishment. The consistency of the
effect was tested by treating lymphoblast lines from nine FD
patients with 50 .mu.M kinetin for 24 hours. All nine lines showed
an increase in WT IKBKAP mRNA with WT:MU ratios ranging from
1.3-1.9 in untreated cells to 5.6-8.6 in kinetin treated cells.
Kinetin was also effective in FD fibroblast lines which exhibited a
dose-dependent increase in WT:MU IKBKAP ratio similar to that seen
in lymphoblasts.
Example 2
MYO5A mRNA Ratio Analysis
[0088] To explore whether the effect of kinetin was specific to
IKBKAP exon 20 or generally increased inclusion of alternatively
spliced exons, splicing of the MYO5A gene was assayed (FIG. 3a).
MYO5A expresses multiple isoforms in fibroblasts and lymphoblasts,
one of which is generated by skipping of exon 31.sup.10. Primers
flanking exon 31 were designed. The ratio of the two MYO5A isoforms
in nine FD lymphoblast cell lines following treatment with 50 .mu.M
kinetin for 72 hours was examined. No significant difference was
observed in the ratio of the two isoforms between the treated and
untreated samples, with average transcript ratios of 2.33 and 2.46,
respectively (FIG. 3b).
Example 3
IKBKAP Nonsense Mediated Decay Analysis
[0089] The IVS20.sup.+6T.fwdarw.C mutation in IKBKAP leads to a
frameshift and a premature termination codon in exon 21, which is
expected to target the mutant transcript for decay via the
nonsense-mediated mRNA decay (NMD) pathway.sup.15. To exclude the
possibility that the observed changes in the WT:MU transcript ratio
in the presence of kinetin were due to increased NMD of the mutant
transcript rather than direct action of kinetin on IKBKAP splicing,
the WT:MU transcript ratio was assayed in the absence of NMD. FD
cells were exposed to cycloheximide, a translation inhibitor, to
inhibit NMD of the mutant transcript.sup.16. FD cells treated with
cyclohexamide alone increased mutant transcript levels thereby
decreasing the WT:MU transcript ratio as expected (FIG. 4).
However, FD cells treated kinetin in the presence of cycloheximide
significantly altered the WT:MU transcript ratio in a manner
similar to that of kinetin treatment alone (FIG. 4). Thus, the
observed decrease in the WT:MU transcript ratio in kinetin-treated
FD cells is not dependent on NMD-mediated destruction of the mutant
transcript, but rather is due to kinetin's action on IKBKAP
splicing.
Example 4
IKBKAP Minigene Analysis
[0090] For studies aimed at understanding the mechanism of action
of kinetin on IKBKAP splicing, both FD and WT minigene constructs
containing the genomic sequence spanning exon 19 to 21 were created
(FIG. 5a). These constructs were transfected into HEK293 cells and
RNA was amplified using vector-specific primers to avoid
amplification of the endogenous IKBKAP message. Evaluation of the
transcripts produced from the WT construct showed that although the
WT transcript is the predominant product, trace levels of exon 20
skipping do occur even in the absence of the FD mutation.
Introduction of the FD mutation into the construct increased exon
20 skipping as predicted. Interestingly, treatment with kinetin
enhanced inclusion of exon 20 not only in the FD construct, but
also in the WT construct (FIG. 5B). In fact, utilization of a
MU-specific PCR assay.sup.4 revealed that exon 20 skipping is
completely corrected in transcripts generated from the WT construct
by kinetin treatment (FIG. 5c). These findings indicated that the
ability of kinetin to enhance splicing efficiency is not dependent
on either the presence of the FD mutation or the wider regulation
of IKBKAP transcription or IKAP protein production, and is likely
due to specific sequence elements in the region of exon 20 that
function to regulate splicing of this particular exon.
Example 5
SMN1 and MYO5A Analysis
[0091] Comparison of SMN1 and SMN2, closely related genes involved
in spinal muscular atrophy (SMA), has shown that a single DNA
change in the coding sequence results in skipping of exon 7 in
SMN2.sup.11,12. It has been reported that aclarubicin treatment can
promote the inclusion of exon 7 in the SMN2 transcript.sup.13.
Aclarubicin was tested on FD cells as part of our original screen
and had no effect on IKBKAP splicing. Similarly, kinetin has no
effect on skipping of exon 7 in SMN2 (Jianhua Zhou). Further,
neither of these drugs promotes inclusion of alternatively spliced
exons in MYO5A.sup.13. Taken together, these studies clearly
indicate that there are multiple distinct mechanisms that
contribute to exon choice during pre-mRNA splicing in both normal
and disease situations. The distinctive tissue-specific splicing
pattern seen in FD and the known importance of tissue-specific
alternative splicing in generating protein diversity add yet
another level of complexity to this important regulatory
process.
Example 6
Clinical Assessment for Treatment of Familial Dysautonomia
[0092] Kinetin Drug Absorption Study. A drug absorption study is
performed on approximately 50 individuals. The individuals are
given a dose of kinetin Serum levels are obtained at various time
points, 0 15 min, 30 min, 60 min and 120 min, to determine
clearance.
[0093] The assessment is an unblinded and longitudinal study where
each patient is his or her own control. However, a blinded
assessment with a placebo control could also be performed. A daily
dose of kinetin is given for individuals diagnosed with FD whose
weight is greater than 25 kg. Dosages are calculated based on drug
absorption assessment. An initial dose is calculated according to
patient's weight and is administered orally, via a gastrointestinal
tube or sublingually, preferably orally. Blood samples will be
taken at baseline (0 min), 15 min, 30 min and then one hour and 2
hours post administration to assess blood level.
[0094] During the study, individuals are assessed at various time
periods, including an initial visit, a visit at 1 month, 6 months,
12 months, 18 months, 24 months and 36 months. Activities that
occur at the various visits include functional assessment,
determination of the wild-type to mutant ratio of mRNA IKAP or
protein IKAP, sensory and gait tests, autonomic (CV and eye) tests
and autonomic (EGG).
[0095] The functional assessment of the individuals is based on a
score from 0 (normal) to 15 (globally severely limited). The
individuals oral intake, crisis frequency, cognitive ability,
speech and gait are assessed.
[0096] In addition, blood tests are conducted to assess the mRNA
IKAP ratio (wildtype:mutant) from isolated lymphoblasts. The ratio
of wild type to mutant protein may also be assessed using methods
well known in the art in combination with the methods described
above. Alternatively or additionally, other tissue types may be
obtained by biopsy, including but not limited to a skin punch test,
to assess the mRNA or protein IKAP wild type to mutant ratio found
in fibroblasts.
[0097] Sensory assessments are also performed. The sensory
assessments may include the following: a histamine test (with
photograph and measurement an appropriate time), a pain test (sharp
vs. dull)*, a temperature assessment (hot and cold thresholds using
Thermotest by Nicolet)*, vibration thresholds (using
biothesiometer)*, and deep tendon reflexes. (Items with * can only
be done in patients over 6 years).
[0098] Autonomic assessments are also performed. The autonomic
assessments may include cardiovascular tests, including tilt test,
heart rate variability, sympathovagal balance, and autonomic
perturbations. More specifically the tilt test includes blood
pressure, mean blood pressure and heart rate while the individual
is first supine and then erect for a short and longer period of
time. The heart rate variability test is performed supine and erect
and uses the Nicolet MMP program. The sympathovagal balance test*
is performed supine and erect and uses ANSAR technology which
provides SDNN and PN50 values (measures of sympathetic and
parasympathetic tone, respectively). The autonomic perturbations*
include deep breathing and valsalva, and tilt to assess
sympathovagal responsiveness (data attained via Atlas technology).
Autonomic assessment also includes an ophthalmologic test--the
Schirmer tear test. In addition, the autonomic assessment includes
gastrointestinal testing comprising a electrogastrogram with water
load.
[0099] The extent of a response to treatment is based on a positive
change in any objective measure of clinical symptom or biochemical
marker during treatment. Such biochemical marker may include a
change in protein or a change in mRNA ratio of wild type to mutant
IKAP.
[0100] Clinical symptoms are assessed according to any method known
in the art including Hilz et al., 1998, Journal Neurology,
Neurosurgery, and Psychiatry.65:338-343; . Pearson and Pytel, 1978,
J Neurol Sci 39: 123-130; Axelrod, 1996, Autonomic and Sensory
Disorders. In: Principles and Practice of Medical Genetics, 3rd
edition, Emory and Rimoin eds. Churchill Livingstone, Edinburgh. pp
397-411;. Axelrod, 2002, Clin Auton Res 12:2-14; Axelrod and
Maayan, Familial Dysautonomia. In: Burg et al, eds, Gellis and
Kagan's Current Pediatric Therapy, 17.sup.th edition WB Saunders,
Philadelphia, 2002, pp 437-441; Brown et al., 2003, Clinical
Science 104:163-9; Maayan et al., 1987, J. Auton Nerv. Syst.
21:51-8; Marthol et al., 2003, Eur. J. Clin. Invest. 33:912-8;
Cuajungco et al., 2003, AM J Hum Genet. 72:749-58.
[0101] Absorption studies and symptom assessment are further
conducted with kinetin in combination with .delta.-tocotrienol
and/or (-)-epigallocatechin gallate.
Example 7
NF1 Minigene Analysis
[0102] For studies aimed at understanding the mechanism of action
of kinetin on NF1 splicing, both NF1 and WT minigene constructs
containing the genomic sequence spanning exons 35 to 37 of the NF1
gene were created. NF1 genomic DNA was amplified from an unaffected
and an NF1 individual using primers at the 5' end of exon 35 and
the 3' end of exon 37. The NF1 individual had a C>T mutation at
nucleotide residue position 6724 of the NF1 cDNA. The amplified
products were cloned into pcDNA3.1/V5-His Topo (Invitrogen) and
sequenced for verification. HEK293 cells were plated and kinetin
was added to the tissue culture media 4 hours later. Minigene
constructs were transiently transfected 12 hours later using
Genejuice (Novagen) as directed by the manufacturer. After 48 hours
RNA was isolated using the RNAeasy kit (Qiagen) and reverse
transcribed using Superscript.TM. II reverse transcriptase
(Invitrogen) as described.sup.4. PCR was performed using vector
specific primers: T7 (TAATACGACTCACTATAGG) (SEQ ID NO:8) and BGHR
(TAGAAGGCACAGTCGAGG) (SEQ ID NO:9), which amplify both WT and MU
transcripts. PCR was performed as follows: 30 cycles of [94.degree.
C., 30 s; 58.degree. C., 30 s; 72.degree. C., 30 s] and products
resolved on a 1.5% agarose gel and visualized by ethidium bromide
staining.
[0103] When treated with kinetin, cells transfected with the NF1
minigene had altered splicing as compared to cells transfected with
the NF1 minigene in the absence of kinetin. One of skill in the art
could determine if the alteration in splicing resulted in more wild
type and/or less mutant splicing to occur using well known
molecular biological techniques.
[0104] All publications and patent applications mentioned in this
specification are indicative of the level of skill of those skilled
in the art to which this invention pertains. All publications and
patent applications are herein incorporated by reference to the
same extent as if each individual publication or patent application
was specifically and individually indicated to be incorporated by
reference. The invention now being fully described, it will be
apparent to one of ordinary skill in the art that many changes and
modifications can be made thereto without departing from the spirit
or scope of the appended claims.
REFERENCES
[0105] 1. Nissim-Rafinia, M. & Kerem, B. Splicing regulation as
a potential genetic modifier. Trends Genet 18, 123-7 (2002). [0106]
2. Slaugenhaupt, S. A. et al. Tissue-specific expression of a
splicing mutation in the IKBKAP gene causes familial dysautonomia.
Am J Hum Genet 68, 598-605 (2001). [0107] 3. Anderson, S. L. et al.
Familial dysautonomia is caused by mutations of the IMP gene. Am J
Hum Genet 68, 753-8 (2001). [0108] 4. Cuajungco, M. P. et al.
Tissue-Specific Reduction in Splicing Efficiency of IKBKAP Due to
the Major Mutation Associated with Familial Dysautonomia. Am J Hum
Genet 72, 749-58 (2003). [0109] 5. Maayan, C., Kaplan, E., Shachar,
S., Peleg, O. & Godfrey, S.
[0110] Incidence of familial dysautonomia in Israel 1977-1981. Clin
Genet 32, 106-8 (1987). [0111] 6. Leyne, M., Mull, J., Gill, S. P.,
Cuajungco, M. P., Oddoux, C., Blumenfeld, A., Maayan, C., Gusella,
J. F., Axelrod, F. B., Slaugenhaupt, S. A. Identification of the
first non-Jewish mutation in Familial Dysautonomia. Am J Med Genet
In Press(2003). [0112] 7. Heemskerk, J., Tobin, A. J. & Bain,
L. J. Teaching old drugs new tricks.
[0113] Meeting of the Neurodegeneration Drug Screening Consortium,
7-8 April 2002, Washington, D.C., USA. Trends Neurosci 25, 494-6
(2002). [0114] 8. Rattan, S. I. & Clark, B. F. Kinetin delays
the onset of ageing characteristics in human fibroblasts. Biochem
Biophys Res Commun 201, 665-72 (1994). [0115] 9. Olsen, A.,
Siboska, G. E., Clark, B. F. & Rattan, S. I.
N(6)-Furfuryladenine, kinetin, protects against Fenton
reaction-mediated oxidative damage to DNA. Biochem Biophys Res
Commun 265, 499-502 (1999). [0116] 10. Lambert, J., Naeyaert, J.
M., Callens, T., De Paepe, A. & Messiaen, L. Human myosin V
gene produces different transcripts in a cell type-specific manner.
Biochem Biophys Res Commun 252, 329-33 (1998). [0117] 11. Lorson,
C. L., Hahnen, E., Androphy, E. J. & Wirth, B. A single
nucleotide in the SMN gene regulates splicing and is responsible
for spinal muscular atrophy. Proc Natl Acad Sci USA 96, 6307-11
(1999). [0118] 12. Monani, U. R. et al. A single nucleotide
difference that alters splicing patterns distinguishes the SMA gene
SMN1 from the copy gene SMN2. Hum Mol Genet 8, 1177-83 (1999).
[0119] 13. Andreassi, C. et al. Aclarubicin treatment restores SMN
levels to cells derived from type I spinal muscular atrophy
patients. Hum Mol Genet 10, 2841-9 (2001). [0120] 14. Blumenfeld,
A. et al. Precise genetic mapping and haplotype analysis of the
familial dysautonomia gene on human chromosome 9q31. Am J Hum Genet
64, 1110-8 (1999). [0121] 15. Frischmeyer, P. A. and Dietz, H. C.
(1999) Nonsense-mediated mRNA decay in health and disease. Hum.
Mol. Genet. 8, 1893-1900. [0122] Noensie, E. N. and Dietz, H. C.
(2001) A strategy for disease gene identification through
nonsense-mediated mRNA decay inhibition. Nat. Biotechnol. 19,
434-439.
Sequence CWU 1
1
10121DNAhuman 1tcaggacttg ctggatcagg a 21221DNAhuman 2ccactggcta
cactcccttc t 21330DNAhuman 3tctggagacg tcatactctg cagtctcagc
30421DNAhuman 4gaatacaatg acagattcca c 21518DNAhuman 5caggctggcc
tcaattgc 18619DNAhuman 6cattacaggc cggcctgag 19721DNAhuman
7cagcttagaa agttacctta g 21819DNAartificialvector 8taatacgact
cactatagg 19918DNAartificialvector 9tagaaggcac agtcgagg
181020DNAartificialnovel 10caaagcttgt attacagact 20
* * * * *