U.S. patent application number 11/879190 was filed with the patent office on 2008-02-28 for g-rich polynucleotides as a novel therapeutic for the treatment of huntington's disease.
Invention is credited to Eric B. Kmiec, Hetal Parekh-Olmedo.
Application Number | 20080051363 11/879190 |
Document ID | / |
Family ID | 37943527 |
Filed Date | 2008-02-28 |
United States Patent
Application |
20080051363 |
Kind Code |
A1 |
Kmiec; Eric B. ; et
al. |
February 28, 2008 |
G-rich polynucleotides as a novel therapeutic for the treatment of
huntington's disease
Abstract
The present invention relates to oligonucleotide compositions
and therapeutic uses thereof to modify protein-protein
interactions. In particular, the invention relates to the use of a
guanidine-rich oligonucleotides to disrupt disease-causing protein
aggregates, for example, Huntington's Disease (HD) protein
aggregates
Inventors: |
Kmiec; Eric B.; (Landenberg,
PA) ; Parekh-Olmedo; Hetal; (Mickleton, NJ) |
Correspondence
Address: |
MCCARTER & ENGLISH, LLP;BASIL S. KRIKELIS
Renaissance Centre
405 N. King Street, 8th Floor
WILMINGTON
DE
19801
US
|
Family ID: |
37943527 |
Appl. No.: |
11/879190 |
Filed: |
July 16, 2007 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
11544278 |
Oct 6, 2006 |
|
|
|
11879190 |
Jul 16, 2007 |
|
|
|
60724085 |
Oct 6, 2005 |
|
|
|
Current U.S.
Class: |
514/44R |
Current CPC
Class: |
C12N 2310/3181 20130101;
C12N 2310/3231 20130101; A61P 43/00 20180101; C12N 2310/16
20130101; C12N 2310/315 20130101; C12N 2310/321 20130101; A61P
25/14 20180101; C12N 2310/321 20130101; C12N 2310/18 20130101; A61P
25/00 20180101; A61P 25/28 20180101; A61P 25/16 20180101; C12N
15/115 20130101; C12N 2310/3521 20130101 |
Class at
Publication: |
514/044 |
International
Class: |
A61K 31/7088 20060101
A61K031/7088; A61P 25/00 20060101 A61P025/00 |
Claims
1. A method of inhibiting or reducing the formation of a protein
aggregate comprising: providing an aptameric oligonucleotide having
from 15 to 50 nucleotides, wherein at least 90% of the nucleotides
are selected from the group consisting of guanosine, and thymidine
nucleotides; and administering an effective amount of the
oligonucleotide to a subject, wherein the oligonucleotide inhibits
the gene expression of the aggregate-forming protein.
2. The method of claim 1, wherein the aptameric oligonucleotide
comprises from 18 to 24 nucleotides, and wherein at least 95% of
the nucleotides are guanosine nucleotides.
3. The method of claim 1, wherein the aptameric oligonucleotide
forms a G-quartet structure and is capable of inhibiting the
aggregation of huntingtin protein (Htt).
4. The method of claim 3, wherein the aptameric oligonucleotide
comprises the nucleotide sequence of SEQ ID NO. 3.
5. The method of claim 1, wherein the aptameric oligonucleotide
comprises the nucleotide sequence of SEQ ID NO. 10.
6. A method of treating or preventing a disease in an individual
related to the formation of a protein aggregate comprising the
steps of: providing an aptameric oligonucleotide having from 15 to
50 nucleotides, wherein at least 90% of the nucleotides are
selected from the group consisting of guanosine, and thymidine
nucleotides; and administering an effective amount of the
oligonucleotide in combination with at least one of a
pharmaceutically acceptable carrier, excipient or both to a
subject, wherein the oligonucleotide inhibits the gene expression
of the aggregate-forming protein.
7. The method of claim 6, wherein the disease is at least one
member selected from the group consisting of Parkinson's Disease,
Alzheimer's Disease, Huntington's Disease, a prion disease, and a
polyglutamine disease.
8. The method of claim 6, wherein the aptameric oligonucleotide
comprises from 18 to 24 nucleotides, and wherein at least 95% of
the nucleotides are guanosine nucleotides.
9. The method of claim 8, wherein the aptameric oligonucleotide
forms a G-quartet structure and is capable of inhibiting the
aggregation of huntingtin protein (Htt).
10. The method of claim 9, wherein the aptameric oligonucleotide
comprises the nucleotide sequence of SEQ ID NO. 3.
11. The method of claim 6, wherein the aptameric oligonucleotide
comprises the nucleotide sequence of SEQ ID NO. 10.
12. A method of inhibiting the expression of Htt in a cell
comprising: administering an effective amount of at least one
aptameric oligonucleotide selected from the group consisting of SEQ
ID NO. 3, and SEQ ID NO. 10, wherein the aptameric oligonucleotide
specifically inhibits the expression of the Htt gene.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S. patent
application Ser. No. 11/544,278, filed Oct. 6, 2006, which claims
the benefit under 35 U.S.C. .sctn.119(e) of U.S. Provisional Patent
Application No. 60/724,085, filed Oct. 6, 2005, entitled: G-Rich
Polynucleotides as a Novel Therapeutic for the Treatment of
Huntington's Disease; the disclosures of which are hereby
incorporated by reference in their entirety.
INCORPORATION BY REFERENCE OF SEQUENCE LISTING
[0002] The present application hereby incorporates by reference, in
its entirety, the Sequence Listing, and identical CRF of the
Sequence Listing filed herewith. The CRF contains nucleic acid
sequences, SEQ. ID NO. 1-7, in file: "GRO_Kmiec.txt;" created: Oct.
4, 2006; OS: MS Windows XP; Software: PatentIn 3.3; size: 6 KB. The
information contained in the Sequence Listing submitted, herewith,
in the instant application is identical to the sequence information
contained in the computer readable form.
FIELD OF THE INVENTION
[0003] The present invention relates to oligonucleotide
compositions and methods of use thereof to modify protein-protein
interactions. In particular, the invention relates to the use of a
guanosine-rich oligonucleotides to disrupt disease-causing protein
aggregates, for example, Huntington's Disease (HD) protein
aggregates.
BACKGROUND
[0004] Huntington's Disease (HD) is an inherited autosomal dominant
genetic disorder caused by expansions of CAG repeats
(polyglutamine-polyQ) at the N-terminus, within exon 1, of the HD
protein. The expansion of CAG repeats results in an increase in the
length of the poly(Q) tract in the huntingtin (Htt) protein, which
leads to protein misfolding and results in changes its solubility
and induces protein aggregation that is the hallmark of HD.
[0005] Pathologically, HD is marked by neuronal tissue degeneration
due to the development of the protein aggregates. Aggregation
occurs in two general phases, nucleation and elongation, and
therefore, agents designed to block either phase are being
considered as potential therapeutics. Recent studies suggest that
mutant Htt can nucleate protein aggregation and interfere with a
multitude of normal cellular functions.
[0006] The extent of polyglutamine expansion is correlated with the
severity of the symptoms and their onset while the pathology of the
disease and neuronal cell death are thought to be associated with
protein misfolding and protein aggregation. These aggregates are
usually seen in the nucleus but can also be found in the cytoplasm.
Protein aggregates develop via a complex biochemical process with
intermediates being visible during the process. PolyQ tracts within
the pathogenic range induce a protein insolubility whereas Htt with
nonpathogenic length maintains a measured degree of solubility.
[0007] Consistent with the aggregate toxicity hypothesis,
inhibition of aggregate formation has been shown to have beneficial
effects on the progression of HD in the R6/2 mouse model. The
implication of the polyQ aggregates in cytotoxicity validates them
as targets for novel therapeutics. Despite the lack of details
surrounding the molecular structure of the polyQ aggregates, high
throughput screening for compounds that inhibit their formation
have produced some promising results. Several compounds, including
Congo Red and Clioquinol, have been reported to inhibit the
aggregation process in the R6/2 mouse model but their neurotoxicity
tempers enthusiasm. Thus, identifying molecules that show efficacy
with minimal toxicity should be an important consideration in the
search for HD therapeutics.
[0008] Much of the focus on developing therapeutics that block
aggregate formation comes from a wealth of data associating HD
pathogenesis with the presence of cellular inclusion bodies. But,
recent evidence from in vitro and in vivo studies suggest that Htt
inclusions may not be toxic to the cell or lead to neuronal
degeneration. In fact, Hayden and colleagues have created an
exciting mouse model that shows no long term effect of Htt
inclusions on behavior or viability. It may be true that inclusion
bodies are neuroprotective and eliminating them may actually
increase the potential for neurotoxicity.
[0009] Intracellular aggregates of Htt have long been considered
phenotypic evidence of the neurodegenerative disorder Huntington's
Disease. It is, however, not clear how the appearance of such
inclusion bodies relates to the pathogenesis of the disease. A
number of model systems have been designed to screen for
therapeutic agents that can inhibit aggregation. Some of these
assays measure the inhibition of fusion protein aggregation,
proteins containing a fragment of Htt (here, GST-Q58-Htn) and a
marker/reporter protein, often eGFP. The Htt component of this
fusion protein harbors an expanded polyQ stretch.
[0010] To date, most therapeutic treatments for HD are merely
palliative. As such, efforts to find a therapy for HD have focused
on agents that disrupt or block the mutant Htt aggregation pathway.
However, there exists an ongoing need in the art for compounds that
are capable of therapeutic intervention at alternative points in
the HD cascade that demonstrate clinically relevant efficacy, are
safe, and that are relatively inexpensive to manufacture.
SUMMARY
[0011] The present invention is based in part on the surprising and
unexpected discovery that certain oligodeoxynucleotides (ODNs) are
capable of inhibiting or ameliorating the cellular events that lead
to HD disease pathology, and therefore, represent a new and useful
class of therapeutics for the treatment or prevention of HD. An
additional advantage is that synthetic oligodeoxnucleotides (ODNs)
provide a model category of reagents that can be produced in highly
purified quantities in a cost-effective way, and because of their
small size are typically well-tolerated.
[0012] Recently, we showed that GROs can inhibit and/or reduce
protein aggregation, for example, the aggregation of Htt that
occurs with HD. See J. Mol. Neurosci. 2004;24(2):257-67. While not
being limited to any particular theory, the inventors theorize that
the GROs may directly interact with the Htt proteins to inhibit
nucleation, elongation, or both.
[0013] There is some support for the notion of a direct interaction
between certain GROs and proteins. Previously, GROs have been shown
to bind directly to STAT3 and disrupt its function by interacting
with regions of the protein that enable dimerization. In another
instance, GROs have been shown to interact with HIV integrase and
block integration of the HIV into the host chromosome. In addition,
it has been reported that treatment of tumor cells with GROs
inhibits cell cycle progression by interfering with DNA
replication, as opposed to normal skin cells that exhibited minimal
disruption of the cell cycle when treated with the same GROs (Xu et
al.). However, no one has previously reported the ability of
certain GROs to disrupt or reduce protein aggregation in general or
Htt protein aggregation specifically.
[0014] Therefore, in certain aspects the invention relates to
guanosine-rich ODNs (GROs) compositions and therapeutic
formulations thereof that are useful for inhibiting and/or reducing
Htt protein aggregation. In additional aspects, the invention
relates to methods of treating or preventing a disease related to
protein aggregation, for example HD, comprising administering a
therapeutically effective amount of a composition comprising a GRO
in combination with at least one of a pharmaceutically acceptable
carrier, excipient or both.
[0015] Additional advantageous features and functionalities
associated with the compositions, methods, and processes of the
present invention will be apparent from the drawings presented
herein, as well as the detailed description which follows. The
publications and other materials used herein to illuminate the
background of the invention, and in particular cases, to provide
additional details respecting the practice, are incorporated by
reference, and for convenience are listed in the appended
bibliography.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1 is a diagram of the biochemical model screening assay
obtained from Wang et al. (2005) and illustrating the steps
involved in the biochemical/immunoblotting assay. The fusion
protein GST-Q58-Htn (20 .mu.g/ml) was mixed with thrombin (0.5
unit/.mu.g protein) for 30 minutes and the mixture centrifuged to
remove aggregated protein. The soluble protein was mixed with an
ODN in a 96-well PCR plate and incubated for 24 hours at room
temperature (RT). SDS was added to a final concentration of 2% and
the mixture heated at 99.degree. C. for 5 minutes. Filtration
through a 0.22 micron acetate cellulose membrane filter was
followed by detection of aggregated Q58-Htn fragment by
immunoblotting with an antibody (HP1) and ECL. Quantitation was
carried out using an ImageQuant program. The blot displays both
positive and negative results--positions lacking a black spot
indicate that aggregation was inhibited by the ODN.
[0017] FIG. 2(A) is the DNA sequence of two G-rich ODNs that form
the G-quartet structure (SEQ ID NOs: 1 and 2). (B) is a dot blot
analysis of T40216 (SEQ ID NO:2) and T30923 (SEQ ID NO:1) activity
on aggregation. The zero (0) hour control represents reactions that
were stopped immediately after addition of the protein; 24-hour
reactions carried out in the absence of the ODN and stopped after
24-hours of incubation; Congo red, level of aggregation 24 hours
after addition of Congo Red (10 .mu.M).
[0018] FIG. 3 is a bar graph depicting aggregation inhibition by
GROs. Here, T40216 (SEQ ID NO:2) and T30923 (SEQ ID NO:1) and Congo
Red are used: Data are presented from five independent reactions,
as shown in (B) for each point with standard deviation. *, denotes
significance p<0.05 as compared to Congo Red (control) as
determined by a one way ANOVA with Tukey's post hoc test.
[0019] FIG. 4 is a dot blot analysis of HDG 20 (SEQ ID NO:3)
activity on aggregation of mutant Htt fragment. HDG 20, a 20-base
monotonic guanosine ODN, was tested in the assay outlined in FIG. 1
(see legend) at the indicated concentration. The reaction was
carried out 4 times in duplicate for 24 hours and a representative
blot from the four independent experiments is shown. 0 hour,
reaction mixture stopped at time zero; 24 hours, control reaction
lacking ODNs. (Inset). The Flow-through from filter binding
reaction containing HDG 20 (20 .mu.M or 40 .mu.M) was placed on
blotting paper, dried and processed as described in the legend to
FIG. 1.
[0020] FIG. 5 is a dot blot analysis of the specificity of various
monotonic 20-mer ODNs in the inhibition of mutant Htt fragment
aggregation. The various monotonic 20-mers were tested for
inhibitory activity in the assay outlined in FIG. 1. HDC, 20-mer
with all Cs (SEQ ID NO:8); HDA, 20-mer with all As (SEQ ID NO:9);
HDT (SEQ ID NO:10), 20-mer with all T's; HDG, 20-mer with all Gs
(SEQ ID NO:3). Four independent experiments were carried out in
duplicate and this blot is most representative of all of the
results. 0 hour, reaction stopped at zero time point; 24 hour,
reaction lacking ODNs, stopped at 24 hours; Congo Red, incubation
with 10 .mu.M of Congo Red for 24 hours.
[0021] FIG. 6 is a bar graph depicting blots carried out to test
monotonic 20-mers. Average aggregation levels, representative of 5
independent experiments with standard deviation and average values
(Series 1) presented. *, denotes significance p<0.05 as compared
to Congo Red (control) as determined by a one way ANOVA with
Tukey's post hoc test.
[0022] FIG. 7 is a CD spectroscopy of HDA (SEQ ID NO:9) and HDG
(SEQ ID NO:3). The CD spectra of 15 .mu.M HDG (heavy solid line),
15 .mu.M T30923 (SEQ ID NO:1) (light solid line) and 15 .mu.M HDA
(dotted line) in 10 mM KCl at 24.degree. C.
[0023] FIG. 8 provides light and dark field microscopic images
showing inhibition of aggregation in HEK 293 cells transfected with
plasmid, pcDNA3.1-72Httexon1-eGFP (p72Q). Series of control
reactions including HEK293 photographed under white light or in
dark field, aggregate formation produced by p72Q and inhibition of
aggregation by Congo red (0.2 .mu.M or 1 .mu.M) added concurrently
with p72Q.
[0024] FIG. 9 provides dark field microscopic images showing HDA
(SEQ ID NO:9) that was co-transfected at the indicated
concentrations with p72Q and the cells were photographed 48 hours
later in dark field.
[0025] FIG. 10 provides light and dark field microscopic images
showing HDG (SEQ ID NO:3) that was co-transfected at the indicated
concentrations with p72Q and the cells were photographed 48 hours
later in dark field.
[0026] FIG. 11 provides dark field microscopic images showing HDG
(SEQ ID NO:3) that was co-transfected with p72Q and the cells
photographed 48 hours later under dark field. The upper left panel
represents a reaction lacking ODN.
[0027] FIG. 12 is a dot blot analysis of various 20-mers having
thymine modifications were tested for inhibitory activity in the
assay outlined in FIG. 1. HDT (SEQ ID NO:10), 20-mer with all T's;
HDG (SEQ ID NO:3), 20-mer with all G's; HDG 20/7 (SEQ ID NO:7),
20-mer with every 7th nucleotide replaced with T; HDG 20/4 (SEQ ID
NO:6), 20-mer with every 4th nucleotide replaced with T HDG 20/3
(SEQ ID NO:5), 20-mer with every 3rd nucleotide replaced with T;
HDG 20/2 (SEQ ID NO:2), 20-mer with every other nucleotide replaced
with T. 0 hour, reaction stopped at zero time point; 24 hour,
reaction lacking ODN and reaction stopped at 24 hours; Congo Red,
incubation with Congo Red (10 .mu.M) for 24 hours.
[0028] FIG. 13 is a FACS measured analysis of inhibition of mutant
Htt fragment aggregation by various ODNs. HEK293 cells were
transfected with pcDNA3.1-72Httexon1-eGFP (p72Q) and the specific
ODN (HDA (SEQ ID NO:9), HDG (SEQ ID NO:3)) at the indicated
concentrations and the degree of aggregation measured by FACS after
48 hours. (A) No plasmid (p72Q); (B) only p72Q, no ODN; (C) p72Q
and 1 .mu.M HDA; (D) p72Q and 2.5 .mu.M HDA; (E) p72Q and 1 .mu.M
HDG; (F) p72Q and 2.5 .mu.M HDG. All ODNs are at final
concentrations in the cell culture reaction. The magnitude of green
fluorescence is measured on the X axis while the number of cells
exhibiting that degree of fluorescence is depicted on the Y
axis.
[0029] FIG. 14 is a line graph showing the viability of PC12 cells
transfected with various HDG (SEQ ID NO:3) concentrations. The PC12
cell line, Htt14A2.6, was transfected with varying amounts of HDG
in Lipofectamine 2000. The Promega CellTiter-Glo Luminescent cell
viability assay was used to analyze the viability of each treatment
as a function of time. One control is a mock-transfected cell
culture containing lipofectamine 2000 but no ODN; whereas the no
treatment control presented here indicates cells that have received
neither ODN nor lipofectamine. Statistics were performed by using
the standard deviation on 27 luminescent readings for every sample
at each time point.
[0030] FIG. 15 depicts the results of quantitative polymerase chain
reaction (qPCR) looking at the effect of HDG20 and HDT20 on
reduction of transcript levels. Cells were transfected with a
plasmid expressing an eGFP protein fused to a mutant huntington
fragment. The results show a clear reduction of eGFP levels with
the addition of HDG20 while the HDT resulted in a much lower
inhibition of message.
[0031] FIG. 16 represents the results obtained when transfecting
cells with a plasmid expressing eGFP fused to a wild type (WT)
huntington fragment. These results show similar reduction of
message as seen in the mutant huntington plasmid using HDG20 and
HDT20. This outcome implies that the huntington message is reduced
regardless of the extent of the polyglutamine repeat region.
[0032] FIG. 17 characterizes the results of a dose response of our
novel compound HDG20. This figure shows an exceptional response in
which a decrease of transcript can be seen at levels ranging from
about 0.75 .mu.g to about 4 .mu.g of oligonucleotide. In similar
experiments looking at a HDT dose response, no significant
reduction of message was seen at levels up to about 4 .mu.g.
DETAILED DESCRIPTION
[0033] The present invention is based on the surprising an
unexpected discovery that certain oligonucleotides are capable of
inhibiting protein aggregation. The invention includes
oligonucleotide compositions useful for research and therapeutic
purposes.
[0034] The term "oligonucleotide" refers generally to, and
interchangeably with nucleic acids, deoxyribonucleotides,
deoxyribonucleic acids, ribonucleotides, and ribonucleic acids, and
polymeric forms thereof, and includes either single- or
double-stranded forms. Also, unless expressly limited, the term
"nucleotide" includes known analogues of natural nucleotides that
have similar binding properties as the reference nucleic acid, for
example, peptide nucleic acids (PNAs). In addition, a particular
nucleotide or nucleic acid sequence includes complementary
sequences, and the sequence explicitly indicated. The term nucleic
acid is generic to the terms "gene," "DNA," "cDNA,"
"oligonucleotide," "RNA," "mRNA," "nucleotide," "polynucleotide,"
and the like. The four nucleotide bases are guanine, cytosine,
thymine, uracil and adenine. Nucleotides are composed of a pentose
sugar, a purine or pyrimidine base, and a phosphate group (i.e.,
adenosine, guanosine, cytidine, uridine, and thymidine).
[0035] As used herein, the term "oligonucleotide" refers to a
series of linked nucleotide residues. A short oligonucleotide
sequence may be based on, or designed from, a genomic or cDNA
sequence and is used to amplify, confirm, or reveal the presence of
an identical, similar or complementary DNA or RNA in a particular
cell or tissue. Oligonucleotides comprise a nucleic acid sequence
having about 10 nt, 50 nt, or 100 nt in length, preferably about 15
nt to 30 nt in length. In certain embodiments of the invention, an
oligonucleotide comprising a nucleic acid molecule less than 100 nt
in length would further comprise at least 6 contiguous nucleotides
of SEQ ID NOS: 1-12.
[0036] As used herein, the terms "GRO", "aptameric GRO", and
"G-rich oligonucleotides" are used interchangeably. Aptameric
oligonucleotide molecules bind a specific target molecule such as
small molecules, proteins, nucleic acids, and even cells, tissues
and organisms. Aptamers offer the utility for biotechnological and
therapeutic applications as they offer molecular recognition
properties similar to antibodies. In addition to their discriminate
recognition, aptamers offer advantages over antibodies as they can
be engineered completely in a test tube, are readily produced by
chemical synthesis, possess desirable storage properties, and
elicit little or no immunogenicity in therapeutic applications.
[0037] Oligonucleotides may be chemically synthesized and may also
be used as probes. Nucleic acid synthesizers are available to
synthesize oligonucleotides of any desired sequence. Certain
oligonucleotide analogs may also be readily synthesized by
modifying the reactants and reaction conditions. For example,
phosphorothioate and methylphosphonate oligonucleotides may be
synthesized using commercially available automated oligonucleotide
synthesizers.
[0038] An oligonucleotide's binding affinity to a complementary
nucleic acid may be assessed by determining the melting temperature
(T.sub.M) of a hybridization complex. The T.sub.M is a measure of
the temperature required to separate the nucleic acid strands of a
hybridization complex. The T.sub.M may be measured by using the
hybridization complex's UV spectrum to assess the degree and
strength of hybridization. During hybridization, base stacking
occurs which reduces the UV absorption of the nucleic acid. By
monitoring UV absorption and the resulting increase in UV
absorption that occurs during strand separation, one may assess the
hybridization affinity of a nucleic acid for its complement.
[0039] The structure and stability of hybridization complexes may
be further assessed using NMR techniques known to those skilled in
the art.
[0040] A vast array of oligonucleotide analogs exist that achieve
the same functionality as naturally occurring oligonucleotides.
There is extensive literature setting forth an almost limitless
variety of modifications that can be used to generate
oligonucleotide analogs. The phosphate, sugar, and/or base moieties
may be modified and/or replaced by the introduction/removal of
chemical groups and/or bonds. Many oligonucleotide analogs have
superior properties to those of naturally occurring
oligonucleotides. Such superior properties include, but are not
limited to, increased hybridization affinity and/or resistance to
degradation.
[0041] "Nucleic acid template," or "parental nucleic acid" refers
to a nucleic acid that has served as a template for a subsequent
step or process. Thus, an mRNA, a cDNA reverse transcribed from an
mRNA, an RNA transcribed from that cDNA, a DNA amplified from the
cDNA, an RNA transcribed from the amplified DNA, etc., are all
derived from the gene and detection of such derived products is
indicative of the presence and/or abundance of the original gene
and/or gene transcript in a sample.
[0042] The term "gene" is used broadly to refer to any segment of
nucleic acid associated with expression of a given RNA or protein.
Thus, genes include regions encoding expressed RNAs (which
typically include polypeptide coding sequences) and, often, the
regulatory sequences required for their expression. Genes can be
obtained from a variety of sources, including cloning from a source
of interest or synthesizing from known or predicted sequence
information, and may include sequences designed to have
specifically desired parameters.
[0043] A "recombinant" nucleic acid is any nucleic acid produced by
an in vitro or artificial (meaning not naturally occurring) process
or by recombination of two or more nucleic acids. The recombinant
nucleic acids and referred to herein are not intended to limit the
scope of the present invention, which one of ordinary skill will
recognize, contemplates the use of any guanosine-rich
oligonucleotide. Nucleic acid modifications include those obtained
by site-specific mutation, shuffling, endonuclease digestion, PCR,
subcloning, methylation, acetylation, chemical modification, and
related techniques.
[0044] Descriptions of the molecular biological techniques useful
to the practice of the invention including mutagenesis, PCR,
cloning, and the like include Berger and Kimmel, GUIDE TO MOLECULAR
CLONING TECHNIQUES, METHODS IN ENZYMOLOGY, volume 152, Academic
Press, Inc., San Diego, Calif. (Berger); Sambrook et al., MOLECULAR
CLONING--A LABORATORY MANUAL (2nd Ed.), Vol. 1-3, Cold Spring
Harbor Laboratory, Cold Spring Harbor, New York, 1989, and CURRENT
PROTOCOLS IN MOLECULAR BIOLOGY, F. M. Ausubel et al., eds., Current
Protocols, a joint venture between Greene Publishing Associates,
Inc. and John Wiley & Sons, Inc.; Berger, Sambrook, and
Ausubel, as well as Mullis et al., U.S. Pat. No. 4,683,202 (1987);
PCR PROTOCOLS A GUIDE TO METHODS AND APPLICATIONS (Innis et al.
eds), Academic Press, Inc., San Diego, Calif. (1990) (Innis);
Arnheim & Levinson (Oct. 1, 1990) C&EN 36-47; Sakamoto, et
al., Laboratory evolution of toluene dioxygenase to accept
4-picoline as a substrate. Appl. Environ. Microbiol. 67:3882-3887
(2001); Lueng, et al., A method for random mutagenesis of a defined
DNA segment using a modified polymerase chain reaction. Technique:
J Methods Cell Molec Biol 1(1):11-15 (1989).
[0045] The term "host cell" includes a cell that might be used to
carry an exogenous nucleic acid, a heterologous nucleic acid, or
expresses a peptide or protein encoded by a heterologous nucleic
acid. A host cell can contain genes that are not found within the
native (non-recombinant) form of the cell, genes found in the
native form of the cell where the genes are modified and
re-introduced into the cell by artificial means, or cells that
contain a nucleic acid endogenous to the cell that has been
artificially modified without removing the nucleic acid from the
cell.
[0046] The terms "degree of similarity" or "identity," in the
context of two or more nucleic acid sequences, refer to two or more
sequences or subsequences that are the same or homologous and have
a specified percentage of nucleotides that are the same, when
compared and aligned for maximum correspondence, as measured using
one of the sequence comparison algorithms such as BLAST, ClustalW,
or other algorithms available to persons of skill or by visual
inspection. For sequence comparison and homology determination,
typically one sequence acts as a reference sequence to which test
sequences are compared. When using a sequence comparison algorithm,
test and reference sequences are input into a computer, subsequence
coordinates are designated, if necessary, and sequence algorithm
program parameters are designated. The sequence comparison
algorithm then calculates the percent sequence identity for the
test sequence(s) relative to the reference sequence, based on the
designated program parameters. Other determinations of homology
include hybridization of nucleic acids under stringent conditions.
The phrase "hybridizing," refers to the binding, duplexing, or
hybridizing of a molecule only to a particular nucleotide sequence
under stringent conditions, including when that sequence is present
in a complex mixture (e.g., total cellular) DNA or RNA.
[0047] A nucleic acid "operon" includes a gene that is situated in
a functional relationship, i.e., operably linked, with other
nucleic acid sequences, for example, a promoter, an enhancer,
termination signals, or another gene if it increases the
transcription of the coding sequence.
[0048] As used herein, "GRO" refers generally to guanosine (or
guanine)-rich oligonucleotides.
[0049] As used herein, "HDG" refers to an oligonucleotide comprised
completely of guanosine (G) nucleotides. (See SEQ ID NO:3).
[0050] As used herein, "HDA" refers to an oligonucleotide comprised
completely of adenosine (A) nucleotides. (See SEQ ID NO:9).
[0051] As used herein, "HDC" refers to an oligonucleotide comprised
completely of cytidine (C) nucleotides. (See SEQ ID NO:8).
[0052] As used herein, "HDT" refers to an oligonucleotide comprised
completely of thymidine (T) nucleotides (See SEQ ID NO:10).
[0053] As used herein, "ODN" refers generally to a synthetic
oligonucleotide of length n, comprising any combination of
nucleotides.
[0054] "Derivatives" are modified nucleic acid sequences formed
from the native compounds either directly, by modification, or by
partial substitution. "Analogs" are nucleic acid sequences or amino
acid sequences that have a structure similar to, but not identical
to, the native compound, e.g. they differ from it in respect to
certain components or side chains. Analogs may be synthetic or
derived from a different evolutionary origin and may have a similar
or opposite metabolic activity compared to wild type.
[0055] Derivatives and analogs may be full length or other than
full length. Derivatives or analogs of the nucleic acids or
proteins of the invention include, but are not limited to,
molecules comprising regions that are substantially homologous to
the nucleic acids or proteins of the invention, in various
embodiments, by at least about 30%, 45%, 70%, 80%, or 95% identity
(with a preferred identity of 80-95%) over a nucleic acid or amino
acid sequence of identical size or when compared to an aligned
sequence in which the alignment is done by a computer homology
program known in the art, or whose encoding nucleic acid is capable
of hybridizing to the complement of a sequence encoding the
proteins of the invention under stringent, moderately stringent, or
low stringent conditions. See e.g. Ausubel, et al., CURRENT
PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York,
N.Y., 1993. Nucleic acid derivatives and modifications include
those obtained by gene replacement, site-specific mutation,
deletion, insertion, recombination, repair, shuffling, endonuclease
digestion, PCR, subcloning, and related techniques.
[0056] In certain embodiments, the invention comprises an isolated
polynucleotide sequence, for example, the isolated aptameric GROs
of SEQ ID NOs: 1-12. By "isolated nucleic acid sequence" is meant a
polynucleotide that is not immediately contiguous with either of
the coding sequences with which it is immediately contiguous (one
on the 5' end and one on the 3' end) in the naturally occurring
genome of the organism from which it is derived. The term therefore
includes, for example, a recombinant DNA which is incorporated into
a vector; into an automatically replicating plasmid or virus; or
into the genomic DNA of a prokaryote or eukaryote, or which exists
as a separate molecule (e.g., a cDNA) independent of other
sequences. The nucleotides can be modified forms of DNA or RNA.
[0057] The present invention relates to the finding that guanosine
(G) rich oligonucleotides (GROs) form functional aptamers, and are
effective inhibitors of protein aggregation, for example, the
aggregation of polyglutamine proteins such as huntingtin protein,
which is associated with Huntington's Disease (HD). As such, the
isolated GROs of the invention have therapeutic potential and can
be used as a treatment for patients with diseases and conditions
resulting from detrimental effects of protein aggregation, for
example, Huntington's disease.
[0058] While not being limited by any particular theory, the
inventors postulate that the beneficial effect observed with the
GROs of the invention may result from the inhibition or slowing of
the aggregation process. The GROs of the invention that possess
aptameric activity may also be beneficial in other amyloid or
neurodegenerative diseases, for example, Alzheimer's Disease,
Parkinson's Disease, spinocerebellar ataxia, and prion diseases.
Moreover, the GROs of the present invention can be used to examine
the relationship between cellular aggregates and toxicity in
various model systems.
[0059] Therefore, in certain aspects the invention relates to
guanosine-rich ODNs (GROs) compositions and therapeutic
formulations thereof that are useful for inhibiting and/or reducing
Htt protein aggregation, for example by a direct-protein
interaction. In additional aspects, the invention relates to
methods of treating or preventing a disease related to protein
aggregation, for example HD, comprising administering to a subject
a therapeutically effective amount of a composition comprising a
GRO in combination with at least one of a pharmaceutically
acceptable carrier, excipient or both.
[0060] Example 4 (and FIGS. 15, 16, and 17) demonstrates the
surprising and unexpected finding that oligodeoxynucleotides, for
example guanosine-rich (SEQ ID NO. 3; herein HDG) and
thymidine-rich (SEQ ID NO. 10; herein HDT) are capable of specific
down regulation of Htt transcript in human cells (HEK293T). Reduced
Htt transcript present in cells treated with the
oligodeoxynucleotides of the invention could be due to reduction in
transcription, reduction in translation, or increased turnover of
mRNA (e.g., decrease stability or enzymatic degradation). While not
being limited to any particular theory, experimental controls
suggest that the down-regulation is likely due to degradation of
Htt transcript.
[0061] This discovery provides even greater potential for
therapeutic intervention for the treatment and/or prevention of HD.
Therefore, in another aspect, the invention relates to
guanosine-rich ODNs (GROs) compositions and therapeutic
formulations thereof that are useful for inhibiting and/or reducing
Htt protein aggregation, for example, by a protein-independent
mechanism. In still another aspect, the present invention relates
to a method for treating or preventing a disease related to protein
aggregation, for example HD, comprising administering to a subject
a therapeutically effective amount of a composition comprising an
oligodeoxynucleotide in combination with at least one of a
pharmaceutically acceptable carrier, excipients or both; and
wherein the oligonucleotide reduces the level of Htt gene
expression.
[0062] GROs form a structure known as a G-quartet which arises from
the association of four adjacent G-bases assembled into a cyclic
conformation. These structures are stabilized by von Hoogstein
hydrogen bonding and by base stacking interactions. These molecules
exhibit a very compact tetra-helical structure which allows them to
interact productively with functionally important protein domains.
The stability of these G-quartets is related to several factors,
including the presence of monovalent cations such as K+ and Na+,
the concentration of G-rich ODNs present, and the sequence of the
G-rich ODNs being used
[0063] In additional aspects the invention relates to ODNs that are
useful as therapeutics in the treatment and prevention of such
diseases, and can also aid in the study of the diseases and their
underlying physiological origins. In particular, the invention
relates to guanosine-rich oligonucleotide compositions and
associated methods of use to inhibit protein aggregates and their
detrimental effects.
[0064] Although factors are known which lead to aggregation of
proteins in the native state, for example, salting out, and
isoelectric precipitation; the majority of cases of protein
aggregation involve the intermolecular association of a
partially-folded or "unfolded" intermediate state of the protein.
The underlying reason is probably that partially-folded
intermediates have hydrophobic patches, which normally pack
together to yield the native state, but which can also interact in
an intermolecular manner to form an aggregate. Diseases where
protein aggregation is causal or an associated symptom and for
which the present invention may be useful for treatment and/or
prevention include Down's syndrome, Alzheimer's disease (AD),
Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), and
prion diseases such as bovine spongiform encephalitis (BSE) and
Creutzfeldt-Jakob Disease (CJD), cystic fibrosis, and the so-celled
polyglutamine diseases (TABLE 1), for example, Huntington's disease
(HD), dentato-rubral and pallido-luysian atrophy (DRPLA) and
several forms of spino-cerebellar ataxia (SCA), also have
intracellular inclusions in regions corresponding to the regions of
neuronal degeneration TABLE-US-00001 TABLE 1 Summary of
Polyglutamine Diseases. Normal Disease Gene Chromosomal Pattern of
repeat repeat Disease name location inheritance Protein length
length Spinobulbar AR Xq13-21 X-linked androgen 9-36 38-62 muscular
atrophy recessive receptor (AR) (Kennedy disease) Huntington
disease HD 4p16.3 autosomal huntingtin 6-35 36-121 dominant
Dentatorubral- DRPLA 12p13.31 autosomal atrophin-1 6-35 49-88
pallidoluysian dominant atrophy (Haw River syndrome)
Spinocerebellar SCA1 6p23 autosomal ataxin-1 6-44 39-82 ataxia type
1 dominant Spinocerebellar SCA2 12q24.1 autosomal ataxin-2 15-31
36-63 ataxia type 2 dominant Spinocerebellar SCA3 14q32.1 autosomal
ataxin-3 12-40 55-84 ataxia type 3 dominant (Machado-Joseph
disease) Spinocerebellar SCA6 19p13 autosomal
.alpha.1.sub.A-voltage- 4-18 21-33 ataxia type 6 dominant dependent
calcium channel subunit Spinocerebellar SCA7 3p12-13 autosomal
ataxin-7 4-35 37-306 ataxia type 7 dominant Spinocerebellar SCA17
6q27 autosomal TATA binding 25-42 45-63 ataxia type 17 dominant
protein
[0065] In certain aspects the invention relates to isolated
oligonucleotide compositions comprising from about 15 to about 50
nucleotides, and having at least 40% guanosine nucleotides. In
certain embodiments the invention comprises oligonucleotides of SEQ
ID NOs: 1-7. These gunosine rich oligonucleotides (GROs) have been
shown to form higher order aggregates, for example, G-quartet
structures, in which the GROs align in a parallel or antiparallel
configuration. (See Biyani and Nisigaki, Gene 364: 130-38 (2005),
incorporated herein by reference in its entirety). While not being
limited to any particular theory, the inventors hypothesize that
the higher-order structures of the GROs of the invention mediate
their efficacy; i.e., inhibiting the aggregation of proteins, for
example, the disease associated polyglutamine proteins. However,
the GROs of the present invention may also be used generally to
inhibit aggregation of other disease related proteins as indicated
above. Therefore, in another aspect the oligonucleotide of the
invention comprises a G-quartet structure. In a preferred
embodiment, the isolated oligonucleotide of the invention comprises
from 18-24 nucleotides, and has at least 95% guanosine
nucleotides.
[0066] In other aspects the isolated GRO of the invention is
disposed in a vector or plasmid nucleic acid for its convenient
cloning, amplification, and/or transcription. In still other
aspects the isolated GRO of the invention is operably linked to one
or more transcription regulatory nucleic acid sequences. In yet
another aspect, the isolated GRO is disposed in a vector or plasmid
nucleic acid, and is operably linked with one or more transcription
regulatory nucleic acid sequences.
[0067] In other aspects, the invention relates to a host cell
comprising the isolated GRO of the invention. In certain
embodiments, the host cell further comprises a vector or plasmid
nucleic acid containing one or more transcription regulatory
nucleic acid sequences operably linked with the GRO sequence of the
invention. The vector or plasmid nucleic acids can be, for example,
suitable for eukaryotic or prokaryotic cloning, amplification, or
transcription. The vector or plasmid nucleic acids can also be
stably integrated into the host cell's genome or maintained
episomally.
[0068] In another aspect, the invention relates to method for
inhibiting and/or reducing the aggregation of proteins. In other
aspects, the invention relates to methods for inhibiting or
reducing the aggregation of polyglutamine proteins, such as those
that cause Huntington's Disease, or Spinocerebellar ataxia. In any
embodiment of these aspects the invention comprises contacting an
protein capable of forming a protein aggregate or a protein
aggregate with an effective amount of a GRO of the invention to
result in the inhibition of protein aggregate formation, the
reduction of protein aggregation, and/or the dissociation of the
components from a protein aggregate.
[0069] In other aspects, the invention relates to methods for
treating and/or preventing a disease or condition in an individual
related to the detrimental effects of protein aggregation. In
certain embodiments, the methods of the invention comprise
administering an effective amount of an isolated GRO in a
pharmaceutically acceptable form to an individual in need thereof.
In certain embodiments, the isolated GRO of the invention is
administered together with a pharmaceutically acceptable carrier,
excipient, adjuvant, amino acid, peptide, polypeptide, chemical
compound, drug, biologically active agent or a combination thereof.
As such, in another aspect the invention relates to therapeutic
compositions comprising the isolated GRO of the invention in a
pharmaceutically acceptable form together with at least one
pharmaceutically acceptable carrier, excipient, adjuvant, amino
acid, peptide, polypeptide, chemical compound, drug, biologically
active agent or a combination thereof.
[0070] In certain embodiments the therapeutic GRO of the invention
is complexed, bound, or conjugated to one or more chemical moieties
to improve and/or modify, for example, bioavailability, half-life,
efficacy, and/or targeting. In certain aspects of this embodiment,
the GRO may be complexed or bound, either covalently or
non-covalently with, for example, cationic molecules, salts or
ions, lipids, glycerides, carbohydrates, amino acids, peptides,
proteins, other chemical compounds, for example, phenolic
compounds, and combinations thereof. In certain aspects the
invention relates to a GRO of the invention conjugated to a
polypeptide, for example, an antibody. In certain embodiments the
antibody is specific for the protein or protein aggregate of
interest and therefore targets the GRO to the protein and/or
protein aggregate.
[0071] The therapeutic GRO of the invention can be administered by
any suitable route recognized by those of skill in the art, for
example, enteral, intravenous, intra-arterial, parenteral, topical,
transdermal, nasal, and the like. In addition, the therapeutic may
be in any pharmaceutically acceptable form such as, for example, a
liquid, lyophilized powder, gel, pill, controlled release capsule,
and the like, which is now known or becomes known to those of skill
in the art.
[0072] Therefore, in one embodiment the polynucleotide composition
of the invention comprises an isolated aptameric oligonucleotide
having from about 15 to about 50 nucleotides, and having at least
40% guanosine nucleotides. In certain embodiments the invention
comprises oligonucleotides of SEQ ID NOs: 1-12. In another
embodiment, the oligonucleotides of the invention are capable of
forming G-quartet structures.
[0073] G-rich DNA and RNA have the ability to form inter- and
intramolecular four-stranded structures, referred to as G-quartets.
(See Biyani and Nisigaki, Gene 364: 130-38 (2005)). G-quartets
arise from the association of four G-bases into a cyclic Hoogsteen
H-bonding parallel or anti-parallel arrangement, and each G-base
makes two hydrogen bonds with its neighbor G-base (N1 to O6 and N2
to N7). G-quartets stack on top of each other to give rise to
tetrad-helical structures. The stability of G-quartet structures
depends on several factors: the presence of the monovalent cations,
the concentration of the G-rich oligonucleotides present, and the
sequence of the G-rich oligonucleotides under study. Potassium with
the optimal size to interact within a G-octamer greatly promotes
the formation of G-quartet structures and increases their
stability. G-quartet oligodeoxynucleotides (GQ-ODNs) have been
suggested to play a critical role in several biological processes
including modulation of telomere activity, inhibition of human
thrombin, HIV infection, HIV-1 integrase activity, human nuclear
topoisomerase 1 activity, and DNA replication in vitro. On the
basis of the structure and mechanism of Stat3 activation,
G-quartet-forming oligonucleotides were developed recently to block
Stat3 activity within cancer cells.
[0074] While there is no hard rule governing what specific
nucleotide sequence will result in the G-quartet structure, they
can usually form with some iteration of a guanosine repeat, for
example, GGTT.sub.n. Thus, as along as the guanosines can come in
contact via parallel or anti parallel positioning, then the
oligonucleotides can form higher-order structures such as the
G-quartet structure. As such, the sequence of the aptameric
oligonucleotide of the invention can be varied in any number of
ways as long as the oligonucleotide comprises from about 15 to
about 50 nucleotides, comprises at least 40% guanosine nucleotides.
In a preferred embodiment, the aptameric oligonucleotides form a
G-quartet structure. In certain embodiments, the invention
comprises an aptameric oligonucleotide of SEQ ID NOs:1-12.
[0075] While not being limited to any particular theory, the
inventors hypothesize that the higher-order structures of the
aptameric GROs of the invention mediate their efficacy; i.e.,
inhibiting the aggregation of proteins, for example, the disease
associated polyglutamine proteins. However, the aptameric GROs of
the present invention may also be used generally to inhibit
aggregation of other disease related proteins as indicated above.
In a preferred embodiment, the isolated aptameric oligonucleotide
of the invention comprises from 18-24 nucleotides, and has at least
95% guanosine nucleotides. In a particularly preferred embodiment,
the invention comprises the GRO of SEQ ID NO:3. By utilizing a
biochemical assay as an initial screen, SEQ ID NO:3 inhibited Htt
aggregation. The monotonic G-ODN of the invention was also able to
improve cell survival in PC12 cells overexpressing a mutant Htt
fragment fusion gene.
[0076] In any of the embodiments described herein, the aptameric
GRO of the invention may comprise one or more modified nucleotides
or nucleotide analogs. Nucleotide modifications can be incorporated
during or after oligonucleotide synthesis, and include
modifications of the nucleobase, the sugar moiety, and/or the
phosphate group.
[0077] Phosphodiester Moiety Analogs. Numerous analogs to the
naturally occurring phosphodiester backbone have been used in
oligonucleotide design. Phosphorothioate, phosphorodithioate, and
methylphosphonate are readily synthesized using known chemical
methods. Because novel nucleotide linkages can be synthesized
manually to form a dimer and the dimer later introduced into the
oligonucleotide via automated synthesis, the range of potential
backbone modifications is as broad as the scope of synthetic
chemistry. For example, the oligonucleotide may be substituted or
modified in its internucleotide phosphate residue with a thioether,
carbamate, carbonate, acetamidate or carboxymethyl ester.
[0078] Unlike the naturally occurring phosphodiester moieties, many
phosphodiester analogs have chiral centers. For example,
phosphorothioates, methylphosphonates, phosphoramidates, and alkyl
phosphotriesters all have chiral centers. One skilled in the art
would recognize numerous other phosphodiester analogs that possess
chiral centers. Because of the importance of stereochemistry in
hybridization, the stereochemistry of phosphodiester analogs can
influence the affinity of the oligonucleotide for its target.
[0079] Most phosophodiester backbone analogs exhibit increased
resistance to nuclease degradation. In an embodiment,
phosphorothioates, methyl phosphonates, phosphorimidates, and/or
phosphotriesters are used to achieve enhanced nuclease resistance.
Increased resistance to degradation may also be achieved by capping
the 5' and/or 3' end of the oligonucleotide. In an embodiment, the
5' and/or 3' end capping of the oligonucleotide is via a 5'-5'
and/or 3'-3' terminal inverted linkage.
[0080] Phosphorothioate oligodeoxynucleotides are relatively
nuclease resistant, water soluble analogs of phosphodiester
oligodeoxynucleotides. These molecules are racemic, but still
hybridize well to their RNA targets. Stein, C., et al. (1991)
Pharmac. Ther. 52:365 384. Phosphorothioate oligonucleotides may be
stereo regular, stereo non-regular or stereo random. A stereo
regular phosphorothioate oligonucleotide is a phosphorothioate
oligonucleotide in which all of the phosphodiester linkages or
phosphorothiodiester linkages polarize light in the same direction.
Each phosphorous in each linkage may be either an S.sub.p or
R.sub.p diastereomer.
[0081] Sugar Moiety Analogs. Oligonucleotide analogs may be created
by modifying and/or replacing a sugar moiety. The sugar moiety of
the oligonucleotide may be modified by the addition of one or more
substituents. For example, one or more of the sugar moieties may
contain one or more of the following substituents:
amino-alkylamino, araalkyl, heteroalkyl, heterocycloalkyl,
aminoalkylamino, O, H, an alkyl, polyalkylamino, substituted silyl,
F, Cl, Br, CN, CF.sub.3, OCF.sub.3, OCN, O-alkyl, S-alkyl, SOMe,
SO.sub.2Me, ONO.sub.2, NH-alkyl, OCH.sub.2CH.dbd.CH.sub.2,
OCH.sub.2CCH, OCCHO, allyl, O-allyl, NO.sub.2, N.sub.3, and
NH.sub.2.
[0082] Modification of the 2' position of the ribose sugar has been
shown in many instances to increase the oligonucleotide's
resistance to degradation. For example, the 2' position of the
sugar may be modified to contain one of the following groups: H,
OH, OCN, O-alkyl, F, CN, CF.sub.3, allyl, O-allyl, OCF.sub.3,
S-alkyl, SOMe, SO.sub.2Me, ONO.sub.2, NO.sub.2, N.sub.3, NH.sub.2,
NH-alkyl, or OCH.dbd.CH.sub.2, OCCH, wherein the alkyl may be
straight, branched, saturated, or unsaturated.
[0083] In addition, the oligonucleotide may have one or more of its
sugars modified and/or replaced so as to be a ribose or hexose
(i.e. glucose, galactose). Further, the oligonucleotide may have
one or more modified sugars. The sugar may be modified to contain
one or more linkers for attachment to other chemicals such as
fluorescent labels. In an embodiment, the sugar is linked to one or
more aminoalkyloxy linkers. In another embodiment, the sugar
contains one or more alkylamino linkers. Aminoalkyloxy and
alkylamino linkers may be attached to biotin, cholic acid,
fluorescein, or other chemical moieties through their amino
group.
[0084] Base Moiety Analogs. In addition, the oligonucleotide may
have one or more of its nucleotide bases substituted or modified.
In addition to adenine, guanine, cytosine, thymine, and uracil,
other bases such as inosine, deoxyinosine, hypoxanthine may be
used. In addition, isoteric purine 2'deoxy-furanoside analogs,
2'-deoxynebularine or 2'deoxyxanthosine, or other purine or
pyrimidine analogs may also be used. By carefully selecting the
bases and base analogs, one may fine tune the binding properties of
the oligonucleotide. For example, inosine nay be used to reduce
hybridization specificity, while diaminopurines may be used to
increase hybridization specificity.
[0085] Adenine and guanine may be modified at positions N3, N7, N9,
C2, C4, C5, C6, or C8 and still maintain their hydrogen bonding
abilities. Cytosine, thymine and uracil may be modified at
positions N1, C2, C4, C5, or C6 and still maintain their hydrogen
bonding abilities.
[0086] Some base analogs have different hydrogen bonding attributes
than the naturally occurring bases. For example, 2-amino-2'-dA
forms three, instead of the usual two, hydrogen bonds to thymine
(T). Examples of base analogs that have been shown to increase
duplex stability include, but are not limited to, 5-fluoro-2'-dU,
5-bromo-2'-dU, 5-methyl-2'-dc, 5-propynyl-2'-dC, 5-propynyl-2'-dU,
2-amino-2'-dA, 7-deazaguanosine, 7-deazadenosine, and
N2-Imidazoylpropyl-2'-dG.
[0087] Pendant Groups. A "pendant group" may be linked to the
oligonucleotide. Pendant groups serve a variety of purposes which
include, but are not limited to, increasing cellular uptake of the
oligonucleotide, enhancing degradation of the target nucleic acid,
and increasing hybridization affinity. Pendant groups can be linked
to any portion of the oligonucleotide but are commonly linked to
the end(s) of the oligonucleotide chain. Examples of pendant groups
include, but are not limited to: acridine derivatives (i.e.
2-methoxy-6-chloro-9-aminoacridine); cross-linkers such as psoralen
derivatives, azidophenacyl, proflavin, and azidoproflavin;
artificial endonucleases; metal complexes such as EDTA-Fe(II),
o-phenanthroline-Cu(I), and porphyrin-Fe(II); alkylating moieties;
nucleases such as amino-1-hexanolstaphylococcal nuclease and
alkaline phosphatase; terminal transferases; abzymes; cholesteryl
moieties; lipophilic carriers; peptide conjugates; long chain
alcohols; phosphate esters; amino; mercapto groups; phenolic
groups, radioactive markers; nonradioactive markers such as dyes;
and polylysine or other polyamines.
[0088] In one embodiment, the aptameric oligonucleotide of the
invention contains at least one nucleotide conjugated to a
carbohydrate, sulfated carbohydrate, or gylcan. Conjugates may be
regarded as a way as to introduce a specificity into otherwise
unspecific DNA binding molecules by covalently linking them to a
selective oligonucleotide or polypeptide.
[0089] Cellular Uptake. To enhance cellular uptake, the
oligonucleotide may be administered in combination with a carrier
or lipid. For example, the oligonucleotide may be administered in
combination with a cationic lipid. Examples of cationic lipids
include, but are not limited to, lipofectin, dotma, dope, DMRIE and
DPPES. The oligonucleotide may also be administered in combination
with a cationic amine such as poly (L-lysine). Oligonucleotide
uptake may also be increased by conjugating the oligonucleotide to
chemical moieties such as transferrin and cholesteryls. In
addition, oligonucleotides may be targeted to certain organelles by
linking specific chemical groups to the oligonucleotide. For
example, linking the oligonucleotide to a suitable array of mannose
residues will target the oligonucleotide to the liver.
[0090] The cellular uptake and localization of oligonucleotides may
be monitored by using labeled oligonucleotides. Methods of labeling
include, but are not limited to, radioactive and fluorescent
labeling. Fluorescently labeled oligonucleotides may be monitored
using fluorescence microscopy and flow cytometry.
[0091] The efficient cellular uptake of oligonucleotides is well
established. For example, when a 20 base sequence phosphorothioate
(PS) oligonulceotide was Injected into the abdomens of mice, either
intraperitoneally (IP) or intravenously (IV). The oligonucleotide
accumulated in the kidney liver, and brain. Chain-extended
oligonucleotides were also observed. Argrawal, S., et al. (1988)
Proc. Natl. Acad. Sci. U.S.A. 85:7079 7083. When the PS
27-oligonucleotide was given by IV to rats, the initial T.sub.1/2
(transit out of the plasma) was 23 min, while the T.sub.1/2beta of
total body clearance was 33.9 hours. The long beta half-life of
elimination demonstrates that dosing could be infrequent and still
maintain effective, therapeutic tissue concentrations. Iverson, P.
(1991) Anti-Cancer Drug Des. 6:531.
[0092] Another aspect of the invention pertains to vectors,
containing an aptameric GRO of the invention, for example, nucleic
acid encoding SEQ ID NOs: 1-12, or derivatives thereof for its
convenient cloning, amplification, and/or transcription. As used
herein, the term "vector" refers to a nucleic acid molecule capable
of transporting another nucleic acid to which it has been "operably
linked." One type of vector is a "plasmid", which refers to a
circular double stranded DNA loop into which additional DNA
segments can be ligated. Another type of vector is a viral vector,
wherein additional DNA segments can be ligated into the viral
genome. Certain vectors are capable of autonomous replication in a
host cell into which they are introduced (e.g., bacterial vectors
having a bacterial origin of replication and episomal mammalian
vectors). Other vectors (e.g., non-episomal mammalian vectors) are
integrated into the genome of a host cell upon introduction into
the host cell, and thereby are replicated along with the host
genome. Moreover, certain vectors are capable of directing the
transcription of sequences to which they are operatively-linked.
Such vectors are referred to herein as "expression vectors". In
general, expression vectors of utility in recombinant DNA
techniques are often in the form of plasmids. In the present
specification, "plasmid" and "vector" can be used interchangeably
as the plasmid is the most commonly used form of vector. However,
the invention is intended to include such other forms of expression
vectors, such as viral vectors (e.g., replication defective
retroviruses, adenoviruses and adeno-associated viruses), and
artificial chromosomes, which serve equivalent functions.
[0093] The recombinant expression vectors of the invention comprise
a nucleic acid of the invention in a form suitable for expression
of the nucleic acid in a host cell, which means that the
recombinant expression vectors include one or more regulatory
sequences, selected on the basis of the host cells to be used for
expression, that is operatively-linked to the nucleic acid sequence
to be transcribed. Within a recombinant expression vector,
"operably-linked" is intended to mean that the nucleotide sequence
of interest is linked to the regulatory sequence(s) in a manner
that allows for transcription and/or expression of the nucleotide
sequence (e.g., in an in vitro transcription/translation system or
in a host cell when the vector is introduced into the host
cell).
[0094] The term "regulatory sequence" is intended to include
promoters, enhancers and other expression control elements (e.g.,
polyadenylation signals). Such regulatory sequences are described,
for example, in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN
ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990).
Regulatory sequences include those that direct constitutive
expression of a nucleotide sequence in many types of host cell and
those that direct expression of the nucleotide sequence only in
certain host cells (e.g., tissue-specific regulatory sequences). It
will be appreciated by those skilled in the art that the design of
the expression vector can depend on such factors as the choice of
the host cell to be transformed, the level of transcription, and/or
expression of protein desired, etc. The expression vectors of the
invention can be introduced into host cells to thereby produce
proteins or peptides, including fusion proteins or peptides,
encoded by nucleic acids as described herein. The recombinant
expression vectors of the invention can be designed for
transcription and/or expression in prokaryotic or eukaryotic cells.
For example, transcription and/or expression in bacterial cells
such as Escherichia coli, insect cells (using baculovirus
expression vectors) yeast cells or mammalian cells. Suitable host
cells are discussed further in Goeddel, GENE EXPRESSION TECHNOLOGY:
METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif (1990).
Alternatively, the recombinant expression vector can be transcribed
and/or translated in vitro, for example using T7 promoter
regulatory sequences and T7 polymerase.
[0095] In another embodiment, the recombinant vector is capable of
directing transcription of the aptameric GRO preferentially in a
particular cell type (e.g., tissue-specific regulatory elements are
used to express the nucleic acid). Tissue-specific regulatory
elements are known in the art. Non-limiting examples of suitable
tissue-specific promoters include the albumin promoter
(liver-specific; Pinkert, et al., 1987. Genes Dev. 1: 268-277),
lymphoid-specific promoters (Calame and Eaton, 1988. Adv. Immunol.
43: 235-275), in particular promoters of T cell receptors (Winoto
and Baltimore, 1989. EMBO J. 8: 729-733) and immunoglobulins
(Banerji, et al., 1983. Cell 33: 729-740; Queen and Baltimore,
1983. Cell 33: 741-748), neuron-specific promoters (e.g., the
neurofilament promoter; Byrne and Ruddle, 1989. Proc. Natl. Acad.
Sci. USA 86: 5473-5477), pancreas-specific promoters (Edlund, et
al., 1985. Science 230: 912-916), and mammary gland-specific
promoters (e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and
European Application Publication No. 264,166).
Developmentally-regulated promoters are also encompassed, e.g., the
murine hox promoters (Kessel and Gruss, 1990. Science 249: 374-379)
and the alpha-fetoprotein promoter (Campes and Tilghman, 1989.
Genes Dev. 3: 537-546).
[0096] In other aspects, the invention relates to a host cell
comprising the isolated aptameric GRO of the invention. In certain
embodiments, the host cell comprises a vector, plasmid or
artificial chromosome nucleic acid containing one or more
transcription regulatory nucleic acid sequences operably linked
with the aptameric GRO sequence of the invention. The vector or
plasmid nucleic acids can be, for example, suitable for eukaryotic
or prokaryotic cloning, amplification, or transcription. In other
embodiments, the invention comprises a plurality of aptameric GRO
sequences linked contiguously as a single polynucleotide chain. In
still other embodiments, the invention comprises a nucleic acid
vector containing a plurality of aptameric GRO sequences linked
contiguously and operably linked with the nucleic acid sequence of
the vector.
[0097] The term "host cell" includes a cell that might be used to
carry a heterologous nucleic acid, or expresses a peptide or
protein encoded by a heterologous nucleic acid. A host cell can
contain genes that are not found within the native
(non-recombinant) form of the cell, genes found in the native form
of the cell where the genes are modified and re-introduced into the
cell by artificial means, or a nucleic acid endogenous to the cell
that has been artificially modified without removing the nucleic
acid from the cell. A host cell may be eukaryotic or prokaryotic.
For example, bacteria cells may be used to carry or clone nucleic
acid sequences or express polypeptides. General growth conditions
necessary for the culture of bacteria can be found in texts such as
BERGEY'S MANUAL OF SYSTEMATIC BACTERIOLOGY, Vol. 1, N. R. Krieg,
ed., Williams and Wilkins, Baltimore/London (1984). A "host cell"
can also be one in which the endogenous genes or promoters or both
have been modified to produce the aptameric GRO of the
invention.
[0098] In another aspect, the invention relates to method for
inhibiting and/or reducing the aggregation of proteins. In other
aspects, the invention relates to methods for inhibiting or
reducing the aggregation of polyglutamine proteins, such as those
that cause Huntington's Disease, or Spinocerebellar ataxia. In any
embodiment of these aspects the invention comprises contacting an
protein capable of forming a protein aggregate or a protein
aggregate with an effective amount of a GRO of the invention to
result in the inhibition of protein aggregate formation, the
reduction of protein aggregation, and/or the dissociation of the
components from a protein aggregate.
[0099] Plasmids disclosed herein are either commercially available,
publicly available on an unrestricted basis, or can be constructed
from available plasmids by routine application of well-known,
published procedures. Many plasmids and other cloning and
expression vectors are well known and readily available, or those
of ordinary skill in the art may readily construct any number of
other plasmids suitable for use. These vectors may be transformed
into a suitable host cell to form a host cell vector system.
Suitable hosts include microbes such as bacteria, yeast, insect or
mammalian organisms or cell lines. Examples of suitable bacteria
are E. coli and B. subtilis. A preferred yeast vector is
pRS426-Gal. Examples of suitable yeast are Saccharomyces and
Pichia. Suitable amphibian cells are Xenopus cells. Suitable
vectors for insect cell lines include baculovirus vectors. Mouse,
rat or human cells are preferred mammalian cells.
[0100] Transformation of a host cell with recombinant DNA may be
carried out by conventional techniques as are well known to those
skilled in the art. By "transformation" is meant a permanent or
transient genetic change induced in a cell following incorporation
of new DNA (i.e., DNA exogenous to the cell). Where the cell is a
mammalian cell, a permanent genetic change is generally achieved by
introduction of the DNA into the genome of the cell. By
"transformed cell" or "host cell" is meant a cell (e.g.,
prokaryotic or eukaryotic) into which (or into an ancestor of
which) has been introduced, by means of recombinant DNA techniques,
a DNA molecule encoding a polypeptide of the invention (i.e., an
INDY polypeptide), or fragment thereof.
[0101] Where the host is prokaryotic, such as E. coli, competent
cells which are capable of DNA uptake can be prepared from cells
harvested after exponential growth phase and subsequently treated
by the CaCl.sub.2 method by procedures well known in the art.
Alternatively, MgCl.sub.2 or RbCl can be used. Transformation can
also be performed after forming a protoplast of the host cell or by
electroporation.
[0102] When the host is a eukaryote, such methods of transfection
with DNA include calcium phosphate co-precipitates, conventional
mechanical procedures such as microinjection, electroporation,
insertion of a plasmid encased in liposomes, or virus vectors, as
well as others known in the art, may be used. Another method is to
use a eukaryotic viral vector, such as simian virus 40 (SV40) or
bovine papilloma virus, to transiently infect or transform
eukaryotic cells. (Eukaryotic Viral Vectors, Cold Spring Harbor
Laboratory, Gluzman ed., 1982). Preferably, a eukaryotic host is
utilized as the host cell as described herein. The eukaryotic cell
may be a yeast cell (e.g., Saccharomyces cerevisiae) or may be a
mammalian cell, including a human cell.
[0103] Mammalian cell systems that utilize recombinant viruses or
viral elements to direct expression may be engineered. For example,
when using adenovirus expression vectors, the nucleic acid
sequences may be ligated to an adenovirus transcription/translation
control complex, e.g., the late promoter and tripartite leader
sequence. This chimeric gene may then be inserted in the adenovirus
genome by in vitro or in vivo recombination. Insertion in a
non-essential region of the viral genome (e.g., region E1 or E3)
will result in a recombinant virus that is viable and capable of
expressing the polypeptides in infected hosts (e.g., Logan &
Shenk, Proc. Natl. Acad. Sci. U.S.A. 81:3655-3659, 1984).
[0104] For long-term, high-yield production of recombinant genes,
stable expression is preferred. Rather than using expression
vectors that contain viral origins of replication, host cells can
be transformed with the cDNA encoding an aptameric GRO controlled
by appropriate expression control elements (e.g., promoter,
enhancer, sequences, transcription terminators, polyadenylation
sites, etc.), and a selectable marker. The selectable marker in the
recombinant plasmid confers resistance to the selection and allows
cells to stably integrate the plasmid into their chromosomes and
grow to form foci, which in turn can be cloned and expanded into
cell lines. For example, following the introduction of foreign DNA,
engineered cells may be allowed to grow for 1 to 2 days in an
enriched media, and then are switched to a selective media. A
number of selection systems may be used, including but not limited
to the herpes simplex virus thymidine kinase (Wigler et al., Cell
11: 233, 1977), hypoxanthine-guanine phosphoribosyltransferase
(Szybalska & Szybalski, Proc. Natl. Sci. U.S.A. 48: 2026,
1962), and adenine phosphoribosyltransferase (Lowy et al., Cell 22:
817, 1980) genes can be employed.
[0105] In other aspects, the invention relates to methods for
treating and/or preventing a disease or condition in an individual
related to the detrimental effects of protein aggregation. In
certain embodiments, the methods of the invention comprise
administering an effective amount of an isolated GRO in a
pharmaceutically acceptable form to an individual in need thereof.
In certain embodiments, the isolated GRO of the invention is
administered together with a pharmaceutically acceptable carrier,
excipient, adjuvant, amino acid, peptide, polypeptide, chemical
compound, drug, biologically active agent or a combination thereof.
As such, in another aspect the invention relates to therapeutic
compositions comprising the isolated GRO of the invention in a
pharmaceutically acceptable form together with at least one
pharmaceutically acceptable carrier, excipient, adjuvant, amino
acid, peptide, polypeptide, chemical compound, drug, biologically
active agent or a combination thereof.
[0106] In certain embodiments the therapeutic GRO of the invention
is complexed, bound, or conjugated to one or more chemical moieties
to improve and/or modify, for example, bioavailability, half-life,
efficacy, and/or targeting. In certain aspects of this embodiment,
the GRO may be complexed or bound, either covalently or
non-covalently with, for example, cationic molecules, salts or
ions, lipids, glycerides, carbohydrates, amino acids, peptides,
proteins, other chemical compounds, for example, phenolic
compounds, and combinations thereof. In certain aspects the
invention relates to a GRO of the invention conjugated to a
polypeptide, for example, an antibody. In certain embodiments the
antibody is specific for the protein or protein aggregate of
interest and therefore targets the GRO to the protein and/or
protein aggregate.
[0107] The efficacy of oligonucleotide therapy is also well
established. For example, when a 24-base sequence PS
oligonucleotide targeted to human c-myb mRNA was infused, through a
miniosmotic pump, into scid mice bearing the human K562 chronic
myeloid leukemia cell line, mean survival times of the mice treated
with the antisense oligonucleotides were six- to eightfold longer
than those of mice untreated or treated with the sense controls or
treated with an oligonucleotide complementary to the c-kit
proto-oncogene mRNA. Ratajczak, et al. (1992) Proc. Natl. Acad.
Sci. U.S.A. 89:11823.
[0108] Therapeutic uses and formulations. The nucleic acids and
proteins of the invention are useful in potential prophylactic and
therapeutic applications implicated in a variety of disorders
including, but not limited to: metabolic disorders, diabetes,
obesity, infectious disease, anorexia, cancer, neurodegenerative
disorders, Huntington's Disease, Alzheimer's Disease, Parkinson's
Disorder, prion diseases (e.g., BSE and CJD), spinocerebellar
ataxia, immune disorders, hematopoictic disorders, and the various
dyslipidemias, metabolic disturbances associated with obesity, the
metabolic syndrome X and wasting disorders associated with chronic
diseases and various cancers, cardiomyopathy, atherosclerosis,
hypertension, congenital heart defects, aortic stenosis, atrial
septal defect (ASD), atrioventricular (A-V) canal defect, ductus
arteriosus, pulmonary stenosis, subaortic stenosis, ventricular
septal defect (VSD), valve diseases, tuberous sclerosis,
scleroderma, lupus erythematosus, obesity, transplantation,
adrenoleukodystrophy, congenital adrenal hyperplasia, prostate
cancer, neoplasm; adenocarcinoma, lymphoma, uterus cancer,
fertility, leukemia, hemophilia, hypercoagulation, idiopathic
thrombocytopenic purpura, immunodeficiencies, graft versus host
disease, AIDS, bronchial asthma, rheumatoid and osteoarthritis,
Crohn's disease; multiple sclerosis, treatment of Albright
Hereditary Ostoeodystrophy, and other diseases, disorders and
conditions of the like.
[0109] Preparations for administration of the therapeutic complex
of the invention 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. Vehicles include
sodium chloride solution, Ringer's dextrose, dextrose and sodium
chloride, lactated Ringer's intravenous vehicles including fluid
and nutrient replenishers, electrolyte replenishers, and the like.
Preservatives and other additives may be added such as, for
example, antimicrobial agents, anti-oxidants, chelating agents and
inert gases and the like.
[0110] The nucleic acid molecules, polypeptides, and antibodies
(also referred to herein as "active compounds") of the invention,
and derivatives, fragments, analogs and homologs thereof, can be
incorporated into pharmaceutical compositions suitable for
administration. Such compositions typically comprise the nucleic
acid molecule, protein, or antibody and a pharmaceutically
acceptable carrier. As used herein, "pharmaceutically acceptable
carrier" is intended to include any and all solvents, dispersion
media, coatings, antibacterial and antifungal agents, isotonic and
absorption delaying agents, and the like, compatible with
pharmaceutical administration. Suitable carriers are described in
the most recent edition of Remington's Pharmaceutical Sciences, a
standard reference text in the field, which is incorporated herein
by reference. Preferred examples of such carriers or diluents
include, but are not limited to, water, saline, finger's solutions,
dextrose solution, and 5% human serum albumin. Liposomes and
non-aqueous vehicles such as fixed oils may also be used. The use
of such media and agents for pharmaceutically active substances is
well known in the art. Except insofar as any conventional media or
agent is incompatible with the active compound, use thereof in the
compositions is contemplated. Supplementary active compounds can
also be incorporated into the compositions.
[0111] A pharmaceutical composition of the invention is formulated
to be compatible with its intended route of administration.
Examples of routes of administration include parenteral, e.g.,
intravenous, intradermal, subcutaneous, oral (e.g., inhalation),
transdermal (i.e., topical), transmucosal, intraperitoneal, and
rectal administration. Solutions or suspensions used for
parenteral, intradermal, or subcutaneous application can include
the following components: a sterile diluent such as water for
injection, saline solution, fixed oils, polyethylene glycols,
glycerine, propylene glycol or other synthetic solvents;
antibacterial agents such as benzyl alcohol or methyl parabens;
antioxidants such as ascorbic acid or sodium bisulfite; chelating
agents such as ethylenediaminetetraacetic acid (EDTA); buffers such
as acetates, citrates or phosphates, and agents for the adjustment
of tonicity such as sodium chloride or dextrose. The pH can be
adjusted with acids or bases, such as hydrochloric acid or sodium
hydroxide. The parenteral preparation can be enclosed in ampoules,
disposable syringes or multiple dose vials made of glass or
plastic.
[0112] Pharmaceutical compositions suitable for injectable use
include sterile aqueous solutions (where water soluble) or
dispersions and sterile powders for the extemporaneous preparation
of sterile injectable solutions or dispersion. For intravenous
administration, suitable carriers include physiological saline,
bacteriostatic water, Cremophor.TM.. (BASF, Parsippany, N.J.) or
phosphate buffered saline (PBS). In all cases, the composition must
be sterile and should be fluid to the extent that easy
syringeability exists. It must be stable under the conditions of
manufacture and storage and must be preserved against the
contaminating action of microorganisms such as bacteria and fungi.
The carrier can be a solvent or dispersion medium containing, for
example, water, ethanol, polyol (for example, glycerol, propylene
glycol, and liquid polyethylene glycol, and the like), and suitable
mixtures thereof The proper fluidity can be maintained, for
example, by the use of a coating such as lecithin, by the
maintenance of the required particle size in the case of dispersion
and by the use of surfactants. Prevention of the action of
microorganisms can be achieved by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
ascorbic acid, thimerosal, and the like. In many cases, it will be
preferable to include isotonic agents, for example, sugars,
polyalcohols such as manitol, sorbitol, sodium chloride in the
composition. Prolonged absorption of the injectable compositions
can be brought about by including in the composition an agent which
delays absorption, for example, aluminum monostearate and
gelatin.
[0113] Sterile injectable solutions can be prepared by
incorporating the active compound (e.g., the therapeutic complex of
the invention) in the required amount in an appropriate solvent
with one or a combination of ingredients enumerated above, as
required, followed by filtered sterilization. Generally,
dispersions are prepared by incorporating the active compound into
a sterile vehicle that contains a basic dispersion medium and the
required other ingredients from those enumerated above. In the case
of sterile powders for the preparation of sterile injectable
solutions, methods of preparation are vacuum drying and
freeze-drying that yields a powder of the active ingredient plus
any additional desired ingredient from a previously
sterile-filtered solution thereof.
[0114] Oral compositions generally include an inert diluent or an
edible carrier. They can be enclosed in gelatin capsules or
compressed into tablets. For the purpose of oral therapeutic
administration, the active compound can be incorporated with
excipients and used in the form of tablets, troches, or capsules.
Oral compositions can also be prepared using a fluid carrier for
use as a mouthwash, wherein the compound in the fluid carrier is
applied orally and swished and expectorated or swallowed.
Pharmaceutically compatible binding agents, and/or adjuvant
materials can be included as part of the composition. 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 or gelatin; an
excipient such as starch or lactose, a disintegrating agent such as
alginic acid, Primogel, or corn starch; a lubricant such as
magnesium stearate or Sterotes; a glidant such as colloidal silicon
dioxide; a sweetening agent such as sucrose or saccharin; or a
flavoring agent such as peppermint, methyl salicylate, or orange
flavoring.
[0115] For oral administration, the pharmaceutical compositions may
take the form of, for example, tablets or capsules prepared by
conventional means with pharmaceutically acceptable excipients such
as binding agents (e.g., pregelatinised maize starch,
polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers
(e.g., lactose, microcrystalline cellulose or calcium hydrogen
phosphate); lubricants (e.g., magnesium stearate, talc or silica);
disintegrants (e.g., potato starch or sodium starch glycolate); or
wetting agents (e.g., sodium lauryl sulphate). The tablets may be
coated by methods well known in the art. Liquid preparations for
oral administration may take the form of, for example, solutions,
syrups, or suspensions, or they may be presented as a dry product
for constitution with water or other suitable vehicle before use.
Such liquid preparations may be prepared by conventional means with
pharmaceutically acceptable additives such as suspending agents
(e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible
fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous
vehicles (e.g., almond oil, oily esters, ethyl alcohol or
fractionated vegetable oils); and preservatives (e.g., methyl or
propyl-p-hydroxybenzoates or sorbic acid). The preparations may
also contain buffer salts, flavoring, coloring, and sweetening
agents as appropriate.
[0116] Preparations for oral administration may be suitably
formulated to give controlled release of the active compound. For
buccal administration the compositions may take the form of tablets
or lozenges formulated in conventional manner. For administration
by inhalation, the compounds for use according to the present
invention are conveniently delivered in the form of an aerosol
spray presentation from pressurized packs or a nebulizer, with the
use of a suitable propellant, e.g., dichlorodifluoromethane,
trichlorofluoromethane, dichlorotetrafluoroethan-e, carbon dioxide
or other suitable gas. In the case of a pressurized aerosol the
dosage unit may be determined by providing a valve to deliver a
metered amount. Capsules and cartridges of e.g. gelatin for use in
an inhaler or insufflator may be formulated containing a powder mix
of the compound and a suitable powder base such as lactose or
starch. The compounds may be formulated for parenteral
administration by injection, e.g., by bolus injection or continuous
infusion. Formulations for injection may be presented in unit
dosage form, e.g., in ampoules or in multi-dose containers, with an
added preservative. The compositions may take such forms as
suspensions, solutions, or emulsions in oily or aqueous vehicles,
and may contain formulatory agents such as suspending, stabilizing,
and/or dispersing agents. Alternatively, the active ingredient may
be in powder form for constitution with a suitable vehicle, e.g.,
sterile pyrogen-free water, before use. The compounds may also be
formulated in rectal compositions such as suppositories or
retention enemas, e.g., containing conventional suppository bases
such as cocoa butter or other glycerides. In addition to the
formulations described previously, the compounds may also be
formulated as a depot preparation. Such long acting formulations
may be administered by implantation (for example subcutaneously or
intramuscularly) or by intramuscular injection. Thus, for example,
the compounds may be formulated with suitable polymeric or
hydrophobic materials (for example as an emulsion in an acceptable
oil) or ion exchange resins, or as sparingly soluble derivatives,
for example, as a sparingly soluble salt.
[0117] For administration by inhalation, the compounds are
delivered in the form of an aerosol spray from pressured container
or dispenser which contains a suitable propellant, e.g., a gas such
as carbon dioxide, or a nebulizer.
[0118] Systemic administration can also be by transmucosal or
transdermal means. For transmucosal or transdermal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the art,
and include, for example, for transmucosal administration,
detergents, bile salts, and fusidic acid derivatives. Transmucosal
administration can be accomplished through the use of nasal sprays
or suppositories. For transdermal administration, the active
compounds are formulated into ointments, salves, gels, or creams as
generally known in the art.
[0119] Principles and considerations involved in preparing such
compositions, as well as guidance in the choice of components are
provided, for example, in Remington: The Science And Practice Of
Pharmacy 19th ed. (Alfonso R. Gennaro, et al., editors) Mack Pub.
Co., Easton, Pa.: 1995; Drug Absorption Enhancement: Concepts,
Possibilities, Limitations, And Trends, Harwood Academic
Publishers, Langhorne, Pa., 1994; and Peptide And Protein Drug
Delivery (Advances In Parenteral Sciences, Vol. 4), 1991, M.
Dekker, New York.
[0120] The active ingredients can also be entrapped in
microcapsules prepared, for example, by coacervation techniques or
by interfacial polymerization, for example, hydroxymethylcellulose
or gelatin-microcapsules and poly-(methylmethacrylate)
microcapsules, respectively, in colloidal drug delivery systems
(for example, liposomes, albumin microspheres, microemulsions,
nano-particles, and nanocapsules) or in macroemulsions. The
formulations to be used for in vivo administration must be sterile.
This is readily accomplished by filtration through sterile
filtration membranes.
[0121] Sustained-release preparations can be prepared. Suitable
examples of sustained-release preparations include semipermeable
matrices of solid hydrophobic polymers containing the antibody,
which matrices are in the form of shaped articles, e.g., films, or
microcapsules. Examples of sustained-release matrices include
polyesters, hydrogels (for example,
poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),
polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic
acid and gamma-ethyl-L-glutamate, non-degradable ethylene-vinyl
acetate, degradable lactic acid-glycolic acid copolymers such as
the LUPRON DEPOT.TM. (injectable microspheres composed of lactic
acid-glycolic acid copolymer and leuprolide acetate), and
poly-D-(-)-3-hydroxybutyric acid. While polymers such as
ethylene-vinyl acetate and lactic acid-glycolic acid enable release
of molecules for over 100 days, certain hydrogels release proteins
for shorter time periods.
[0122] In one embodiment, the active compounds are prepared with
carriers that will protect the compound against rapid elimination
from the body, such as a controlled release formulation, including
implants and microencapsulated delivery systems. Biodegradable,
biocompatible polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Methods for preparation of such formulations will
be apparent to those skilled in the art. The materials can also be
obtained commercially from Alza Corporation and Nova
Pharmaceuticals, Inc. Liposomal suspensions (including liposomes
targeted to infected cells with monoclonal antibodies to viral
antigens) can also be used as pharmaceutically acceptable carriers.
These can be prepared according to methods known to those skilled
in the art, for example, as described in U.S. Pat. No.
4,522,811.
[0123] It is especially advantageous to formulate oral or
parenteral compositions in dosage unit form for ease of
administration and uniformity of dosage. Dosage unit form as used
herein refers to physically discrete units suited as unitary
dosages for the subject to be treated; each unit containing a
predetermined quantity of active compound calculated to produce the
desired therapeutic effect in association with the required
pharmaceutical carrier. The specification for the dosage unit forms
of the invention are dictated by and directly dependent on the
unique characteristics of the active compound and the particular
therapeutic effect to be achieved, and the limitations inherent in
the art of compounding such an active compound for the treatment of
individuals.
[0124] The nucleic acid molecules of the invention can be inserted
into vectors and used as gene therapy vectors. Gene therapy vectors
can be delivered to a subject by, for example, intravenous
injection, local administration (see, e.g., U.S. Pat. No.
5,328,470) or by stereotactic injection (see, e.g., Chen, et al.,
1994. Proc. Natl. Acad. Sci. USA 91: 3054-3057). The pharmaceutical
preparation of the gene therapy vector can include the gene therapy
vector in an acceptable diluent, or can comprise a slow release
matrix in which the gene delivery vehicle is imbedded.
Alternatively, where the complete gene delivery vector can be
produced intact from recombinant cells, e.g., retroviral vectors,
the pharmaceutical preparation can include one or more cells that
produce the gene delivery system. The pharmaceutical compositions
can be included in a container, pack, or dispenser together with
instructions for administration.
[0125] A therapeutically effective dose refers to that amount of
the therapeutic complex sufficient to result in amelioration or
delay of symptoms. Toxicity and therapeutic efficacy of such
compounds can be determined by standard pharmaceutical procedures
in cell cultures or experimental animals, e.g., for determining the
LD50 (the dose lethal to 50% of the population) and the ED50 (the
dose therapeutically effective in 50% of the population). The dose
ratio between toxic and therapeutic effects is the therapeutic
index and it can be expressed as the ratio LD50/ED50. Compounds
that exhibit large therapeutic indices are preferred. While
compounds that exhibit toxic side effects may be used, care should
be taken to design a delivery system that targets such compounds to
the site of affected tissue in order to minimize potential damage
to uninfected cells and, thereby, reduce side effects. The data
obtained from the cell culture assays and animal studies can be
used in formulating a range of dosage for use in humans. The dosage
of such compounds lies preferably within a range of circulating
concentrations that include the ED50 with little or no toxicity.
The dosage may vary within this range depending upon the dosage
form employed and the route of administration utilized. For any
compound used in the method of the invention, the therapeutically
effective dose can be estimated initially from cell culture assays.
A dose may be formulated in animal models to achieve a circulating
plasma concentration range that includes the IC50 (i.e., the
concentration of the test compound which achieves a half-maximal
inhibition of symptoms) as determined in cell culture. Such
information can be used to more accurately determine useful doses
in humans. Levels in plasma may be measured, for example, by high
performance liquid chromatography.
[0126] Pharmaceutical compositions may be formulated in
conventional manner using one or more physiologically acceptable
carriers or excipients. Thus, the compounds and their
physiologically acceptable salts and solvates may be formulated for
administration by inhalation or insufflation (either through the
mouth or the nose) or oral, buccal, intravenous, intraperitoneal,
parenteral or rectal administration.
[0127] Also disclosed according to the present invention is a kit
or system utilizing any one of the methods, selection strategies,
materials, or components described herein. Exemplary kits according
to the present disclosure will optionally, additionally include
instructions for performing methods or assays, packaging materials,
one or more containers which contain an assay, a device or system
components, or the like.
[0128] In an additional aspect, the present invention provides kits
embodying the complex and methods of using disclosed herein. Kits
of the invention optionally include one or more of the following:
(1) polypeptide or nucleic acid components described herein; (2)
instructions for practicing the methods described herein, and/or
for operating the selection procedure herein; (3) one or more
detection assay components; (4) a container for holding nucleic
acids or polypeptides, other nucleic acids, transgenic plants,
animals, cells, or the like and, (5) packaging materials.
[0129] While preferred embodiments of the invention have been shown
and described herein, it will be understood that such embodiments
are provided by way of example only. Numerous variations, changes
and substitutions will occur to those skilled in the art without
departing from the spirit of the invention in view of the present
description and examples. Accordingly, it is intended that the
appended claims cover all such variations as fall within the spirit
and scope of the invention.
EXAMPLES
[0130] The original observation that ODNs bearing random sequences
reduced Htt aggregate formation prompted a closer examination of a
potential role for ODNs in HD therapy. We chose to utilize a
biochemical/immunochemical assay system that enables rapid
screening of compounds/molecules for the inhibition of Htt
aggregation. In this test, ODNs (40 .mu.m) were mixed with purified
mutant Huntington for 24 hours and then passed through a cellulose
acetate membrane filter (0.2 .mu.m). The percentage of aggregates
remaining on the filter was detected by immunochemistry using a
primary HD-antibody and a secondary anti-rabbit antibody conjugated
to alkaline phosphatase. Included in these experiments was a
positive control, Congo Red. Two ODNs, HD3S/53 (all DNA, 53-mer)
and HDR/25NS (all RNA, 25-mer), were found to be effective
inhibitors. Fundamentally, the results established that ODNs can be
used to inhibit Huntington aggregation. These molecules however
ranged in size up to 53 bases and some were found to be unstable in
cells, showing little inhibitory activity. This work was published
and the paper is incorporated by reference herein in its entirety
(Parekh-Olmedo, et al., Modified Single-Stranded ODNs inhibit
Aggregate Formation and Toxicity Induced by Expanded Polyglutamine,
Journal of molecular Science, Vol. 24, pp. 257-267, (2004)). Having
established that ODNs have potential as a possible HD therapeutic,
we turned our attention toward screening ODNs with specific, yet
simple, sequences. As a starting point, we designed 20-mers of
monomeric sequences (all Gs, Ts, Cs or As) and passed them through
the biochemical assay described above. The three ODNs, referred to
herein as Huntington's Disease (HD) A, G, T, or C oligonucleotides:
HDA (SEQ ID NO:9), HDG (SEQ ID NO:3), HDT (SEQ ID NO:10) and HDC
(SEQ ID NO:8), respectively.
[0131] As shown in FIG. 6, HDA, HDC and HDT (SEQ ID NOs: 8, 9, and
10) are unable to inhibit aggregate formation in the biochemical
assay at either 20 .mu.M or 40 .mu.M. In sharp contrast, HDG (SEQ
ID NO:3) was remarkably efficient in blocking aggregation rivaling
Congo Red in activity. In some cases, no aggregates were retained
on the filter falling below our capacity to detect them. This
result was repeated numerous times (>10) and was judged to be
robust and reproducible. Thus, we pursued the HDG molecule as a
possible therapeutic for HD by examining its activity at various
doses in the biochemical assay. As seen in FIG. 4, low levels of
HDG exhibited high levels of inhibition activity, confirming our
earlier results. This dose curve was extremely reproducible with
1-5 .mu.M concentrations producing a near complete inhibition of
Htt aggregation. We were unable to detect any Htt aggregate
inhibition catalyzed by HDA, HDT and HDC at similar levels.
[0132] When the concentrations exceeded 60 .mu.M; we observed small
but nonreproducible positive and negative effects (data not shown).
Thus, we ended this line of experimentation and focused once again
on the basic G-rich ODN, HDG (SEQ ID NO:3).
[0133] Agents demonstrating positive effects in any biochemical
assay must be capable of inhibiting Htt aggregation in cells. One
of the most versatile and robust test systems utilizes the human
embryonic kidney cell line, HEK293T cells.
[0134] A well-established biochemical assay was used to examine
GROs blockage of aggregation. Molecules T40216 (SEQ ID NO:2) and
T30923 (SEQ ID NO:1), GROs that are known to form intermolecular
G-quartets were found to be effective inhibitors of aggregation.
Both of these GROs, adopt conventional G-quartet structure with the
G residues (quartets) in the center and a loop domain at the top
and bottom. The GROs of the invention, including the preferred HDG,
which exhibits the highest level of activity in the aggregation
assays, can also adopt a stable G-quartet structure and further
studies to elucidate the details of the G-quartet structure adopted
by HDG are currently being performed. This molecule of the
invention also may prevent or delay neurotoxicity in PC12
cells.
[0135] In one of the embodiments, aptaperic GRO, HDG (SEQ ID NO:3),
is unique among monotonic ODNs containing 20 bases. None of the
related 20-mers, HDA, HDC or HDT (SEQ ID NOs: 8-10) show
reproducible inhibitory activity in either the biochemical or
cell-based assays. Furthermore, HDG displays a dose response with
concentrations as low as 1 .mu.M exhibiting substantial levels of
aggregate reduction. For example, HDG is effective when added at
the start of the Q58-Htn aggregation reaction but much less so when
added after the process has begun. Without being limited to any
particular theory, the inventors speculate that HGD is likely most
effective at blocking the nucleation phase of aggregation rather
than the elongation phase.
[0136] HDG (SEQ ID NO:3) was also found to be quite active in
blocking aggregation of the Httexon1-eGFP fusion protein
aggregation in HEK293 cells. In this system, the fusion protein is
produced from an expression plasmid and co-transfection with HDG
was found to prevent the appearance of green fluorescent foci in a
dose-dependent inhibition. Importantly, the well-known aggregation
inhibitor, Congo Red, was used as a positive control and displayed
effects similar to HDG. MTT
(3,[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide)
viability assays reveal no cell toxicity or negative effects on
cell growth as a function of ODN addition (data not shown). This
result is not surprising since ODNs used as antisense or antigene
therapy have been found to be practically inert in human cells with
regard to cytotoxicity. A number of clinical trials using ODNs have
taken place and while the efficacy of such treatments may be
questioned, significant adverse effects on cells or patients were
not observed. The lack of serious side effects from ODNs is a
virtue in the development of these molecules for use in HD
patients. For example, while the effective levels for GRO activity
of the GROs of the present invention are higher than those used for
traditional pharmaceuticals, ODNs are particularly well-tolerated
in humans. The levels presented herein are not unusual and levels
exceeding 50 mg/kg have been found to be both efficacious and
nontoxic in various antisense therapies. The higher amounts may be
required because delivery to target cells or penetration into the
cells may be less efficient than other drug treatments.
[0137] The aptameric GROs of the invention were modified in order
to determine and confirm the potency of the G-quartet structure of
the GROs, including HDG (SEQ ID NO:3). Using a type of reverse
genetics strategy, we created several "mutant" HDGs; HDG 20/7 (SEQ
ID NO:7) wherein each 7th G was replaced with a T, HDG 20/3 (SEQ ID
NO:5) wherein each 3rd G was replaced with a T and HDG 20/2 (SEQ ID
NO:4) wherein every other G was substituted with a T residue. None
of these molecules were found to be effective inhibitors of
aggregation. The results presented in FIG. 11 most clearly
illustrate the importance of the HDG G-quartet structure while
support for this notion is also gained when T30923 (SEQ ID NO:1)
could not fully substitute for HDG in the HEK293 assay as presented
in Example 3.
[0138] G-quartets formed within GROs have also been shown to
inhibit protein dimerization of such molecules as STAT3. They exert
their activity by binding to specific domains within STAT3 with a
high degree of precision. Since mutant Htt aggregation relies on a
nucleation phase in which the mutant protein begin to assemble, HDG
(SEQ ID NO:3) could block the transition between nucleation and
elongation as aggregation (dimerization) begins. Alternatively, HDG
could block other enzymes involved in the development of the
pathogenic phenotype, such as caspases which cleave the native
protein perhaps producing a toxic fragment. Bates and colleagues
have shown that certain aptameric GROs can bind to nucleolin in a
variety of cancer cells with a high degree of specificity. In all
of these cases, direct interactions with cellular proteins would be
required.
[0139] As described above, we have shown that G-rich
oligonucleotides, most preferably the HDG (SEQ ID NO:3) having a
length of approximately 18-25 G-residues, and more preferably, 20 G
residues, inhibits the aggregation process in a mutant Huntington
protein. Functional ODNs which inhibit Huntington aggregation
include the following aptameric GROs: TABLE-US-00002 HDG (SEQ ID
NO. 3) 5' - GGG GGG GGG GGG GGG GGG GG-3' GRO26B (SEQ ID NO. 11) 5'
- GGT GGT GGT GGT TGT GGT GGT GGT GG-3' GRO29A (SEQ ID NO. 12) 5' -
TTT GGT GGT GGT GGT TGT GGT GGT GGT GG-3'
[0140] In other aspects the present invention comprises a random
screening process for finding active ODNs which inhibit protein
aggregation. These G-rich ODNs (GROs) are known to possess
aptameric activity, and we believe this aptameric activity is
important for the non-specific binding of the ODN to the protein.
GROs are capable of interacting with numberous cellular proteins
owing to their polyanionic character or specific secondary
structure. These aptameric GROs form quadruplex structures which
are stabilized by G-quartets. We believe they have therapeutic
potential for protein aggregation-related diseases. Current results
show the level of aggregation is minimal compared to controls which
contain no aptameric GRO and are normalized to 100%
aggregation.
[0141] Although the invention is illustrated and described herein
with reference to specific embodiments, the invention is not
intended to be limited to the details shown. Rather, various
modifications may be made in the details within the scope and range
of equivalents of the claims and without departing from the
invention.
Experimental Examples
[0142] Variations in the structure of the ODNs. The aptameric GRO
molecule, HDG (SEQ ID NO:3), is 20 bases in length, and is composed
of all G nucleotides. While it exhibits robust activity thus so
far, we cannot assume that HDG is the most optimal vector. Thus, we
changed HDG in a methodical fashion and tested variants in the
biochemical screen. The chart below outlines strategic plans and
the rationale for modifying HDG: TABLE-US-00003 Variable Specific
Changes Rationale A. Length 20.fwdarw.18.fwdarw.16 . . . 4 bases,
Shorter ODNs may be length are changed in steps of 2 more capable
transiting the BBB B. Composition Guanosine DNA bases are RNA can
adopt similar tested first and 2'-O-methyl structural changes in
tracts RNA bases (all G's) are of G residues. 2'-O-methyl tested in
turn RNA is more stable than unmodified RNA C. Sequence All G
residue tracts are G-quartet structures are interrupted with Ts at
various also known to form with T intervals i.e. residues at
3.sup.rd and 4.sup.th GGGTGGGTGGGT etc. positions: some with
greater stability D. Chemical Modifications G residues are linked
with LNA or PNA bases may phosphodiester bonds. HDG assist in
crossing the BBB is changed to contain LNA, or more readily and PNA
bases and/or phosphorothioate linkages phosphorothioate linkages
protect against nuclease (PS) digestion
[0143] Each of these alterations was evaluated for biological
activity using a matrix of conditions, but, the primary
discriminatory screen involved length since eventually smaller
molecules will be more likely to penetrate the BBB (see below).
Once several short active molecules were identified, other
modifications were tested, including the incorporation of T
residues into the G-rich sequence, linkage groups and base
chemistries. As a standard, however, HDG was processed through all
of the assays. The most active GROs were analyzed in
time-of-addition experiments wherein the ODN was added at periodic
intervals following the initialization of protein aggregation.
[0144] Active GROs and/or related GROs that were identified were
re-screened at various dosages in a range of 10-2 .mu.M through 102
.mu.M to generate an IC50. Each assay group was tested in
triplicate with the blots scanned and quantitated using Image Quant
Software. Of particular interest were GROs with PNA linkages
because of their potential advantage in crossing the BBB. We
focused on synthesizing GROs with various number of PNAs and
conducted a methodical, systematic analyses of PNA (and
LNA)-modified GROs. The baseline for all assays was established
using two control samples; Congo Red and I to set positive and
negative boundaries. These control samples have been used routinely
in previous screens and have proven to be quite robust in
establishing the parameters and validating the assay system. IC50
was obtained by graphing the % aggregate reduction and the
concentration (.mu.M) on a log scale. Standard deviation, SEM means
and P values will be automatically calculated for each point using
a program from Prism 3.0 software, one way ANNOVA and Tukey's
posttest for multiple comparisons, respectively.
[0145] Structural analyses of G-rich ODNs exhibiting inhibitory
activity: structure/function relationships. GROs are believed to
fold into a stable (G-quartet) secondary structure. G-quartets are
formed between the N1 to O6 and N2 to N7 positions of adjacent
guanosines; such interactions, amid a string of Gs which stack in a
coordinated fashion giving rise to tetrad helices. Secondary
structures form under favorable ionic conditions that include an
environment rich in K+ ions; but can form in the presence of other
monovalent cations. Importantly, the cellular concentration of KCl
is 140 mM, clearly above the required minimum concentration
conducive for G-quartet assembly.
[0146] We analyzed the structure of each GRO that exhibited
significant activity in the biochemical assay. Circular dichroism
was used to analyze structure including HDG (SEQ ID NO:3) at a
concentration of 15 .mu.M in 10 .mu.M KCl and 20 mM Li3PO4 at pH7
at 24.degree. C. The spectrophotometer used was a JASCO J-500A
spectropolarimeter which allowed us to obtain data in molar
ellipticity (deg.cm2dmol-1). For each ODN, we utilized 5-10 scans
and integrated all scans to determine the most probable structural
profile. The GROs were synthesized by Integrated DNA Technologies
(IDT, Coralville, Iowa) and quantitated by the manufacture. GROs
were tested at 264 nm with a minimum of 240 nm as ellipticities at
264 nm and 240 nm respectively are highly characteristic of a
G-quartet structure. The following ODNs which are known to form
these structures were used as positive controls: T30923 (SEQ ID
NO:1), 5'-GGGTGGGTGGGTGGGT-3', and T40216 (SEQ ID NO:2),
5'-GGGGGTGGGGGTGGGGTGGGGGT-3'. For negative controls, the following
ODNs, which have are known not to form a G-quartet, NS-ODN,
5'-TGCCGGATCAAGCGCTACCA-3' and the poly A monomer of 20 bases in
length, HDA (SEQ ID NO:9), were utilized. By generating CD spectra
profiles of the G-quartets, with GROs known to adopt that structure
we were able to compare and even correlate our candidate GROs
exhibiting strong activity in the aggregation assays with the
degree of G-quartet formation. By coupling this information we were
able to gain some fundamental insight into the structure of the
efficacious molecules.
[0147] Structure/function analyses were also be carried out using
PAGE. In this assay, G-rich ODNs were electrophoresed through a 19%
native acrylamide gel matrix and stained with SYBR Gold, a protocol
that enables visualization of the degree of secondary structure in
the sample. Such a procedure is most useful in analyzing potential
variation that could occur among preparations of GROs obtained from
the manufacturer. It does not, however, measure G-quartet assembly.
We also compared GROs that exhibit high levels of activity to those
that do not, with a goal correlating secondary structure with
increased or decreased levels of inhibitory activity, with the long
term goal of identifying a "structural marker" for active GROs.
Secondary structures were detected and quantified after staining
with a Typhoon Image using a 532 nm green filter directly from the
imaged gel.
Example 2
[0148] Biochemical analyses of GROs. We chose to utilize a
biochemical/immunochemical assay system that enables rapid
screening of compounds/molecules for the inhibition of aggregation.
In this test, ODNs were mixed with purified mutant Htt fragment for
24 hours and then passed through a cellulose acetate membrane
filter. The percentage of aggregates remaining on the filter was
detected by immunochemistry using a primary Htt-antibody and a
secondary anti-rabbit antibody conjugated to peroxidase. Signals
from the SDS insoluble aggregates were scanned and quantified. A
diagram of this assay, established by Wang et al. (2005), is
presented in FIG. 1. In all preparations of the mutant protein,
thrombin-directed cleavage of GST-Q58Htn was allowed to proceed for
45 minutes prior to the addition of the GRO. This cleavage
generates an amino terminal polyglutamine fragment consisting of
171 amino acids of the human huntingtin with tract of 58 glutamine
residues. The fragment is fused to GST to enable purification. We
will utilize the Wang et al terminology, GST-Q58-Htn to designate
the protein used in this assay. The mixture was centrifuged to
remove any aggregates that had already formed. Western blot
analyses have shown that >95% of the GST-Q58-Htn is cleaved to
completion by the thrombin. This parameter is an important control
for our study since a variety of agents are known to block the
enzymatic cleavage reaction directed by thrombin.
[0149] Two known GROs were tested for inhibitory activity in the
biochemical assay described above. ODN T30923 (SEQ ID NO:1) and ODN
T40216 (SEQ ID NO:2) were used as aptamers to inhibit the function
of STAT3 protein. Both of these molecules have been determined by
Circular Dichroism (CD) and NMR to have an intramolecular G-quartet
structure, and similar CD spectra were seen by our lab (see below).
The sequence of each is provided in FIG. 2A; T30923 contains
(GGGT).sub.4, 16 bases in length while T40216 contains
(GGGGGT).sub.4, 24 bases in length.
[0150] To analyze the inhibition of aggregation by GROs, a
biochemical assay was employed (FIG. 1). The fusion protein
GST-Q58-Htn was incubated for 45 minutes at room temperature with
thrombin (1 U/1 .mu.g protein) at a concentration of 10 .mu.g/ml in
a buffer of 50 mM Tris-HCl, pH 8, 100 mM NaCl, 2.5 mM CaCl2, and 1
mM EDTA, to cleave between the huntingtin and GST. As indicated by
Wang et al., this fragment consists of the amino terminal 171 amino
acids with a tract of 58 glutamine residues fused to GST. The
protein mix was then centrifuged at 30,000.times.g at 4.degree. C.
for 35 minutes to remove any aggregates that had already formed.
The protein was added to wells containing 0.5-60 .mu.M GROs or
control ODNs, 10 .mu.M Congo Red, or no treatment in the buffer
detailed above with 100 mM KCl replacing NaCl. The 0-hour control
was stopped immediately and after 24 hours incubation at room
temperature the remaining reactions were stopped by adding 10%
SDS/50 mM 2-mercaptoethanol and heating to 99.degree. C. for five
minutes. The mixture was diluted in 1.times.PBS and then filtered
through a cellulose acetate membrane (Osmonics) using the
Easy-Titer ELIFA system (Pierce) followed by a 2% SDS wash. After
blocking in 5% milk/1.times.PBS-0.05% Tween, the membrane was
incubated with a specific anti-huntingtin antibody (HP1, 1:1000
dilution), followed by incubation with a peroxidase-conjugated goat
anti-rabbit antibody (Sigma, 1:40,000 dilution) and
chemiluminescence reagent (ECL-Plus, Amersham). Signals from the
aggregates retained on the filter were scanned and quantified using
ImageQuant image analysis software (Molecular Dynamics). Aggregates
were quantified by optical density and statistical differences were
determined by one way ANOVA with Tukey's post hoc analysis using
Statistical Package for the Social Sciences (SPSS). Significance
was determined by a p<0.05 as compared to Congo Red
(control).
[0151] Three control reactions, designated 0-hour, 24-hour and
Congo Red (FIG. 2B), were repeated for each experiment. The 0-hour
control displays the amount of aggregation at the start of the
reaction, usually none. The 24-hour control reflects the amount of
GST-Q58-Htn aggregation when no inhibitor is added to the mixture.
The third control displays the degree of aggregation formed in the
presence of Congo Red, a known inhibitor functioning as the
positive control in the series. As shown in FIG. 2B, both T40216
(SEQ ID NO:2) and T30923 (SEQ ID NO:1) are capable of inhibiting
GST-Q58-Htn aggregation with a dose response visible in the samples
with T30923. FIG. 3 represents the results of five experiments
conducted in duplicate, followed by quantitation using ImageQuant
analytical software. A statistically significant difference is
observed between each GRO and the 24-hour control in each
experiment.
[0152] The effect of GROs on aggregation prompted an examination of
the activity of a monotonic guanosine ODN (HDG; SEQ ID NO:3)
because this molecule can also form a G-quartet. We chose 20 bases
as a compromised length of T30923 (SEQ ID NO:1) (16 bases) and
T40216 (SEQ ID NO:2) (24 bases) to establish the HDG series. When a
dose range of HDG was tested in the biochemical assay, inhibition
of aggregation Q58-Htn fragment was readily observed (FIG. 4). A
significant decrease was seen at 1 .mu.M, a much lower final
concentration than the inhibitory level found with either T30923 or
T40216. HDG was found to be unique in its inhibitory activity
compared to other monotonic ODNs. Huang et al. demonstrated that
the flow-through fraction of reactions containing inhibitors of
aggregation is comprised predominantly of monomeric Htt fragments.
To verify that the flow-through in reactions bearing HDG 20
contains mutant Htt fragments, we captured this fraction and placed
it on blotting paper. Stacked membranes to capture monomers using
the same antibody used to detect aggregates. As seen in the inset
for FIG. 4, a positive reaction was observed indicating the
presence of mutant Htt fragment. When 20-mers of A (HDA; SEQ ID
NO:9), T (HDT; SEQ ID NO:10) or C (HDC; SEQ ID NO:8) were tested at
20 .mu.M and 40 .mu.M, no inhibition of aggregation was observed
(FIG. 5). Quantitation after scanning revealed a large,
statistically significant difference in the activity of HDG
compared to any of the other monotonic ODNs (FIG. 6). Taken
together, our results suggest that HDG, a 20-mer containing all G
residues, is a powerful inhibitor of aggregation of Q58-Htn
fragment based on the results of the immunoblotting assay.
[0153] CD measures differences in the absorbance of right-handed
and left-handed circularly polarized light and can be used to
investigate DNA helicity. G-quadruplexes can exist as antiparallel
monomers, dimers or tetramers or as parallel tetramers.
Traditionally, antiparallel conformations are characterized by a
positive ellipticity maximum at 295 nm and a negative minimum at
265 nm. In contrast, the parallel conformation is characterized by
a positive maximum at 264 nm and a negative minimum at 240 nm;
however, recent results have shown some antiparallel structures to
have some positive maximums at 264 nm and negative minimums at 240
nm. HDG (SEQ ID NO:3) was characterized by CD in order to gain a
perspective view of its structure. HDG was analyzed along with HDA
(SEQ ID NO:9) and T30923 (SEQ ID NO:1) at 15 .mu.M in 10 mM KCl and
at 24.degree. C. CD spectropolarity was determined using an AVIV
Model 202 spectrometer with an effective range of analysis from 200
nm to 320 nm (FIG. 7). HDA has an unusual maximum absorbance at 220
nm with a smaller positive absorbance at 260 nm. T30923 and HDG,
however, exhibit maximum positive absorbances at 264 nm and
negative minimums at 241 nm, a distinct profile that matches
closely with molecules known to adopt G-quartet structures. HDG is
a more effective inhibitor of GST-Q58-Htn aggregation than T30923
which is known to adopt a dimer basket G-quartet conformation [see
7] suggesting that HDG's structure is a more active conformation in
our assays.
Example 3
[0154] Inhibition of aggregate formation in HEK293 cells. Since HDG
(SEQ ID NO:3) exhibited strong inhibitory activity of GST-Q58-Htn
in a biochemical assay, we tested this molecule in a cell-based
assay. Human embryonic kidney cells, HEK293T, were grown in low
glucose DMEM supplemented with 10% FBS. Cells were seeded at
0.5-1.times.10.sup.6 cells/well on 6-well plates. The cells were
transfected with 1 .mu.g of the plasmid pcDNA3.1-72Httexon1-eGFP
(p72Q) and 150-750 nM GRO or control ODN using 2.5 .mu.l
Lipofectamine 2000 (Invitrogen). Forty-eight hours after
transfection cells were viewed to determine the approximate number
of green fluorescent foci using an Olympus IX50 microscope.
pcDNA3.1-72Httexon1-eGFP (p72Q) is a construct that contains a
fusion gene uniting the first exon of the HD gene containing a
polyQ repeat of 72 codons and the eGFP gene. This fragment of
huntingtin differs from GST-Q58-Htn in both length of polyglutamine
stretch and that it is fused to eGFP rather than GST. When
transfected into HEK293 cells, the gene is expressed and aggregates
appear within 12 hours, reflected by the appearance of discrete
green foci. Cells were photographed first under white light to
verify that equal numbers of cells were present for each treatment
and a representative sample is shown. eGFP foci were then imaged in
the presence or absence of plasmid p72Q. In FIG. 8, green
fluorescent foci are evident when p72Q is present but a significant
reduction is seen in cells that have also received Congo Red.
Importantly, inhibition of aggregate formation is only partially
inhibited when a lower dosage of Congo Red is present,
demonstrating a dose-dependent effect. In FIG. 9, a cell population
in which HDA (SEQ ID NO:9) was co-transfected with p72Q is
presented; HDA appears to have had little effect on the number of
aggregates formed in these cells. As is the case in the biochemical
assay (FIG. 5), HDA does not appear to inhibit aggregate formation
in HEK293 cells. FIG. 10 illustrates the effect of HDG on aggregate
formation. The white light photograph again reveals that HDG has no
detectable toxic effect on cells or cell growth at 750 nM (top left
panel), but a clear dose effect is seen on the number of aggregates
when the level of HDG is increased (bottom panels). These
observations confirm results obtained in the biochemical assay
using HDG as the inhibitor. Finally, in FIG. 11, a panel of
photographs reveals once again that HDG is an effective inhibitor
of aggregate formation but that this positive activity can be
reduced significantly when T residues are inserted at the 7th and
14th position of the HDG 20-mer (HDG 20/7; SEQ ID NO:7), every
third base of the HDG 20-mer (HDG 20/3; SEQ ID NO:5) or every other
base of the HDG 20-mer (HDG 20/2; SEQ ID NO:4). To validate these
results and to further explore the relationship between the
cell-based, and biochemical assays, we assayed HDG 20/7, HDG 20/4
and HDG 20/3 individually for activity in the immunoblot assay (see
FIG. 2B). These results confirm the low level of activity observed
for HDG 20/7, HDG 20/3 and HDG 20/2 respectively in the cell-based
assay (FIG. 12). The correlation between the results obtained in
the cell-based and immunoblot assay reveal a similar mode of action
for the ODNs. We have preliminary evidence that the reduction in
aggregates observed in the cell-based assay can be confirmed when
aggregates isolated from transfected cells are passed through the
immunoblot assay.
[0155] Aggregate reduction in response to the addition of HDG (SEQ
ID NO:3) can also be seen using FACS analysis as the readout.
Again, HEK293 cells were transfected with p72Q with or without HDG
(or HDA; SEQ ID NO:9) and the reactions were allowed to proceed for
48 hours. The cells were then processed for FACS and measured for
green fluorescence. The Y axis reflects the degree or intensity of
fluorescence. As seen in FIG. 13A, the background is gated at the
far left of the graphic whereas expression of p72Q produces a sharp
peak of green fluorescence near the right edge of the profile (FIG.
13B). This peak represents aggregated Htt-eGFP, scored by FACS as
cells containing high intensity eGFP (aggregates). In FIGS. 13C and
13D, the profile of cells treated with HDA is represented and
little detectable change is observed in the peak at the far right
edge. Even as the level of HDA is increased from 1 .mu.M to 2.5
.mu.M, no significant reduction in aggregates is observed. In
contrast, cells treated with HDG exhibit a very different profile
(FIGS. 13E and 13F) as the peak representing aggregates is
diminished in a dose-dependent fashion. Thus, taken together, the
data suggest that HDG can inhibit aggregation formation in HEK293
cells expressing the Htt-eGFP fusion protein from plasmid p72Q.
[0156] Finally, a derivative PC12 cell line, Htt14A2.6, was used to
measure the capacity of HDG to improve cellular viability. This
neuronal cell line is used as a standard in the field for studying
the survival phenotype associated with aggregate formation. In this
assay, a truncated form of Htt exon 1 (103Q) fused to enhanced
green fluorescent protein (eGFP) is induced to express by addition
of muristerone to the culture. After induction, cell viability
decreases rapidly between 48 hours and 72 hours, respectively, as
measured by a CellTiter-Glo Luminescent cell viability assay, as
shown in FIG. 14. The addition of increasing doses of HDG (SEQ ID
NO:3) (0.4 -1.6 .mu.g/.mu.l) appears to arrest the drop in
viability providing some level of neuroprotection. The differences
in these are statistically significant and a larger, survival study
is underway to confirm and/or expand upon these results.
[0157] Circular dichroism spectroscopy. Circular dichroism spectra
of 15 .mu.M ODN samples in 10 mM KCl were recorded on an Aviv model
202 spectrometer. Measurements were performed at 24.degree. C.
using a 0.1 cm path-length quartz cuvette (Hellma). The CD spectra
were obtained by taking the average of two scans made at 1 nm
intervals from 200 to 320 nm and subtracting the baseline value
corresponding to that of buffer alone. Spectral data are expressed
in units of millidegree.
[0158] PC12 viability assay. Rat pheochromocytoma cells, PC12, were
grown in high glucose DMEM with 10% horse serum and 5% FBS while
under selection with G418 (0.05 mg/mL) and Zeocin (0.1 mg/mL)
(Invitrogen). This cell line, Htt14A2.6, expresses a truncated form
of expanded repeat Htt exon 1 protein containing 1-17 amino acids
and 103 polyglutamine tract fused to eGFP. The promoter was induced
with muristerone resulting in the expression of the Htt exon 1 with
expanded 103 CAG polyglutamine (103Q) region. Cells were seeded at
3.times.104 cells/well on a 24-well plate and transfected with a
ratio of 0.8 .mu.g HDG 20 to 2 .mu.L Lipofectamine 2000
(Invitrogen) depending on the desired HDG concentration. After a
4-hour treatment, the transfection media was removed, whole media
was added for 1-hour, and then the cells were induced using 5 .mu.M
muristerone for 24 hours. The Promega CellTiter-Glo Luminescent
cell viability assay was then used. The control cells using only
Lipofectamine 2000 were counted and plated at 2.times.104 cells in
at least 6 wells of a 96-well plate. The same volume of cells used
in this control at 24-hours, was used in the following treatments
at that time point and the remaining 48 and 72-hour time points.
After the cells were replated, an equal amount of cell viability
substrate was added to each well, according to protocol. After the
substrate is added, the plate was placed on a rocker for 2 mins
then incubated for 10 mins. Finally, the plate was read 3 times per
treatment on a Victor3V 1420 Multilabel counter and analyzed using
the Wallac 1420 software.
Example 4
[0159] Employment of quantitative polymerase chain reaction to
infer transcript levels of mutant and wild type huntingtin.
Real-time quantitative polymerase chain reaction (RT-qPCR), is an
adaptation of PCR in which the real time expression of transcript
level can be accurately and precisely measured. In order to utilize
this technique for the purpose of looking at huntingtin transcript
levels a Qiagen RNeasy.TM. mini kit was used in order to isolate
RNA from human cells. With the intention of performing qPCR, cells
were cultured with a huntingtin expressing plasmid with or without
treatment of oligonucleotides. This was accomplished by the
utilization of human embryonic kidney cells, HEK293T. HEK293T cells
were originally developed by Michelle Calos at Stanford University
and stably express the large T antigen of SV40. These cells are
grown in low glucose Dulbecco's Modified Eagle's Medium, DMEM,
media supplemented with penicillin and streptomycin as wells as 10%
fetal bovine serum. Cells were plated in 6-well plate at 0.3 to
0.7.times.10.sup.6 cells per ml. The number of cells that are
originally plated differs due to the passage number of the HEK293T
cells, in light of the fact that later passages grow at a faster
rate then lower passage numbers.
[0160] The cells are plated and allowed to adhere for 24 hours.
After the HEK293T cells had attached to the plate and are
approximately 50 to 90 percent confluent they are transfected with
2.5 .lamda. Lipofectamine.TM. 2000. Lipofectamine.TM. 2000
transfection reagent was purchased by Invitrogen and provides high
transfection efficiency for HEK cells. The HEK293T cells were
transfected with 1 .mu.g of plasmid that expresses the mutant
huntingtin fragment that contained a polyglutamine repeat of 72 or
a wild type fragment containing a polyglutamine repeat of 25 as
well as 0.5 .mu.g to 2.5 .mu.g of our GRO or control
oligonucleotide. The plasmid is designated pcDNA3.1
-72httexon-1-egfp and expresses a fusion protein of huntingtin that
contains a polyglutamine expansion, 25 or 72, fused to eGFP on the
C terminal end of the protein. Transfection of the HEK293T cells by
the plasmid pcDNA3.1-72httexon-1-egfp, pQ72, at 24 hours post
plating results in efficient transfection that can be used for
QPCR.
[0161] After HEK293T cells have been incubated for 48 hours in an
incubator at 37 degrees Celsius, RNA is isolated from each well.
Using a pipette, cells from each individual well are resuspended
and detached from the surface by simply pipetting up and down.
These cells are place in 15 ml tubes and spun at 3,750 rpm in an
eppendorf centrifuge 5804. Once the cells have been pelleted, the
pellets are resuspended in a mixture of 350 .lamda. of resuspension
lysis buffer (RLT) and 3.5 .lamda. of .beta.-mercaptoethanol. This
cell lysate was then applied to a QIAshredder spin column and
placed in a 2 ml collection tube.
[0162] The lysate was spun at 13,000 rpm for 2 minutes in an
eppendorf centrifuge 5415D. The cell homogenized lysate was then
mixed with equal volume of 70% ethanol solution in order to
precipitate the RNA/DNA out of solution. This mixture was then
applied to an RNeasy.TM. spin column and spun at 13,000 rpm for 15
seconds in order to apply the RNA/DNA mixture to the filter. The
flow through was discarded and the RNeasy.TM. spin column was
washed with buffer (RW1). The sample is then treated with DNase
(Qiagen RNase-Free Dnase I) for 15 minutes at room temperature to
remove any genomic DNA contamination. An addition wash and spin is
then required before 2 additional washes are performed with an
ethanol rich wash buffer (RPE), used to wash and maintain RNA
stability. The column is finally incubated with DepC treated
H.sub.2O for 5 minutes then spun at 13,000 rpm for one minute to
elute RNA into a fresh 1.5 ml eppendorf tube. This is repeated one
additional time and then the RNA quantity is determined by use of a
NanoDrop ND-1000 spectrophotometer.
[0163] Reverse transcriptase was used performed on the previously
isolated RNA by invitrogen using a Superscript.TM. First-strand
synthesis system for RT-PCR. 1 ug of RNA was used to be converted
to first strand cDNA by use of the following procedure. 1 ug of RNA
was combined with random hexamers and dNTP's for a final volume of
10 microliters then denatured for 5 minutes at 65 degrees C. cDNA
synthesis mix was then prepared: 10.times.RT buffer, 25 mM MgCL2,
0.1M DTT, RNase OUT.TM., and superscript II RT, and incubated with
the denatured sample. cDNA synthesis was performed and the reaction
was terminated as according to the manufactures protocol.
[0164] RT-qPCR amplification was performed using primers against
eGFP and using Beta-2-microglobulin, a component of major
histocompatibility complex, class I molecules as well as
.beta.-actin, one of six different actin isoforms, as endogenous
controls. SYBR.RTM. Green PCR Master Mix from AB Applied Biosystems
was combined with the eGFP primers and the previously prepared RT
sample and added to applied Biosystems MicroAmp.TM. optical 96-well
reaction plates. These places are then added to an Applied
Biosystems 7300 Real time PCR system and the data was collected
using applied biosystems sequence detection software version
1.3.1.
REFERENCES
[0165] The following references are incorporated herein by
reference in their entirety for all purposes. [0166] 1. Landles C,
Bates G P: Huntingtin and the molecular pathogenesis of
Huntington's disease. Fourth in molecular medicine review series.
EMBO Rep. 2004, 5:958-963. [0167] 2. DiFiglia M, Sapp E, Chase K O,
Davies S W, Bates G P, Vonsattel J P, Aronin N:
[0168] Aggregation of huntingtin in neuronal intranuclear
inclusions and dystrophic neurites in brain. Science 1997,
277:1990-1993. [0169] 3. Scherzinger E, Sittler A, Schweiger K,
Heiser V, Lurz R, Hasenbank R, Bates GP, Lehrach H, Wanker E E:
Self-assembly of polyglutamine-containing huntingtin fragments into
amyloid-like fibrils: implications for Huntington's disease
pathology. Proc.Natl.Acad.Sci. U.S.A 1999, 96:4604-4609. [0170] 4.
Scherzinger E, Lurz R, Turmaine M, Mangiarini L, Hollenbach B,
Hasenbank R, Bates G P, Davies S W, Lehrach H, Wanker E E:
Huntingtin-encoded polyglutamine expansions form amyloid-like
protein aggregates in vitro and in vivo. Cell 1997, 90:549-558.
[0171] 5. Sanchez I, Mahike C, Yuan J: Pivotal role of
oligomerization in expanded polyglutamine neurodegenerative
disorders. Nature 2003, 421:373-379. [0172] 6. Slow E J, Graham R
K, Osmand A P, Devon R S, Lu G, Deng Y, Pearson J, Vaid K, Bissada
N, Wetzel R, Leavitt B R, Hayden M R: Absence of behavioral
abnormalities and neurodegeneration in vivo despite widespread
neuronal huntingtin inclusions. Proc.Natl.Acad.Sci.U.S.A 2005,
102:11402-11407. [0173] 7. Jing N, Li Y, Xu X, Sha W, Li P, Feng L,
Tweardy D J: Targeting Stat3 with G-quartet oligodeoxynucleotides
in human cancer cells. DNA Cell Biol. 2003, 22:685-696. [0174] 8.
Jing N, De Clercq E, Rando R F, Pallansch L, Lackman-Smith C, Lee
S, Hogan M E:
[0175] Stability-activity relationships of a family of G-tetrad
forming oligonucleotides as potent HIV inhibitors. A basis for
anti-HIV drug design. J.Biol.Chem. 2000, 275:3421-3430. [0176] 9.
Mazumder A, Neamati N, Ojwang J O, Sunder S, Rando R F, Pommier Y:
Inhibition of the human immunodeficiency virus type 1 integrase by
guanosine quartet structures. Biochemistry 1996, 35:13762-13771.
[0177] 10. Sen D, Gilbert W: A sodium-potassium switch in the
formation of four-stranded G4-DNA. Nature 1990, 344:410-414. [0178]
11. Arrasate M, Mitra S, Schweitzer E S, Segal M R, Finkbeiner S:
Inclusion body formation reduces levels of mutant huntingtin and
the risk of neuronal death. Nature 2004, 431:805-810. [0179] 12.
Kim M, Lee H S, Laforet G, McIntyre C, Martin E J, Chang P, Kim T
W, Williams M, Reddy P H, Tagle D, Boyce F M, Won L, Heller A,
Aronin N, DiFiglia M: Mutant huntingtin expression in clonal
striatal cells: dissociation of inclusion formation and neuronal
survival by caspase inhibition. J.Neurosci. 1999, 19:964-973.
[0180] 13. Saudou F, Finkbeiner S, Devys D, Greenberg M E:
Huntingtin acts in the nucleus to induce apoptosis but death does
not correlate with the formation of intranuclear inclusions. Cell
1998, 95:55-66. [0181] 14. Klement I A, Skinner P J, Kaytor M D, Yi
H, Hersch S M, Clark H B, Zoghbi H Y, Orr H T: Ataxin-1 nuclear
localization and aggregation: role in polyglutamine-induced disease
in SCA1 transgenic mice. Cell 1998, 95:41-53. [0182] 15. Stenoien D
L, Cummings C J, Adams H P, Mancini M G, Patel K, DeMartino G N,
Marcelli M, Weigel N L, Mancini M A: Polyglutamine-expanded
androgen receptors form aggregates that sequester heat shock
proteins, proteasome components and SRC-1, and are suppressed by
the HDJ-2 chaperone. Hum.Mol.Genet. 1999, 8:731-741. [0183] 16.
Bowman A B, Yoo S Y, Dantuma N P, Zoghbi H Y: Neuronal dysfunction
in a polyglutamine disease model occurs in the absence of
ubiquitin-proteasome system impairment and inversely correlates
with the degree of nuclear inclusion formation. Hum.Mol.Genet.
2005, 14:679-691. [0184] 17. Parekh-Olmedo H, Wang J, Gusella J F,
Kmiec E B: Modified single-stranded oligonucleotides inhibit
aggregate formation and toxicity induced by expanded polyglutamine.
J.Mol.Neurosci. 2004, 24:257-267. [0185] 18. Huang C C, Faber P W,
Persichetti F, Mittal V, Vonsattel J P, MacDonald M E, Gusella J F:
Amyloid formation by mutant huntingtin: threshold, progressivity
and recruitment of normal polyglutamine proteins. Somat. Cell Mol.
Genet. 1998, 24:217-233. [0186] 19. Wang J, Gines S, MacDonald M E,
Gusella J F: Reversal of a full-length mutant huntingtin neuronal
cell phenotype by chemical inhibitors of polyglutamine-mediated
aggregation. BMC.Neurosci. 2005, 6:1. [0187] 20. Macaya R F,
Schultze P, Smith F W, Roe J A, Feigon J: Thrombin-binding DNA
aptamer forms a unimolecular quadruplex structure in solution.
Proc.Natl.Acad.Sci. U.S.A 1993, 90:3745-3749. [0188] 21. Heiser V,
Scherzinger E, Boeddrich A, Nordhoff E, Lurz R, Schugardt N,
Lehrach H, Wanker E E: Inhibition of huntingtin fibrillogenesis by
specific antibodies and small molecules: implications for
Huntington's disease therapy. Proc.Natl.Acad.Sci. U.S.A 2000,
97:6739-6744. [0189] 22. Hardin C C, Henderson E, Watson T, Prosser
J K: Monovalent cation induced structural transitions in telomeric
DNAs: G-DNA folding intermediates. Biochemistry 1991, 30:4460-4472.
[0190] 23. Balagurumoorthy P, Brahmachari S K: Structure and
stability of human telomeric sequence. J.Biol.Chem. 1994,
269:21858-21869. [0191] 24. Balagurumoorthy P, Brahmachari S K,
Mohanty D, Bansal M, Sasisekharan V: Hairpin and parallel quartet
structures for telomeric sequences. Nucleic Acids Res. 1992,
20:4061-4067. [0192] 25. Apostol B L, Kazantsev A, Raffioni S,
Illes K, Pallos J, Bodai L, Slepko N, Bear J E, Gertler F B, Hersch
S, Housman D E, Marsh J L, Thompson L M: A cell-based assay for
aggregation inhibitors as therapeutics of polyglutamine-repeat
disease and validation in Drosophila. Proc.Natl.Acad.Sci. U.S.A
2003, 100:5950-5955. [0193] 26. Yang W, Dunlap J R, Andrews R B,
Wetzel R: Aggregated polyglutamine peptides delivered to nuclei are
toxic to mammalian cells. Hum.Mol.Genet. 2002, 11:2905-2917. [0194]
27. Chen S, Berthelier V, Hamilton J B, O'Nuallain B, Wetzel R:
Amyloid-like features of polyglutamine aggregates and their
assembly kinetics. Biochemistry 2002, 41:7391-7399. [0195] 28.
Zakian V A: Telomeres: beginning to understand the end. Science
1995, 270:1601-1607. [0196] 29. Sun D, Thompson B, Cathers B E,
Salazar M, Kerwin S M, Trent J O, Jenkins T C, Neidle S, Hurley L
H: Inhibition of human telomerase by a G-quadruplex-interactive
compound. J.Med.Chem. 1997, 40:2113-2116. [0197] 30. Fedoroff O Y,
Salazar M, Han H, Chemeris V V, Kerwin S M, Hurley L H: NMR-Based
model of a telomerase-inhibiting compound bound to G-quadruplex
DNA. Biochemistry 1998, 37:12367-12374. [0198] 31. Bates G:
Huntingtin aggregation and toxicity in Huntington's disease. Lancet
2003, 361:1642-1644. [0199] 32. Ross C A, Poirier M A, Wanker E E,
Amzel M: Polyglutamine fibrillogenesis: the pathway unfolds.
Proc.Natl.Acad.Sci. U.S.A 2003, 100:1-3. [0200] 33. Dapic V,
Abdomerovic V, Marrington R, Peberdy J, Rodger A, Trent J O, Bates
P J:
[0201] Biophysical and biological properties of quadruplex
oligodeoxyribonucleotides. Nucleic Acids Res. 2003, 31:2097-2107.
[0202] 34. Jing N, Sha W, Li Y, Xiong W, Tweardy D J. Rational drug
design of G-quartet DNA as anti-cancer agents. Curr Pharm Des.
2005;11(22):2841-54. Review. [0203] 35. Jing N, Li Y, Xiong W, Sha
W, Jing L, Tweardy D J. G-quartet oligonucleotides: a new class of
signal transducer and activator of transcription 3 inhibitors that
suppresses growth of prostate and breast tumors through induction
of apoptosis. Cancer Res. 2004 Sep. 15;64(18):6603-9. [0204] 36.
Biyani and Nisigaki, Structural Characterization of Ultra-Stable
Higher-Ordered Aggregates Generated by Novel Guanine-rich DNA
Sequences. Gene 2005; 364: 130-38.
Sequence CWU 1
1
12 1 16 DNA Artificial Chemically Synthesized 1 gggtgggtgg gtgggt
16 2 24 DNA Artificial Chemically Synthesized 2 gggggtgggg
gtgggggtgg gggt 24 3 20 DNA Artificial Chemically Synthesized 3
gggggggggg gggggggggg 20 4 20 DNA Artificial Chemically Synthesized
4 gtgtgtgtgt gtgtgtgtgt 20 5 20 DNA Artificial Chemically
Synthesized 5 ggtggtggtg gtggtggtgg 20 6 20 DNA Artificial
Chemically Synthesized 6 gggtgggtgg gtgggtgggt 20 7 20 DNA
Artificial Chemically Synthesized 7 ggggggtggg gggtgggggg 20 8 20
DNA Artificial Chemically Synthesized 8 cccccccccc cccccccccc 20 9
20 DNA Artificial Chemically Synthesized 9 aaaaaaaaaa aaaaaaaaaa 20
10 20 DNA Artificial Chemically Synthesized 10 tttttttttt
tttttttttt 20 11 26 DNA Artificial Chemically Synthesized 11
ggtggtggtg gttgtggtgg tggtgg 26 12 29 DNA Artificial Chemically
Synthesized 12 tttggtggtg gtggttgtgg tggtggtgg 29
* * * * *