HUNTINGTON'S DISEASE

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. provisional patent application Ser. No. 62/455,302, filed February 6, 2017, and titled "DNAzyme Based Methods and Compositions for Treating Huntington's Disease." The entire disclosure of the above- identified priority application is hereby fully incorporated herein by reference.

STATEMENT OF GOVERNMENT SUPPORT

[0002] This invention was made with government support under grant number CA107183 awarded by National Institutes of Health. The government has certain rights in the invention.

TECHNICAL FIELD

[0003] The present disclosure relates generally to treating Huntington's disease, and more particularly to the use of DNAzymes to reduce production of mutant huntingtin protein in subjects with Huntington's disease.

BACKGROUND

[0004] Huntington's disease (HD) is an inherited, neurodegenerative disorder that is characterized by profound affective, cognitive, behavioral, and motor dysfunctions. Affecting males and females at the same frequency, the mean age of HD onset is around 40 although it can be as early as 4 and as late as 80 years of age. Epidemiologic studies show that, in the US, there are about 30,000 HD patients and that there are about 150,000 people at risk of developing the disease. Neuron loss is progressive, and the dysfunction and loss of neurons account for the cognitive and motor decline.

[0005] The basis of HD is a CAG repeat expansion to >35 CAG in the gene that codes for a ubiquitous protein known as huntingtin, resulting in an abnormally long polyglutamine tract in the protein N-terminus. Synthesis of the mutant protein leads to protein mis-folding and protein aggregation in the affected individual. This in turn is believed to induce the neuronal disorders and other complications in HD. [0006] At present, no treatments are available to alter the progressive course of HD. Rather, treatment is currently aimed at symptom management, as certain medications can lessen some symptoms of movement and psychiatric disorders. Potential treatments, such as treatment with the drug geldanamycin, may attempt to mitigate the effects of protein aggregation. Other potential treatments target the role of apoptosis in the clinical manifestations of the disease. For example, the compounds zVAD-fmk and minocycline, both of which inhibit caspase activity, have been shown to slow disease manifestation in mice. The drug remacemide has also been tested in small human trials, but unfortunately no statistically significant improvements were seen in neuron function in these trials. Likewise, in studies using Coenzyme Q, there was no significant change in the rate of decline of total functional capacity.

[0007] A need thus exists for methods to treat HD. More particularly, a need exists to effectively silence or reduce the expression of mutant huntingtin protein in individuals with Huntington's disease.

SUMMARY

[0008] In certain example aspects, provided is a method of modulating cellular expression of mutant huntingtin protein, the method comprising contacting a cell with at least one DNA oligonucleotide having binding specificity for a target region of a messenger ribonucleotide (mRNA) encoding the mutant huntingtin protein. For example, the cell can be exposed to the DNA oligonucleotide. The DNA oligonucleotide, for example, can include a nucleotide sequence that is identical to the any one of the nucleotide sequences set forth as SEQ ID NOS: l-4. In certain example aspects, the DNA oligonucleotide can include a nucleotide sequence that is about 80%, 85%, 90%, 95%, 98%, or 99% or more identical to the any one of the nucleotide sequences set forth as any one of SEQ ID NOS: 1-4.

[0009] In certain example aspects, the method for modulating the cellular expression of the mutant huntingtin protein includes reducing or inhibiting the expression of the mutant huntingtin protein. In certain example aspects, the DNA oligonucleotide is a catalytic antisense polynucleotide. In certain example aspects, the DNA oligonucleotide is a DNAzyme. The DNA oligonucleotide can also be modified, for example, to increase the stability of the DNA oligonucleotide. In certain example aspects, contacting the cell with the DNA oligonucleotide results in introduction of the DNA oligonucleotide into the contacted cell. In certain example aspects, the target region of the mRNA includes a mutant huntingtin exon 1 mRNA. In certain example aspects, contacting the cell with the DNA oligonucleotide results in a reduction in mutant huntingtin protein and mRNA.

[0010] In certain example aspects, provided is a method for treating Huntington's disease in a subject. In certain example aspects, a subject having Huntington's disease can be selected. In certain example aspects, the subject can be tested to determine whether the subject has Huntington's disease before administering the at least one DNA oligonucleotide. The treatment method includes, for example, administering to a subject at least one DNA oligonucleotide having binding specificity for a target region of a messenger ribonucleotide (mRNA) encoding the mutant huntingtin protein. In certain example aspects, a therapeutically effective amount of one or more DNA oligonucleotides are administered to the subject. The DNA oligonucleotide, for example, can include a nucleotide sequence that is identical to any one of the nucleotide sequences set forth as SEQ ID NOS: l-4. In certain example aspects, the DNA oligonucleotide can include a nucleotide sequence that is about 80%, 85%, 90%, 95%, 98%, or 99% or more identical to the any one of the nucleotide sequences set forth as any one of SEQ ID NOS: l-4.

[0011] In certain example aspects, the method of treatment includes reducing or inhibiting the expression of the mutant huntingtin protein in the subject. In certain example aspects, the administered DNA oligonucleotide is a catalytic antisense polynucleotide. In certain example aspects, the administered DNA oligonucleotide is a DNAzyme. The administered DNA oligonucleotide can also be modified, for example, to increase the stability of the DNA oligonucleotide before the DNA oligonucleotide is administered. In certain example aspects, the target region of the mRNA includes a mutant huntingtin exon 1 mRNA.

[0012] In certain example embodiments, provided is an enzymatic DNA molecule that includes a polynucleotide sequence having binding specificity for a target region of a messenger ribonucleotide (mRNA) encoding a mutant huntingtin protein. The DNA molecule, for example, can be a catalytic antisense polynucleotide. The enzymatic DNA molecule, for example, can include a nucleotide sequence that is identical to the any one of the nucleotide sequences set forth as SEQ ID NOS: l-4. In certain example aspects, the enzymatic DNA molecule can include a nucleotide sequence that is about 80%, 85%, 90%, 95%, 98%, or 99% or more identical to the any one of the nucleotide sequences set forth as any one of SEQ ID NOS: l-4. In certain example aspects, the enzymatic DNA molecule can include a catalytic region that is identical to or 80%, 85%, 90%, 95%, 98%, or 99% identical to the nucleotide sequence set forth as SEQ ID NO: 7. In certain example aspects, the enzymatic DNA molecule includes a modification that increases the stability of the DNA molecule. For example, the enzymatic DNA molecule can include an inverted deoxythymidine at the 3' end of the DNA molecule. In certain example aspects, the target region of the mRNA includes a mutant huntingtin exon 1 mRNA.

[0013] In certain example aspects, provided is a composition that includes a DNA oligonucleotide having binding specificity for a target region of a messenger ribonucleotide (mRNA) encoding a mutant huntingtin protein. The DNA oligonucleotide can be a catalytic antisense polynucleotide, such as a DNAzyme. The DNA oligonucleotide, for example, can include a nucleotide sequence that is identical to the any one of the nucleotide sequences set forth as SEQ ID NOS: l-4. In certain example aspects, the DNA oligonucleotide can include a nucleotide sequence that is about 80%, 85%, 90%, 95%, 98%, or 99% or more identical to the any one of the nucleotide sequences set forth as any one of SEQ ID NOS: l-4. In certain example aspects, the DNA oligonucleotide can include a catalytic region that is identical to or 80%, 85%, 90%, 95%, 98%, or 99% identical to the nucleotide sequence set forth as SEQ ID NO: 7. In certain example aspects, the DNA oligonucleotide includes a modification that increases the stability of the DNA oligonucleotide, such as an inverted deoxythymidine at the 3' end of the DNA oligonucleotide. In certain example aspects, the target region of the mRNA includes a mutant huntingtin exon 1 mRNA.

[0014] In certain example aspects, the methods and compositions described herein may rely on and/or include a DNA oligonucleotide that is identical to— or 80%, 85%, 90%, 95%, 98%, or 99% identical— to the nucleic acid sequences set forth as either SEQ ID NOS: 5-6. For example, the compositions described herein may include a DNA oligonucleotide that has the same sequence as SEQ ID NO: 6 or SEQ ID NO: 7 or that shares 80%, 85%, 90%, 95%, 98%, or 99% identity with these sequences. In such examples aspects, the DNA oligonucleotide can include a catalytic region that is identical to - or 80%, 85%, 90%, 95%, 98%, or 99% identical to - the nucleotide sequence set forth as SEQ ID NO: 7.

[0015] These illustrative features and aspects are mentioned not to limit or define the disclosure, but to provide examples to aid understanding thereof. Additional embodiments are discussed in the Detailed Description, and further description is provided there.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate certain aspects of the instant invention and, together with the description, serve to explain, without limitation, the principles of the invention.

[0017] FIG. 1 is schematic diagram illustrating binding of a DNAzyme to a corresponding sequence in a target mRNA via the binding domain (step 1), followed by cleavage of the target mRNA by the catalytic domain (step 2), in accordance with certain example embodiments.

[0018] FIG. 2 is an image of a polyacrylamide-urea gel showing the effect of six DNAzymes on mRNA for human mutant exon 1 of the huntingtin protein, in accordance with certain example embodiments. Six different DNAzymes (abbreviated DNZ) were constructed against the part of the huntingtin (Htt) mRNA transcript derived from human mutant exon 1, and tested in vitro for their ability to cleave mutant exon 1 mRNA. These include DNZ1 (SEQ ID NO: l), DNZ2 (SEQ ID NO:2), DNZ3 (SEQ ID NO:5), DNZ4 (SEQ ID NO:6), DNZ5 (SEQ ID NO: 3), and DNZ6 (SEQ ID NO: 4). Lane 2 shows the band for mutant Htt exon 1 signal. DNZ3 and DNZ4 (lanes 6 and 7) did not cleave exon 1 RNA, as there were no cleavage products evident. Although DNZ1 and DNZ2 (lanes 4 and 5) did cleave exon 1 RNA, the efficiency was low. By contrast, DNZ5 and DNZ6 (lanes 8 and 9) cleaved exon 1RNA with high efficiency, as shown by the near complete absence of the exon 1 RNA band, and its replacement by two lower MW cleavage products (arrows). Note that each effective DNAzyme cleaves mutant exon 1 mRNA at a different site, yielding differences in cleavage product size.

[0019] FIG. 3 is a graph depicting the results of DNZ6 administration on weight drop in wild-type mice and R6/2 transgenic mice, in accordance with certain example embodiments. R6/2 transgenic mice are transgenic for the promoter and exon 1 of a human HD gene with about 125 CAG repeats. Weight gain in PBS-treated WT mice, PBS-treated R6/2 mice, low-dose DNZ6-treated mice, beginning with the initiation of intraperitoneal injections at day 32 of age and ending at day 75 are shown. Mice were injected 5 out of every 7 days. Note that low dose DNZ6 prevented the weight drop seen in R6/2 mice that begins around day 65 of age. [0020] FIG. 4A is a graph showing the results of low dose DNZ6 administration on the clasping motor abnormality in R6/2 transgenic mice as compared to wild-type (WT) mice, in accordance with certain example embodiments. Clasping was scored on a 0 (absent) to 3 (maximal) scale in vehicle-treated (n=3) and low-dose DNZ6 treated (n=4) R6/2 mice, beginning at 32 days of age. Clasping was evident in both R6/2 groups by the age of 50 days and increased in severity progressively. All PBS (vehicle) treated R6/2 mice showed maximal clasping severity by 75 days, but clasping was less severe in low dose DNZ6 R6/2 mice after 75 days. Note that WT mice do not clasp and score at zero.

[0021] FIG. 4B is a graph showing the results of high dose DNZ6 administration on the clasping motor abnormality in R6/2 transgenic mice, in accordance with certain example embodiments. Clasping was scored on a 0 (absent) to 3 (maximal) scale in vehicle-treated (n=3) and high dose DNZ6 treated (n=4) R6/2 mice, beginning 36 days of age. As shown, clasping was reduced in high-dose DNZ6 mice already at 45 days of age, and was significant after 57 days of age.

[0022] FIG. 5 is a graph showing the results of low and high dose DNZ6 administration on open field motor performance in R6/2 transgenic mice and wild-type (WT) mice, in accordance with certain example embodiments. More particularly, the benefit in open field of low dose (n=7) versus high dose DNZ6 (n=16) compared to vehicle (n=15) in R6/2 mice treated daily from 36 to 70 days of age was examined. High dose DNZ6 mice were not statistically different than WT mice treated with vehicle (n=6) in distance traveled, maximum speed, turn rate, turn radius or arena center avoidance. By contrast, both low dose and vehicle-treated R6/2 mice generally performed significantly more poorly (asterisks) or trended so (ampersand) compared to vehicle-treated WT mice - showing reduced distance traveled, reduced speed, increased turn rate (a sign of HD- like hyperkinesis), reduced turning ability and increased anxiety. Thus, high dose DNZ6 rescued R6/2 deficits and abnormalities in open field.

[0023] FIG. 6 is a series of confocal laser-scanning photomicrographs showing the results of high does DNZ6 administration on the formation of neuronal intranuclear aggregates in R6/2 transgenic mice, in accordance with certain example embodiments. Neuronal intranuclear aggregates of mutant huntingtin protein were visualized using a red fluorophore. The aggregates are evident as 2-3 μιη wide balls. Shown are cerebral cortex layers 2-4 (L2-4), cerebral cortex layers 5-6 (L5-6), and striatum from a PBS (vehicle) treated and a high dose DNZ6-treated R6/2 mouse. Note that the neuronal intranuclear inclusion ("Nil's") are fewer and smaller in the high dose DNZ6-treated R6/2 mouse.

[0024] FIG. 7 is a graph showing the results of high dose DNZ6 administration on Nil abundance, size, and overall mutant huntingtin protein burden in cerebral cortex and striatum of R6/2 transgenic mice, in accordance with certain example embodiments. More particularly, quantification of Nil abundance, size, and overall mutant huntingtin burden (area covered by Nils) in R6/2 mice treated daily with PBS (vehicle) (n=3) and R6/2 mice treated daily with high dose DNZ6 (n=2) from 5 to 10 weeks of age was examined. As shown, high dose DNZ6 reduces the abundance, size and overall brain burden of mutant huntingtin protein aggregates (Nils) in cerebral cortex and striatum.

[0025] FIG. 8 is a graph showing the results of daily systemic DNZ6 administration on mutant Htt mRNA production in brains of R6/2 transgenic mice, in accordance with certain example embodiments. More particularly, PCR was used to measure the effect of daily high dose DNZ6 on message for the mutant Htt exon-lexpressed in the brains of R6/2 mice treated daily with high dose DNZ6 (n=10) from 5 to 10 weeks of age compared to mice treated with vehicle (n=10). Mutant huntingtin message (Htt) relative to beta-actin expression was significantly (asterisk) reduced by DNZ6 treatment to about 50% of that in vehicle-treated R6/2 mice. The reductions in message for mutant Htt are consistent with, and explain, the reduction in mutant protein Nils observed by immunolabeling. In contrast, daily DNZ6 had no effect on message for endogenous mouse Htt (n=3 per group), demonstrating that DNZ6 is specific for human Htt.

[0026] FIG. 9 is a graph showing survival rate of mice following the administration of a DNZ6, in accordance with certain example embodiments. More particularly, during studies assessing benefit of either low or high dose DNZ6 compared to vehicle in R6/2 mice, some mice died before reaching their behavioral, histological, or biochemical endpoints. This allowed survival assessment in R6/2 mice treated daily from 50 to 76 days of age with vehicle (n=22) or DNZ6 (n=23). As shown, even with this limited age range and sample, DNZ6 administration appeared to provide a survival benefit, with more of the control R6/2 mice dying by 76 days of age than the DNZ6-treated mice.

[0027] FIG. 10 is an image of a 6% polyacrylamide-urea gel showing the effect of different solvents on activity of DNZ6 toward human Htt exon I mRNA, in accordance with certain example embodiments. As shown, DNZ6 is stable and active in a variety of solvents, in accordance with certain example embodiments. DNZ6 was incubated for 2 hr or over night in saline, PBS from different sources, or in water and tested in vitro for its ability to cleave mutant exonl mRNA. Note that contrary to inactive DNZ molecules i.e. scrambled Arm, scrambled active site and sense DNZ, DNZ 6 effectively cleaves mutant exon 1 mRNA, yielding different cleavage products (arrow) and solvents do not have any effect on the activity of DNZ toward cleaving Htt RNA.

[0028] FIG. 11 is a graph showing the effect of DNZ6 administration on rotarod open field performance, in accordance with certain example embodiments. Wild-type (WT) mice were treated with PBS (n=12) or high dose DNZ6 in PBS (n=7) from 36 to 66 days of age, and then tested behaviorally. DNZ6 had no significant effect on rotarod (RR) performance or on such open field parameters as distance traveled, maximum speed, number of stops of anxiety (i.e. avoiding the arena center).

[0029] FIG. 12 is a graph showing the effect of DNZ6 administration on serum inflammatory markers in wild-type (WT) mice, in accordance with certain example embodiments. Serum was collected from 4 of the WT mice treated with PBS and 4 of the WT mice treated with high dose DNZ6 after behavioral testing. As shown, ELISA analysis revealed no significant increase in the inflammatory markers IL6, IL-Ιβ, IFNy, MCP1, TNFa and IgG2a in the DNZ6-treated mice compared to PBS mice.

[0030] FIG. 13 is a pair of images showing liver histology in DNZ6 treated wild-type mice, in accordance with certain example embodiments. Mice were treated with PBS alone (left panel) or high dose DNZ6 (right panel) in PBS from day 36 to day 66. All major organs were collected, sectioned and analyzed for cytotoxicity by H&E staining. The results show no liver toxicity with chronic DNZ6 treatment.

[0031] FIG. 14 is a graph showing the time-dependent tissue distribution profile for systemically (i.p.) administered DNZ6 in healthy mice, in accordance with certain example embodiments. The systemic distribution of DNAzyme in mice [ ³⁵ S]- was determined by injecting naked [^^S]-DNZ6 via i.p. into healthy mice, and the amount, of radioactivity present in blood and different organs was measured as a function of time. As shown, [ ³⁵ S]-DNZ6 is distributed to all major organs and the majority of [ ³⁵ S]-DNZ6 is cleared in the first 24 hours following administration. Tissue distribution of [ ³⁵ S]-DNZ6 presented in average percentages revealed the order of accumulation to be Liver>Kidney> Lung> Heart=Brain>Spleen. [0032] FIG. 15 is a graph showing the elimination profile of DNZ6 in healthy mice, in accordance with certain example embodiments. During studies assessing the pharmacokinetics of DNZ6 in normal healthy mice, the rate of clearance of DNZ6 was also determined as a function of time. As shown, DNZ6 is mainly cleared in both the urine and feces, with the majority cleared via urine in the first 24 hours with approximately 45% of DNZ6 cleared via urine in the first 72 hours.

DETAILED DESCRIPTION OF THE EXAMPLE EMBODIMENTS

[0033] The embodiments described herein can be understood more readily by reference to the following detailed description, examples, and claims, and their previous and following description. Before the present system, devices, compositions and/or methods are disclosed and described, it is to be understood that this invention is not limited to the specific systems, devices, and/or compositions methods disclosed unless otherwise specified, as such can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting.

[0034] The following description of the invention is provided as an enabling teaching of the invention in its best, currently known aspect. Those skilled in the relevant art will recognize that many changes can be made to the aspects described, while still obtaining the beneficial results of the present invention. It will also be apparent that some of the desired benefits of the present invention can be obtained by selecting some of the features of the present invention without utilizing other features. Accordingly, those who work in the art will recognize that many modifications and adaptations to the present invention are possible and can even be desirable in certain circumstances and are a part of the present invention. Thus, the following description is provided as illustrative of the principles of the present invention and not in limitation thereof.

Overview

[0035] Provided herein are methods and compositions for treating Huntington's disease. For example, provided are compositions that include DNA oligonucleotides that have targeting specificity for RNA encoding mutant huntingtin protein. When introduced into a cell, such DNA oligonucleotides reduce expression of the mutant huntingtin protein in a cell. For example, the DNA oligonucleotide, functioning as an enzymatic DNA molecule or "DNAzyme," cleaves the RNA encoding the mutant mRNA protein, thereby rendering the mRNA incapable of expression. That is, the cleavage event renders the RNA non-functional and reduces or abrogates protein expression from that RNA. Hence, synthesis of the mutant huntingtin protein is selectively reduced or inhibited, in accordance with the methods and compositions described herein. By silencing or reducing the expression of the mutant huntingtin protein, the methods and compositions describe herein can be used to treat Huntington's disease, including adult and juvenile onset Huntington's disease.

[0036] More particularly, DNAzymes are catalytically active single-stranded DNA molecules that can be targeted to bind to and cleave specific disease-related mRNAs, and thus decrease the level of disease-related or disease-causing protein. DNAzymes typically include two binding domains flanking a central catalytic domain, which consists of a specific sequence of 15 deoxynucleotides that is employed in all DNAzymes (FIG. 1). In contrast, the binding domains that flank the catalytic domain are variable and can be engineered to target DNAzyme action to the mRNA responsible for a specific protein. This variability thus provides the potential for specificity for binding to a target mRNA of interest by Watson-Crick base-pairing (FIG. 1).

[0037] After successful cleavage of a target mRNA molecule, the DNAzyme-RNA- complex dissociates, and the RNA cleavage products are further degraded by endogenous intracellular ribonuclease enzymes. The DNAzyme molecules are then available for subsequent binding and cleavage of additional RNA molecules. Hence, a relatively low concentration of DNAzymes is sufficient to efficiently reduce levels of specific disease- causing proteins and thereby abate disease.

[0038] In certain examples, provided herein are DNAzymes that cleave RNA coding for specific protein products related to Huntington's disease. For example, provided are DNAzymes that recognize the mRNA sequence encoding exon 1 of a human HD gene. Such DNAzymes act to cleave mRNA, thereby rendering the cleaved mRNA incapable of producing the mutant huntingtin protein. As such, provided herein is DNAzyme technology to reduce production of mutant huntingtin protein, the causal agent in Huntington's disease (HD).

[0039] The approaches described herein are advantageous over other methods currently under investigation, including antisense, shRNA, or siRNA technology as a gene therapy. For example, because DNAzymes are catalytically active, their dose- response efficacy is better than for antisense oligonucleotides (ASOs). Further, DNAzymes are stable and can be delivered repeatedly by systemic injections, while the ASOs require the riskier approach of intrathecal delivery.

[0040] Further, because systemic DNAzyme therapy can be discontinued at any time, adverse side effects can be more readily curtailed than with other gene therapy approaches. For example, viral delivery of knockdown constructs, as is the case for shRNA therapy, is permanent and any adverse side effects cannot be easily halted. Finally, because HD affects diverse body organs— including heart, muscle, pancreas and liver— systemic DNAzyme treatment, as described herein, may improve overall health in HD victims.

Summary of Terms

[0041] The invention will now be described in detail by way of reference only using the following definitions and examples. Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described. It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described, as these may vary.

[0042] The headings provided herein are not limitations of the various aspects or embodiments of the invention, which can be understood by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification as a whole.

[0043] As used herein, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise.

[0044] Ranges can be expressed herein as from "about" one particular value, and/or to "about" another particular value. When such a range is expressed, another aspect includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. [0045] As used herein, the terms "optional" or "optionally" mean that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0046] Terms used herein, such as "example," "exemplary," or "exemplified," are not meant to show preference, but rather to explain that the aspect discussed thereafter is merely one example of the aspect presented.

[0047] Additionally, as used herein, relative terms, such as "substantially," "generally," "approximately," "about," and the like are used herein to represent an inherent degree of uncertainty that can be attributed to any quantitative comparison, value, measurement, or other representation. These terms are also utilized herein to represent the degree by which a quantitative representation can vary from a stated reference without resulting in a change in the basic function of the subject matter at issue. In certain example embodiments, the term "about" is understood as within a range of normal tolerance in the art for a given measurement, for example, such as within 2 standard deviations of the mean. In certain example embodiments, depending on the measurement "about" can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein can be modified by the term about.

[0048] As used herein, "significance" or "significant" relates to a statistical analysis of the probability that there is a non-random association between two or more entities. To determine whether or not a relationship is "significant" or has "significance," statistical manipulations of the data can be performed to calculate a probability, expressed as a "p- value." Those p-values that fall below a user-defined cutoff point are regarded as significant. In one example, a p-value less than or equal to 0.05, in another example less than 0.01, in another example less than 0.005, and in yet another example less than 0.001, are regarded as significant.

[0049] As used herein, the term "enzymatic DNA molecule" is used to describe a DNA-containing molecule that is capable of functioning as an enzyme. The term "enzymatic DNA molecule" is inclusive of the terms "DNAzyme," "deoxyribozyme," and "catalytic DNA molecule," which terms should all be understood to include enzymatically active portions thereof. The term "enzymatic DNA molecule," as used herein, also includes DNA molecules that have complementary sequences in a substrate binding domain or region to a specified oligoribonucleotide target or substrate. Such molecules also have an enzymatic activity, which is active to specifically cleave an oligoribonucleotide substrate. That is, the enzymatic DNA molecule is capable of cleaving the oligoribonucleotide substrate intermolecularly. The complementarity functions to allow sufficient hybridization of the enzymatic DNA molecule to the substrate oligoribonucleotide at a target region to allow the intermolecular cleavage of the substrate to occur. While one-hundred percent (100%) complementarity is preferred, complementarity in the range of 70, 75, 80, 85, 90, 95, or 100% is also useful and contemplated with the various aspects and embodiments described herein.

[0050] As used herein, the phrase "target RNA" or "target region" of an RNA refers to an RNA molecule (for example, an mRNA molecule encoding the mutant huntingtin gene product) that is a target for downregulation. Similarly, the phrase "target site" refers to a sequence within a target RNA that is "targeted" for cleavage mediated by an enzymatic DNA molecule that contains sequences within its substrate binding domains that are complementary to the target site. Similarly, the phrase "target cell" refers to a cell that expresses a target RNA and into which an enzymatic DNA molecule is intended to be introduced. A target cell is in some embodiments a cell in a subject. For example, a target cell can comprise a cell that expresses mutant huntingtin protein gene.

[0051] The term "expression," "cellular expression," or "cellular gene expression" generally refers to the cellular processes by which a biologically active polypeptide is produced from a DNA sequence and exhibits a biological activity in a cell. As such, gene expression involves the processes of transcription and translation that can be regulated by post-transcriptional and post-translational processes, which influence a biological activity of a gene or gene product. These processes include, for example, RNA synthesis, processing, and transport, as well as polypeptide synthesis, transport, and post- translational modification of polypeptides. Additionally, processes that affect protein- protein interactions within the cell can also affect gene expression as defined herein.

[0052] As used herein, the term "modulate" refers to a change in the expression level of a gene or RNA molecules encoding one or more proteins or protein subunits, or activity of one or more proteins or protein subunits is up regulated or down regulated, such that expression, level, or activity is greater than or less than that observed in the absence of the modulator. For example, the term "modulate" can mean "inhibit", "suppress," or "activate", but the use of the word "modulate" is not limited to this definition.

[0053] As used herein, the terms "inhibit," "suppress," "down regulate," "reduce," "silence," and grammatical variants thereof are used interchangeably and refer to an activity whereby gene expression of and/or a level of an RNA encoding one or more gene products, such as the mutant huntingtin protein, is reduced below that observed in the absence of the DNA oligonucleotides described herein. In certain example embodiments, inhibition with a DNA oligonucleotide described herein results in a decrease in the steady state level of a target RNA, such as an mRNA. For example, inhibition with the DNA oligonucleotide described herein results in a decrease in the steady state of mRNA transcripts encoding mutant huntingtin protein.

[0054] In certain example embodiments, inhibition with a DNA oligonucleotide described herein results in an expression level of a target gene, such as the mutant huntingtin gene, that is below that level observed in the presence of an inactive or attenuated DNA oligonucleotide that is unable to modulate an inhibitory response. In certain example embodiments, inhibition of gene expression with a DNA oligonucleotide described herein is greater in the presence of the DNA oligonucleotide than in its absence of the DNA oligonucleotide. In certain example embodiments, inhibition of gene expression is associated with an enhanced rate of degradation of the mRNA encoded by the gene (for example, by enzymatic cleavage mediated by a DNAzyme described herein).

[0055] As used herein, the terms "gene" and "target gene" refer to a nucleic acid that encodes an RNA, for example, nucleic acid sequences including, but not limited to, structural genes encoding a polypeptide. The target gene can be a gene derived from a cell, an endogenous gene, a transgene, or exogenous genes such as genes of a pathogen, for example a virus, which is present in the cell after infection thereof. The cell containing the target gene can be derived from or contained in any organism, for example a plant, animal, protozoan, virus, bacterium, or fungus. The term "gene" also refers broadly to any segment of DNA associated with a biological function. As such, the term "gene" encompasses sequences including but not limited to a coding sequence, a promoter region, a transcriptional regulatory sequence, a non-expressed DNA segment that is a specific recognition sequence for regulatory proteins, a non-expressed DNA segment that contributes to gene expression, a DNA segment designed to have desired parameters, or combinations thereof. A gene can be obtained by a variety of methods, including cloning from a biological sample, synthesis based on known or predicted sequence information, and recombinant derivation of an existing sequence.

[0056] As used herein, the "huntingtin gene" (or "Hit") is the nucleic acid sequence encoding the huntingtin protein. The "normal" or "wild-type" huntingtin gene has a CAG trinucleotide sequence repeated about 10-35 times within the gene. The "mutant" huntingtin gene, for example, has 36 or more CAG trinucleotide repeats. For example, subjects carrying the mutant huntingtin gene may have 36 to more than 120 CAG repeats. Subjects with 36 to 39 CAG repeats may or may not develop the signs and symptoms of Huntington' s disease, while people with 40 or more repeats almost always develop symptomology.

[0057] As is understood in the art, a gene comprises a coding strand and a non-coding strand. As used herein, the terms "coding strand" and "sense strand" are used interchangeably, and refer to a nucleic acid sequence that has the same sequence of nucleotides as an mRNA from which the gene product is translated. As is also understood in the art, when the coding strand and/or sense strand is used to refer to a DNA molecule, the coding/sense strand includes thymidine residues instead of the uridine residues found in the corresponding mRNA. Additionally, when used to refer to a DNA molecule, the coding/sense strand can also include additional elements not found in the mRNA including, but not limited to promoters, enhancers, and introns. Similarly, the terms "template strand" and "antisense strand" are used interchangeably and refer to a nucleic acid sequence that is complementary to the coding/sense strand. Further, as used herein, the following abbreviations shall have the following meanings: "A" shall mean Adenine; "bp" shall mean base pairs; "C" shall mean Cytosine; "G" shall mean Guanine; "T" shall mean Thymine; and "U" shall mean Uracil.

[0058] As used herein, the terms "complementarity" and "complementary" refer to a nucleic acid that can form one or more hydrogen bonds with another nucleic acid sequence by either traditional Watson-Crick or other non-traditional types of interactions. Similarly, the phrase "percent complementarity" or the like refer to the percentage of contiguous residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary, respectively). The terms "100% complementary," "fully complementary," or "perfectly complementary," indicate that all of the contiguous residues of a nucleic acid sequence can hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence.

[0059] As used herein, "identity" or "sequence identity" or the like refer to, in the context of a sequence, the similarity between two nucleic acid sequences, or two amino acid sequences, and is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. Example levels of sequence identity include, for example, 80%, 85%, 90%, 95%, 98% or more sequence identity to a given sequence, e.g., the coding sequence for any one of the inventive polypeptides, as described herein.

[0060] Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith & Waterman Adv. Appl. Math. 2: 482, 1981; Needleman & Wunsch J. Mol. Biol. 48: 443, 1970; Pearson & Lipman Proc. Natl. Acad. Sci. USA 85: 2444, 1988; Higgins & Sharp Gene 73: 237-244, 1988; Higgins & Sharp CABIOS 5: 151-153, 1989; Corpet et al. Nuc. Acids Res. 16, 10881-90, 1988; Huang et al. Computer Appls. In the Biosciences 8, 155-65, 1992; and Pearson et al. Meth. Mol. Bio. 24, 307-31, 1994. Altschul et al. (J. Mol. Biol. 215:403- 410, 1990), presents a detailed consideration of sequence alignment methods and homology calculations.

[0061] The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al. J. Mol. Biol. 215:403-410, 1990) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, MD) and on the Internet, for use in connection with the sequence analysis programs that include, for example, the suite of BLAST programs, such as BLASTN, BLASTX, and TBLASTX, B LAS TP and TBLASTN.

[0062] Sequence searches are typically carried out using the BLASTN program when evaluating a given nucleic acid sequence relative to nucleic acid sequences in the GenBank DNA Sequences and other public databases. The BLASTX program is preferred for searching nucleic acid sequences that have been translated in all reading frames against amino acid sequences in the GenBank Protein Sequences and other public databases. Both BLASTN and BLASTX are run using default parameters of an open gap penalty of 11.0, and an extended gap penalty of 1.0, and utilize the BLOSUM-62 matrix. (See, e.g., Altschul, S. F., et al., Nucleic Acids Res. 25:3389-3402, 1997).

[0063] In certain example embodiments, a preferred alignment of selected sequences in order to determine "% identity" between two or more sequences, is performed using for example, the CLUSTAL-W program in MacVector version 13.0.7, operated with default parameters, including an open gap penalty of 10.0, an extended gap penalty of 0.1, and a BLOSUM 30 similarity matrix.

[0064] As used herein, the term "non-nucleotide" refers to any group or compound which can be incorporated into a nucleic acid chain in the place of one or more nucleotide units, including either sugar and/or phosphate substitutions, and allows the remaining bases to exhibit their enzymatic activity. The group or compound is typically abasic, in that it does not typically contain a commonly recognized nucleotide base, such as adenine (A), guanine (G), cytosine (C), thymine (T), or uracil (U), and therefore lacks a base at the 1 '-position.

[0065] The term "nucleotide" is used herein as recognized in the art to include natural bases (standard), and modified bases well known in the art. Such bases are generally located at the position of a nucleotide sugar moiety. Nucleotides generally comprise a base, sugar, and a phosphate group. The nucleotides can be unmodified or modified at the sugar, phosphate, and/or base moiety, (also referred to interchangeably as nucleotide analogs, modified nucleotides, non-natural nucleotides, non-standard nucleotides, and other; see e.g., Usman et al., 1996; PCT International Publication NOS. WO 92/07065 and WO 93/15187, all incorporated by reference herein in their entirety). There are several examples of modified nucleic acid bases known in the art as summarized by Limbach et al., 1994. Some of the non-limiting examples of base modifications that can be introduced into nucleic acid molecules include, inosine, purine, pyridin-4-one, pyridin- 2-one, phenyl, pseudouracil, 2,4,6-trimethoxy benzene, 3-methyl uracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidines (e.g., 5-methylcytidine), 5-alkyluridines (e.g., ribo thymidine), 5-halouridine (e.g., 5-bromouridine), 6-azapyrimidines and 6- alkylpyrimidines (e.g. 6-methyluridine), propyne, and others (Burgin et al., 1996; Uhlman & Peyman, 1990). By "modified bases" in this aspect is meant nucleotide bases other than adenine, guanine, cytosine, and uracil at position or their equivalents. [0066] As used herein, the term "DNA" refers to a molecule comprising at least one deoxyribonucleotide residue. A "deoxyribonucleotide," is a nucleotide without a hydroxyl group and instead a hydrogen at the 2' position of a β-D-deoxyribofuranose moiety. The term encompasses double stranded DNA, single stranded DNA, DNAs with both double stranded and single stranded regions, isolated DNA such as partially purified DNA, essentially pure DNA, synthetic DNA, recombinantly produced DNA, as well as altered DNA, or analog DNA, that differs from naturally occurring DNA by the addition, deletion, substitution, and/or modification of one or more nucleotides. Such modifications can include addition of non-nucleotide material, such as to the end(s) of the DNA or internally, for example at one or more nucleotides of the DNA. The modifications can be for the purpose of increasing stability of the DNA molecule.

[0067] Nucleotides in the DNA molecules described herein can also comprise nonstandard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These modified DNAs can be referred to as analogs or analogs of a naturally occurring DNA. As used herein, a "DNA molecule" is a polymeric chain of single- or double-stranded nucleotides and also referred to herein as "oligonucleotide" and "polynucleotide." Thus, the terms DNA molecule, oligonucleotide, and polynucleotide are used herein interchangeably and the use of one term or another is not intended to limit the described molecule, e.g., to a particular number of nucleotides polymerized.

[0068] As used herein, the term "RNA" refers to a molecule comprising at least one ribonucleotide residue. A "ribonucleotide" is a nucleotide with a hydroxyl group at the 2' position of a β-D-ribofuranose moiety. The terms encompass double stranded RNA, single stranded RNA, RNAs with both double stranded, and single stranded regions, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as altered RNA, or analog RNA, that differs from naturally occurring RNA by the addition, deletion, substitution, and/or modification of one or more nucleotides. Such modifications can include addition of non-nucleotide material, such as to the end(s) of an RNA or internally, for example at one or more nucleotides of the RNA.

[0069] As used herein, the phrase "double stranded region" refers to any region of a nucleic acid molecule that is in a double stranded conformation via hydrogen bonding between the nucleotides including, but not limited to, hydrogen bonding between cytosine and guanosine, adenosine and thymidine, adenosine and uracil, and any other nucleic acid duplex as would be understood by one of ordinary skill in the art. The length of the double stranded region can vary from about four consecutive base pairs to several thousand base pairs. In certain example embodiments, the double stranded region is at least five base pairs, while in certain example embodiments between 5 and 30 base pairs, and in certain example embodiments between 5 and 15 base pairs. In certain example embodiments, the length of the double stranded region is selected from 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15 base pairs. In certain example embodiments, the double stranded region comprises a first strand comprising a ribonucleotide sequence that corresponds to a coding strand of Htt gene and a second strand comprising a deoxyribonucleotide sequence as described herein that is complementary to the first strand, and wherein the first strand and the second strand hybridize to each other to form the double-stranded molecule.

[0070] As used herein, "administration" or "administering" refers to the introduction of a composition into a subject by a chosen route. For example, if the chosen route is injection, the compositions described herein may be administered by intraperitoneal or intravenous injection. Administration can be effected or performed using any of the various methods and delivery systems known to those skilled in the art. The administering can be performed, for example, intravenously, orally, via implant, transmucosally, transdermally, topically, intramuscularly, intra-articularly, subcutaneously, or extracorporeally. In certain example embodiments, nucleic acid or nucleic acid complexes, such as complexes including nucleic acids and lipids, can be locally administered to relevant tissues ex vivo, or in vivo through injection, infusion pump (e.g. an osmotic infusion pump) or stent, with or without their incorporation into biopolymers.

[0071] As used herein, the term "amino acid" is an organic compound containing an amino group and a carboxylic acid group. A peptide or polypeptide contains two or more amino acids. For purposes herein, amino acids include the twenty naturally-occurring amino acids, non-natural amino acids and amino acid analogs (i.e., amino acids wherein the a-carbon has a side chain).

[0072] As used herein, "polypeptide" as used herein, refers to any polymeric chain of amino acids. The terms "peptide" and "protein" are used interchangeably with the term polypeptide and also refer to a polymeric chain of amino acids. The term "polypeptide" encompasses native or artificial proteins, protein fragments and polypeptide analogs of a protein sequence. A polypeptide may be monomeric or polymeric, and may include a number of modifications. Generally, a peptide or polypeptide is greater than or equal to 2 amino acids in length, and generally less than or equal to 40 amino acids in length.

[0073] The term "biodegradable linker," as used herein, refers to a nucleic acid or non-nucleic acid linker molecule that is designed as a biodegradable linker to connect one molecule to another molecule. The term "biodegradable," as used herein, refers to degradation in a biological system, for example enzymatic degradation or chemical degradation.

[0074] The term "biologically active molecule," as used herein, refers to compounds or molecules that are capable of eliciting or modifying a biological response in a system. Non-limiting examples of biologically active molecules provided by the presently disclosed subject matter include therapeutically active molecules such as antibodies, hormones, antivirals, peptides, proteins, chemotherapeutics, small molecules, vitamins, co-factors, nucleosides, nucleotides, oligonucleotides, enzymatic nucleic acids, antisense nucleic acids, triplex forming oligonucleotides, 2,5-A chimeras, DNAzymes, siRNA, dsRNA, allozymes, aptamers, decoys, and analogs thereof.

[0075] As used herein, "carrier" refers to conventional pharmaceutically acceptable carriers. Remington's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, PA, 19th Edition (1995), for example, describes compositions and formulations suitable for pharmaceutical delivery of the compositions disclosed herein. In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (e.g., powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.

[0076] Example carriers include excipients or stabilizers that are nontoxic to the cell, tissue, mammal, or subject being exposed thereto at the dosages and concentrations employed. Often the pharmaceutically acceptable carrier is an aqueous pH buffered solution. Examples of pharmaceutically acceptable carriers also include, without limitation, buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as Tween®, polyethylene glycol (PEG), and Pluronics®.

[0077] As used herein, "conjugated" means covalently attached (e.g. via a crosslinking agent). "Coupled" or "bound" means that members of a binding pair are associated, noncovalently, as through a plurality of charged intereactions (ionic bonds) and non-ionic or hydrophobic interactions including VanDerWaals forces such that the bound members retain separate molecular entity.

[0078] As used herein, "effective amount" or "suitable amount" or "therapeutically effective amount" refers to an amount of a substance sufficient to effect the beneficial or desired clinical or biochemical results. An effective amount can be administered one or more times. For example, an effective amount of a composition as described herein is an amount that is sufficient to modulate the expression of mutant huntingtin protein, whether in a single dose or in multiple doses. For example, the therapeutically effective amount of the composition may inhibit or decrease the expression of mutant huntingtin protein. In other example embodiments, the therapeutically effective amount of the composition may reduce the appearance of mutant huntingtin protein aggregates in a tissue.

[0079] As used herein, "label" refers to a detectable compound or composition that is conjugated or coupled directly or indirectly to another molecule to facilitate detection of that molecule. Specific, non-limiting examples of labels include fluorescent tags, chemiluminescent tags, haptens, enzymatic linkages, and radioactive isotopes. In the context of a labeled oligonucleotide, a label includes, for example, a moiety via which an oligonucleotide can be detected or purified.

[0080] As used herein, the terms "purified" or "isolated" refer to biological or synthetic molecules that are removed from their natural or synthetic environment and are isolated or separated and are free from other components with which they are naturally associated. The term "purified" does not require absolute purity. Rather, this term is intended as a relative term. Thus, for example, a purified or "substantially pure" protein preparation is one in which the protein referred to is more pure than the protein in its natural environment within a cell or within a production reaction chamber (as appropriate). As used in the context of a nucleic acid molecule, "isolated" indicates that the nucleic acid molecule exists apart from its native environment and is not a product of nature. An isolated DNA molecule can exist in a purified form or can exist in a non- native environment such as a transgenic host cell. For a synthetic nucleic acid molecule, the nucleic acid can be purified or isolated, for example, and still include components or impurities generated from the synthesis reaction.

[0081] As used herein, a "subject" refers to an animal, including a vertebrate. The vertebrate may be a mammal, for example, such as a human. The subject may be a human patient. A subject may be a patient suffering from or suspected of suffering from a disease or condition and may be in need of treatment or diagnosis or may be in need of monitoring for the progression of the disease or condition. The subject may also be in on a treatment therapy that needs to be monitored for efficacy. In certain example embodiments, a subject includes a subject suffering from Huntington's disease.

[0082] The terms "treating" or "treatment" refer to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop. The term "ameliorating," with reference to a disease or pathological condition, refers to any observable beneficial effect of the treatment. The beneficial effect can be evidenced, for example, by a delayed onset of clinical symptoms of the disease in a susceptible subject, a reduction in severity of some or all clinical symptoms of the disease, a slower progression of the disease, an improvement in the overall health or well- being of the subject, or by other parameters well known in the art that are specific to the particular disease. A "prophylactic" treatment is a treatment administered to a subject who does not exhibit signs of a disease or exhibits only early signs for the purpose of decreasing the risk of developing pathology.

[0083] As used herein, production by recombinant methods by using recombinant DNA methods refers to the use of the well-known methods of molecular biology for expressing proteins encoded by cloned DNA. For example, standard techniques may be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures may be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See, e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)), which is incorporated herein by reference for any purpose.

[0084] As used herein, "vector" (or plasmid) refers to discrete DNA elements that are used to introduce heterologous nucleic acid into cells for either expression or replication thereof. The vectors typically remain episomal, but can be designed to effect integration of a gene or portion thereof into a chromosome of the genome. Also contemplated are vectors that are artificial chromosomes, such as bacterial artificial chromosomes, yeast artificial chromosomes and mammalian artificial chromosomes. Selection and use of such vehicles are well known to those of skill in the art.

[0085] As used herein, an "expression vector" includes vectors capable of expressing DNA that is operatively linked with regulatory sequences, such as promoter regions, that are capable of effecting expression of such DNA fragments. Such additional segments can include promoter and terminator sequences, and optionally can include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, and the like. Expression vectors are generally derived from plasmid or viral DNA, or can contain elements of both. Thus, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, a phage, recombinant virus or other vector that, upon introduction into an appropriate host cell, results in expression of the cloned DNA. Appropriate expression vectors are well known to those of skill in the art and include those that are replicable in eukaryotic cells and/or prokaryotic cells and those that remain episomal or those, which integrate into the host cell genome. As used herein, vector also includes "virus vectors" or "viral vectors." Viral vectors are engineered viruses that are operatively linked to exogenous genes to transfer (as vehicles or shuttles) the exogenous genes into cells.

[0086] The term "promoter" or "promoter region" refer to a nucleotide sequence within a gene that is positioned 5' to a coding sequence of a same gene and functions to direct transcription of the coding sequence. The promoter region includes a transcriptional start site, and can additionally include one or more transcriptional regulatory elements. As those skilled in the art will appreciate, different promoters have different combinations of transcriptional regulatory elements. Whether or not a gene is expressed in a cell is dependent on a combination of the particular transcriptional regulatory elements that make up the gene's promoter and the different transcription factors that are present within the nucleus of the cell.

[0087] As such, promoters are often classified as "constitutive", "tissue-specific", "cell-type-specific", or "inducible", depending on their functional activities in vivo or in vitro. For example, a constitutive promoter is one that is capable of directing transcription of a gene in a variety of cell types. Example constitutive promoters include the promoters for the following genes which encode certain constitutive or "housekeeping" functions: hypoxanthine phosphoribosyl transferase (HPRT), dihydrofolate reductase (DHFR; (Scharfmann et al., 1991), adenosine deaminase, phosphoglycerate kinase (PGK), pyruvate kinase, phosphoglycerate mutase, the β-actin promoter (see, e.g. Williams et al., 1993), and other constitutive promoters known to those of skill in the art. "Tissue- specific" or "cell-type- specific" promoters, on the other hand, direct transcription in some tissues and cell types but are inactive in others. Example tissue-specific promoters include the PSA promoter (Yu et al., 1999; Lee et al., 2000), the probasin promoter (Greenberg et al., 1994; Yu et al., 1999), and the MUC1 promoter (Kurihara et al., 2000), as well as other tissue-specific and cell-type specific promoters known to those of skill in the art. When used in the context of a promoter, the term "linked" as used herein refers to a physical proximity of promoter elements such that they function together to direct transcription of an operably linked nucleotide sequence

[0088] By the term "host cell," it is meant a cell that contains a vector and supports the replication, and/or transcription or transcription and translation (expression) of the expression construct. Host cells can be prokaryotic cells, such as E. coli or Bacillus subtilus, or eukaryotic cells such as yeast, plant, insect, amphibian, or mammalian cells. In general, host cells are prokaryotic, e.g., E. coli.

Example Embodiments

Enzymatic Nucleic Acid Molecules

[0089] FIG. 1 provides a schematic diagram illustrating binding of an enzymatic nucleic acid molecule, such as a DNAzyme, to a corresponding sequence in a target RNA via the binding domain (step 1), in accordance with certain example embodiments. The binding of the DNAzyme is followed by cleavage of the target RNA by the catalytic domain (step 2). That is, the enzymatic nucleic acid molecule acts by first binding to a target nucleic acid, such as a target RNA. The catalytic domain then cleaves the target RNA molecule. As used herein, the "catalytic domain" of the enzymatic DNA molecule includes, for example, that portion/region of the enzymatic nucleic acid molecule essential for cleavage of a nucleic acid substrate.

[0090] To recognize the target RNA, binding of the enzymatic nucleic acid molecule to the target RNA occurs through the substrate binding domain(s) of the enzymatic nucleic acid molecule. The "substrate binding domain," for example, includes that portion/region of an enzymatic DNA molecule that exhibits binding specificity for a target nucleic acid, also referred to as a substrate. As illustrated in FIG. 1, the substrate binding domain is typically located in close proximity to the catalytic domain of the enzymatic DNA molecule. Thus, the enzymatic DNA molecule first recognizes the target RNA and then binds a target RNA through complementary base-pairing. Once bound to the correct site, the enzymatic nucleic acid molecule acts enzymatically to cut the target RNA (FIG. 1). Strategic cleavage of such a target RNA destroys its ability to direct synthesis of an encoded protein. After an enzymatic nucleic acid has bound and cleaved its RNA target, it is released from that RNA to search for another target and can repeatedly bind and cleave new targets.

[0091] In certain example embodiments, the enzymatic DNA molecules provided herein are enzymatic DNA molecules that include a polynucleotide sequence having binding specificity for a target region of an mRNA encoding all or a portion of mutant huntingtin protein. For example, the enzymatic DNA molecules can be designed to target the huntingtin (Htt) transcript derived from human mutant exon 1, as described herein. By targeting the huntingtin (Htt) transcript derived from human mutant exon 1, for example, such enzymatic DNA molecules cleave the transcript via the enzymatic activity of the DNA molecule. As those skilled in the art will appreciate, human mutant exon 1 will possess a CAG repeat region that exceeds about 35 repeats, and thereby be disease- causing. The human mutant exon 1 otherwise can be identical to wild-type, normal human Htt exon 1.

[0092] In certain example embodiments, the DNA oligonucleotide sequence is a DNAzyme. In certain example embodiments, the enzymatic DNA molecule comprises a catalytic domain flanked on each side by substrate binding domains, each substrate binding domain having binding specificity for a distinct nucleotide sequence of the target region of the target RNA as described herein. In certain example embodiments, the enzymatic DNA molecule has a nucleotide sequence comprising the sequence set forth as any one of SEQ ID NOS: 1-4. In certain example embodiments, the enzymatic DNA molecule has a nucleotide sequence that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of the sequences set forth as SEQ ID NOS: 1-4.

[0093] In certain example embodiments, the enzymatic DNA molecule provided herein includes a conserved core catalytic domain flanked on each side by a substrate binding domain. Each of the binding substrate domains, for example, interact with the target RNA at a target region of the RNA through base-pairing interactions, as described herein. In certain example embodiments, the conserved core comprises one or more conserved sequences. For example, the catalytic domain includes about 5 to about 25 nucleotides. In other example embodiments, the catalytic domain includes about 12 to about 17 nucleotides. In other example embodiments, the catalytic domain includes about 15 bases. For example, the catalytic domain can include about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides. In certain example embodiments, the enzymatic DNA molecule comprises a catalytic core having the sequence set forth as SEQ ID NO: 7 (i.e., ggctagctacaacga). In certain example embodiments, the catalytic core comprises a nucleic acid sequence that 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth as SEQ ID NO: 7.

[0094] Additionally or alternatively, in certain example embodiments the conserved core can comprise a substitution in a conserved sequence, but wherein intramolecular interactions are preserved by the substitution. Additionally or alternatively, the enzymatic DNA molecule can further comprise a "spacer" region (or sequence) between either or both of the regions (or sequences) involved in base pairing. Additionally or alternatively, the conserved core can be "interrupted" at various intervals by one or more less-conserved variable or "spacer" nucleotides. In such embodiments, the enzymatic function and binding specificity of the enzymatic DNA molecule is retained.

[0095] The substrate binding domain of the enzymatic DNA molecule described herein typically comprises two nucleotide sequences flanking the catalytic domain, and typically each substrate binding domain contains a sequence of about 4 to about 30 nucleotides, such as about 8-12 nucleotides. In certain example embodiments, substrate binding domain contains a sequence of about 6 to about 15 nucleotides, which are capable of hybridizing to a complementary sequence of bases within the substrate nucleic acid giving the enzymatic DNA molecule its high sequence specificity. For example, the substrate binding domain includes 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 nucleotides. In certain example embodiments, the substrate binding domains comprise the flanking sequences beyond where the catalytic domain (SEQ ID NO: 7) is located within the DNA molecule. That is, in certain example embodiments the substrate binding domains exclude the catalytic domain (SEQ ID NO: 7).

[0096] Synthesizing the enzymatic DNA having the sequences described herein, for example, allows one to alter the sequence specificity of an enzymatic nucleic acid molecule. In certain example embodiments, the catalytic domain may optionally contain stem-loop structures in addition to the nucleotides required for catalytic activity.

[0097] In certain example embodiments, the enzymatic nucleic acid molecules described herein can have substrate binding domains that are contiguous or noncontiguous and can be varying lengths. For example, the length of each substrate binding domain may be greater than or equal to four nucleotides, such as 5-30 nucleotides. In certain example embodiments, the length of each substrate binding domain is 5-15 nucleotides long, such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 nucleotides. If two substrate binding domains are chosen, the design can optionally be such that the length of the binding domains are symmetrical (i.e., each of the binding domains is of the same length (such as seven and seven nucleotides, eight and eight nucleotides, or nine and nine nucleotides long) or asymmetrical (i.e., the binding domains are of different length, such as six and three nucleotides, three and six nucleotides long, four and five nucleotides long, four and six nucleotides long, four and seven nucleotides long, and the like).

[0098] In certain example embodiments, the enzymatic DNA molecules disclosed herein may also include those with altered substrate binding domains. For example, the altered binding domains confer unique sequence specificities on the enzymatic DNA molecule including such binding domains. The exact nucleotide bases present in the substrate binding domain determine, for example, the nucleotides sequence at which cleavage will take place. Cleavage of the substrate nucleic acid occurs, for example, within the target region determined by the specificity of the substrate binding domain. This cleavage leaves a 2', 3', or 2', 3 '-cyclic phosphate group on the substrate cleavage sequence and a 5' hydroxyl on the nucleotide that was originally immediately 3' of the substrate cleavage sequence in the original substrate. Cleavage can be redirected to a site of choice by changing the bases present in the substrate binding domain, such as during synthesis of the enzymatic DNA molecule

[0099] In certain example embodiments, the terms "binding specificity," "corresponds to," "corresponding to," and grammatical variations thereof refer to the substrate binding domain being identical to or complementary to (i.e., able to base-pair with) a portion of its substrate. Such identity or complementarity can be 100%, but can be less if desired. For example, as few as 75% of the bases can be identical or base- paired in some embodiments over a given stretch of sequences in the substrate binding domain, and as few as 90% of the bases can be identical or base-paired in other embodiments. In some embodiments, 95%, 96%, 97%, 98%, or 99% of the bases can be identical or base-paired. That is, in certain example embodiments, these domains contain sequences within an enzymatic nucleic acid molecule that are intended to bring enzymatic DNA molecule and target together through complementary base-pairing interactions.

[00100] The enzymatic function of an enzymatic DNA molecule described herein has significant advantages. For example, as an enzyme, the concentration of enzymatic DNA molecules necessary to affect a therapeutic treatment is lower. This advantage reflects the ability of the enzymatic DNA molecules to act enzymatically. Thus, a single enzymatic DNA molecule can cleave many molecules of target RNA. In addition, the enzymatic DNA molecule is a highly specific inhibitor, with the specificity of inhibition depending not only on the base-pairing mechanism of binding to the target RNA, but also on the mechanism of target RNA cleavage. Single mismatches, or base-substitutions, near the site of cleavage can be chosen to completely eliminate catalytic activity of enzymatic nucleic acid molecules.

Synthesis of Nucleic Acid Molecules

[00101] The enzymatic DNA molecules provided herein, including those identified in SEQ ID NOS. 1-4, may be synthesized and purified by any method known to those skilled in the art. This includes those enzymatic DNA molecules that share 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of the sequences set forth as SEQ ID NOS: 1-4. In certain example embodiments, the enzymatic DNA molecule provided herein can be synthesized outside of a target cell before introduction of the DNA molecule into the target cell. For example, the synthesis can be performed either mechanically (i.e., using a DNA synthesis machine) or using recombinant techniques.

[00102] As those skilled in the art will appreciate, mechanical synthesis of nucleic acids greater than 100 nucleotides in length may can be difficult, and the cost of such molecules may be prohibitive or undesirable. As used herein, small nucleic acid motifs ("small" referring to nucleic acid motifs in some embodiments no more than 100 nucleotides in length, in some embodiments no more than 80 nucleotides in length, and in some embodiments no more than 50 nucleotides in length; e.g., individual DNA oligonucleotide sequences or DNA sequences synthesized in tandem) can be used for exogenous delivery. The simple structure of these molecules increases the ability of the nucleic acid to invade targeted regions of RNA structure. Exemplary molecules of the presently disclosed subject matter are chemically synthesized, and others can similarly be synthesized.

[00103] In certain example embodiments, provided is a vector for producing the enzymatic DNA molecules described herein. For example, the vector includes a sequence encoding one more of the DNA molecules described herein. Further provided is a method of producing one more of the DNA molecules described herein including culturing a cell having therein a vector comprising a sequence encoding the one more of the DNA molecules under conditions permitting the expression of the nucleic acid molecule by the cell. Methods of culturing cells in order to permit expression and conditions permitting expression are well known in the art. For example see Sambrook et al., "Molecular Cloning: A Laboratory Manual," Second Edition (1989), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Such methods can optionally comprise a further step of recovering the nucleic acid product.

[00104] Oligonucleotide sequences, including those of the enzymatic DNA molecules described herein, may also be synthesized using other protocols known in the art. See, e.g., Caruthers et al, 1992; PCT International Publication No. WO 99/54459; Wincott et al., 1995; Wincott & Usman, 1997; Brennan et al., 1998; and U.S. Pat. No. 6,001,311, each of which is expressly incorporated herein by reference in its entirety. In certain example embodiments, the synthesis of oligonucleotides makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5 '-end, and phosphoramidites at the 3 '-end. In a non-limiting example, small-scale syntheses can be conducted on an Applied Biosystems™ 3400 DNA Synthesizer (Applied Biosystems Inc., Foster City, Calif., United States of America) along with standard protocols associated therewith. In certain example embodiments, deprotection of the DNA-based oligonucleotides is performed in accordance with methods generally known to those skilled in the art. In certain example embodiments, the enzymatic nucleic acid molecules described herein can be synthesized separately and joined together post-synthetically, for example, by ligation (PCT International Publication No. WO 93/23569; Shabarova et al., 1991; Bellon et al., 1997), or by hybridization following synthesis and/or deprotection.

[00105] In certain example embodiments, recombinant techniques can be used to synthesize an enzymatic DNA molecule, which can thereafter be purified from the source and transferred to a target cell. As those skilled in the art will appreciate, there are many techniques for the synthesis of DNA molecules in recombinant cells, and any such technique can be used in the practice of the presently disclosed subject matter. One such general strategy for synthesizing a DNA molecule includes cloning a DNA sequence downstream of a bacterial or yeast origin of replication and introducing the recombinant molecule into a cell in which the origin of replication is competent to direct replication of the cloned sequence. This can be accomplished using a plasmid constructed for this purpose.

Optimizing Activity of Nucleic Acid Molecules

[00106] In certain example embodiments, the enzymatic DNA molecules disclosed herein can combine and/or include one or more modifications or mutations including additions, deletions, and substitutions. Additionally or alternatively, such mutations or modifications can be generated using methods that produce random or specific mutations or modifications. These mutations or modifications can, for example, change the length of, or alter the nucleotide sequence of, a loop, a spacer region or the substrate binding domain or add one or more non-nucleotide moieties to the molecule to increase stability, for example. In certain example embodiments, one or more mutations within one catalytically active enzymatic DNA molecule can be combined with the mutation(s) within a second catalytically active enzymatic DNA molecule to produce a new enzymatic DNA molecule containing the mutations of both molecules.

[00107] It is also to be understood that an enzymatic DNA molecule described herein can comprise enzymatically active portions (e.g. catalytic domains) of a DNAzyme or can comprise a DNAzyme with one or more mutations, e.g., with one or more substrate binding domain sequences or spacers absent or modified, as long as such deletions, additions, or modifications do not adversely impact the molecule's ability to perform as an enzyme.

[00108] In certain example embodiments, mutations can be introduced in the enzymatic DNA molecule by altering the length of the substrate binding domains of the enzymatic DNA molecule. The substrate binding domains of the enzymatic DNA molecule have binding specificity for and associate with a complementary sequence of bases within a target region of a substrate nucleic acid sequence. Methods of altering the length of the recognition domains are known in the art and include direct synthesis and PCR, for example.

[00109] Alteration of the length of the recognition domains of an enzymatic DNA molecule can have a desirable effect on the binding specificity of the enzymatic DNA molecule. For example, an increase in the length of the substrate binding domains can increase binding specificity between the enzymatic DNA molecule and the complementary base sequences of a target region in a substrate polynucleotide, or can enhance recognition of a particular sequence in a hybrid substrate. Additionally or alternatively, an increase in the length of the substrate binding domains can also increase the affinity with which the DNA molecule binds to the polynucleotide substrate. In various example embodiments, these altered substrate binding domains in the enzymatic DNA molecule confer increased binding specificity and affinity between the enzymatic DNA molecule and its substrate, however, it may decrease catalytic efficiency of the DNAzyme. Therefore, one of skill in the art will appreciate that alteration of the length of the recognition domains is a balance of optimal binding and catalytic activity.

[00110] The therapeutic nucleic acid molecules described herein, such as the enzymatic DNA molecules (including DNAzymes) delivered or administered exogenously, are optimally stable within cells until translation of the target RNA has been inhibited long enough to reduce the levels of the undesirable protein. This period of time varies between hours to days depending upon the disease state. Although DNAzymes as described herein are considered advantageous over RNA based molecules in that DNAzymes are less sensitive to degradation, in some embodiments it is desirable to further increase stability of the DNAzymes of the presently disclosed subject matter. Improvements in the chemical synthesis of nucleic acid molecules described in the presently disclosed subject matter and in the art (Wincott et al., 1995; Caruthers et al., 1992) have expanded the ability to modify nucleic acid molecules by introducing nucleotide modifications to enhance their nuclease stability. Hence, in certain example embodiments, the enzymatic DNA molecules described herein can be modified extensively to enhance stability. For example, the enzymatic DNA molecules may be modified with nuclease resistant groups including, for example, to 2'-amino, 2'-C-allyl, 2'-fluoro, 2'-0-methyl, 2'-H (for a review see Usman & Cedergren, 1992; Usman et al., 1994).

[00111] Enzymatic DNA molecules having chemical modifications that maintain or enhance enzymatic activity are also provided. Such nucleic acid molecules are generally more resistant to nucleases than unmodified nucleic acid molecules. Thus, in a cell and/or in vivo the activity may not be significantly lowered. As exemplified herein, such enzymatic nucleic acid molecules are useful in a cell and/or in vivo even if activity over all is reduced 10-fold. Such enzymatic nucleic acid molecules herein are said to "maintain" the enzymatic activity.

[00112] As those skilled in the art will appreciate, chemically synthesizing nucleic acid molecules incorporating various modifications (e.g. to base, sugar, and/or phosphate moieties) can reduce the degradation of the nucleic acid molecules by nucleases present in biological fluids, and can thus can increase the potency of therapeutic nucleic acid molecules {see e.g., PCT International Publication NOS. WO 92/07065, WO 93/15187, and WO 91/03162; U.S. Pat. NOS. 5,334,711 and 6,300,074; Perrault et al., 1990; Pieken et al., 1991; Usman & Cedergren, 1992; and Burgin et al., 1996; all of which are expressly incorporated by reference herein in their entirety). Each of the above references describe various chemical modifications that can be made to the base, phosphate, and/or sugar moieties of the nucleic acid molecules described herein. Modifications can be employed to enhance the efficacy of the disclosed nucleic acid molecules in cells and/or in vivo.

[00113] There are several examples in the art describing sugar, base, and phosphate modifications that can be introduced into nucleic acid molecules with significant enhancement in their nuclease stability and efficacy. For example, oligonucleotides can be modified to enhance their stability and/or enhance biological activity by modification with nuclease resistant groups, for example, 2'-amino, 2'-C-allyl, 2'-fluoro, 2'-0-methyl, 2'-0-allyl, 2'-H, nucleotide base modifications (reviewed in Usman & Cedergren, 1992; Usman et al., 1994; Burgin et al., 1996). Sugar modification of nucleic acid molecules have been extensively described in the art (see PCT International Publication NOS. WO 92/07065, WO 93/15187, WO 98/13526, and WO 97/26270; U.S. Pat. NOS. 5,334,711 ; 5,716,824; and 5,627,053; Perrault et al., 1990; Pieken et al., 1991 ; Usman & Cedergren, 1992; Beigelman et al., 1995; Karpeisky et al., 1998; Earnshaw & Gait, 1998; Verma & Eckstein, 1998; Burlina et al., 1997; all of which are incorporated by reference herein in their entirety). Such publications describe general methods and strategies to determine the location of incorporation of sugar, base, and/or phosphate modifications and the like into nucleic acid molecules without modulating catalysis. In view of such teachings, similar modifications can be used as described herein to modify the enzymatic DNA molecules of the presently disclosed subject matter so long as the ability of the DNA molecules to maintain binding specificity to the substrate and catalytic activity is not significantly inhibited.

[00114] As those skilled in the art will appreciate, while chemical modification of an oligonucleotide by internucleotide linkages with phosphorothioate and/or 5'- methylphosphonate linkages improves stability, excessive modifications can cause toxicity or decreased activity. Therefore, when designing nucleic acid molecules, the number of these internucleotide linkages should be minimized. Reducing the concentration of these linkages should lower toxicity, resulting in increased efficacy and higher specificity of these molecules.

[00115] In certain example embodiments, universal bases can also be employed in the nucleic acids of the presently disclosed subject matter. The term "universal base" as used herein refers to nucleotide base analogs that form base pairs with each of the natural DNA/RNA bases with little discrimination between them. Non-limiting examples of universal bases include C-phenyl, C-naphthyl and other aromatic derivatives, inosine, azole carboxamides, and nitroazole derivatives such as 3-nitropyrrole, 4-nitroindole, 5- nitroindole, and 6-nitroindole as known in the art (see, for example, Loakes, 2001).

[00116] In certain example embodiments, provided are conjugates and/or complexes of enzymatic DNA molecules. Such conjugates and/or complexes can be used to facilitate delivery of the DNA molecules into a biological system, such as a cell. The conjugates and complexes provided by the presently disclosed subject matter can impart therapeutic activity by transferring therapeutic compounds across cellular membranes, altering the pharmacokinetics of, and/or modulating the localization of nucleic acid molecules of the presently disclosed subject matter. [00117] The presently disclosed subject matter also encompasses the design and synthesis of novel conjugates and complexes for the delivery of molecules, including, but not limited to, small molecules, lipids, phospholipids, nucleosides, nucleotides, nucleic acids, antibodies, toxins, negatively charged polymers, alkyl groups, and other polymers, for example proteins, peptides, hormones, carbohydrates, polyethylene glycols, or polyamines, across cellular membranes. In general, the transporters described are designed to be used either individually or as part of a multi-component system, with or without degradable linkers. These compounds are expected to improve delivery and/or localization of nucleic acid molecules of the enzymatic DNA molecules described herein into a number of cell types originating from different tissues, in the presence or absence of serum (see U.S. Pat. No. 5,854,038). Conjugates of the molecules described herein can be attached to biologically active molecules via linkers that are biodegradable, such as biodegradable nucleic acid linker molecules.

[00118] In certain example embodiments, the enzymatic DNA molecules provided herein include a biodegradable linker, such as to a biologically active molecule. The biodegradable linker is designed, for example, such that its stability can be modulated for a particular purpose, such as delivery to a particular tissue or cell type. The stability of a nucleic acid-based biodegradable linker molecule can be modulated by using various chemistries, for example combinations of ribonucleotides, deoxyribonucleotides, and chemically modified nucleotides, such as 2'-0-methyl, 2'-fluoro, 2'-amino, 2'-0-amino, 2'-C-allyl, 2'-0-allyl, and other 2'-modified or base modified nucleotides. The biodegradable nucleic acid linker molecule can be a dimer, trimer, tetramer or longer nucleic acid molecule, for example, an oligonucleotide of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length, or can comprise a single nucleotide with a phosphorus-based linkage, for example, a phosphoramidate or phosphodiester linkage. The biodegradable nucleic acid linker molecule can also comprise nucleic acid backbone, nucleic acid sugar, or nucleic acid base modifications.

[00119] Nucleic acid molecules (e.g., enzymatic DNA molecules such as DNAzymes) delivered or administered exogenously are intended to be stable within cells until the level of the target RNA has been reduced sufficiently. The nucleic acid molecules are resistant to nucleases in order to function as effective intracellular therapeutic agents. Improvements in the chemical synthesis of nucleic acid molecules described in the presently disclosed subject matter and in the art have expanded the ability to modify nucleic acid molecules by introducing nucleotide modifications to enhance their nuclease stability as described above.

[00120] In certain example embodiments, the enzymatic DNA molecules described herein are modified to comprise one or more 5' and/or 3 '-cap structures. As used herein, the "cap structure" refers to chemical modifications that have been incorporated at either terminus of the oligonucleotide (See, e.g., U.S. Pat. No. 5,998,203, which is hereby expressly incorporated by reference herein in its entirety). As those skilled in the art will appreciate, such terminal modifications protect the nucleic acid molecule from exonuclease degradation, and can help in delivery and/or localization within a cell. The cap modification can be present at the 5 '-terminus (5 '-cap) or at the 3 '-terminal (3 '-cap), or can be present on both termini. In non-limiting examples, the 5 '-cap may include: an inverted abasic residue (moiety); 4 ',5 '-methylene nucleotide; l-(beta-D-erythrofuranosyl) nucleotide, 4'-thio nucleotide; carbocyclic nucleotide; 1,5-anhydrohexitol nucleotide; L- nucleotides; alpha-nucleotides; modified base nucleotide; phosphorodithioate linkage; threo-pentofuranosyl nucleotide; acyclic 3 ',4'-seco nucleotide; acyclic 3,4-dihydroxybutyl nucleotide; acyclic 3,5-dihydroxypentyl nucleotide, 3 '-3 '-inverted nucleotide moiety; 3 '- 3 '-inverted abasic moiety; 3 '-2 '-inverted nucleotide moiety; 3 '-2 '-inverted abasic moiety; 1,4-butanediol phosphate; 3 '-phosphoramidate; hexylphosphate; aminohexyl phosphate; 3 '-phosphate; 3 '-phosphorothioate; phosphorodithioate; or bridging or non-bridging methylphosphonate moiety.

[00121] In certain example embodiments, the 3 '-cap includes: an inverted deoxynucleotide, such as for example inverted deoxythymidine, 4 ',5 '-methylene nucleotide; l-(beta-D-erythrofuranosyl) nucleotide; 4'-thio nucleotide, carbocyclic nucleotide; 5 '-amino-alkyl phosphate; l,3-diamino-2-propyl phosphate; 3-aminopropyl phosphate; 6-aminohexyl phosphate; 1,2-aminododecyl phosphate; hydroxypropyl phosphate; 1,5-anhydrohexitol nucleotide; L-nucleotide; alpha-nucleotide; modified base nucleotide; phosphorodithioate; threo-pentofuranosyl nucleotide; acyclic 3 ',4'-seco nucleotide; 3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentyl nucleotide, 5 '-5 '-inverted nucleotide moiety; 5 '-5 '-inverted abasic moiety; 5'-phosphoramidate; 5'- phosphorothioate; 1,4-butanediol phosphate; 5 '-amino; bridging and/or non-bridging 5'- phosphoramidate, phosphorothioate and/or phosphorodithioate, bridging or non bridging methylphosphonate and 5'-mercapto moieties (see generally Beaucage & Iyer, 1993; incorporated by reference herein). [00122] In certain example embodiments, the presently disclosed subject matter includes modified enzymatic DNA molecules with phosphate backbone modifications comprising one or more phosphorothioate, phosphorodithioate, methylphosphonate, phosphotriester, morpholino, amidate carbamate, carboxymethyl, acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal, thioformacetal, and/or alkylsilyl, substitutions. For a review of oligonucleotide backbone modifications, see Hunziker & Leumann, 1995 and De Mesmaeker et al., 1994.

[00123] In connection with 2'-modified nucleotides as described herein, by "amino" is meant 2'— NH2 or 2'-0— NH2, which can be modified or unmodified. Such modified groups are described, for example, in U.S. Pat. NOS. 5,672,695 and 6,248,878, which are both expressly incorporated by reference in their entirety.

[00124] Various modifications to enzymatic DNA molecule nucleic acid structure can be made to enhance the utility of these molecules. Such modifications will enhance shelf- life, half-life in vitro, stability, and/or ease of introduction of such oligonucleotides to the target site (for example, to enhance penetration of cellular membranes, and confer the ability to recognize and bind to targeted cells).

Methods & Use

[00125] Provided herein are methods and compositions that are useful for treating Huntington's disease. The methods for treating Huntington's disease, for example, make use of the compositions and formulations described herein. For example, provided are methods for modulating the cellular expression of mutant huntingtin protein. Such methods include, for example, contacting a cell with at least one DNA oligonucleotide having binding specificity for the RNA encoding the mutant protein, thereby exposing the cell to the oligonucleotide. Contacting the cell, for example, results in introduction of the DNA oligonucleotide in the cell. That is, contacting the cell with the DNA oligonucleotide allows the DNA oligonucleotide to enter the cell and exert its function.

[00126] In certain example embodiments, a DNA oligonucleotide can be introduced into the cell by contacting the cell with the DNA oligonucleotide, such as in conjunction with a lipid carrier. For example, the DNA oligonucleotides described herein can be added directly to a cell, or can be complexed with cationic lipids, packaged within liposomes, or otherwise delivered to target cells or tissues. Once inside the cell, the DNA oligonucleotide can exert its catalytic activity on the mRNA encoding the mutant huntingtin protein, thereby modulating the expression the mutant huntingtin protein. By modulating the expression of the mutant huntingtin protein, the enzymatic DNA oligonucleotides can thus be used to treat Huntington's disease.

[00127] In certain example embodiments, provided are methods of treating Huntington's disease in a subject. The methods include, for example, selecting a subject having Huntington's disease. For example, a subject, such as a human subject, may be selected based on symptom presentation and/or family history alone, thereby making the subject a candidate for treatment with the methods and compositions described herein. In certain example embodiments, the subject, such as a human subject, may additionally undergo medical testing to determine and/or to confirm that the subject in fact has (or likely has) Huntington's disease, thereby making the subject a candidate for treatment with the methods and compositions described herein. For example, the subject may undergo genetic testing, imaging studies, cognitive testing, blood and/or tissue testing, or other testing known in the art to determine that the subject has or likely has Huntington's disease. Following selection of the subject, for example, the subject is administered at least one DNA oligonucleotide having binding specificity for a target region of a messenger ribonucleotide (mRNA) encoding the mutant huntingtin protein.

[00128] In certain example embodiments, use of the DNA oligonucleotides described herein may lead to better treatment of the disease progression by affording the possibility of combination therapies (e.g., multiple enzymatic DNA molecules targeted to different genes; nucleic acid molecules coupled with known small molecule modulators; or intermittent treatment with combinations of molecules, including different motifs and/or other chemical or biological molecules). The treatment of subjects with enzymatic DNA molecules can also include combinations of different types of nucleic acid molecules, such as ribozymes, allozymes, antisense, 2,5-A oligoadenylate, decoys, aptamers etc.

[00129] In certain example embodiments, the methods include modulating cellular expression of mutant huntingtin protein by contacting a cell with more than one DNA oligonucleotide, each having binding specificity for a target region of a distinct mRNA encoding the mutant huntingtin protein. For example, one or more enzymatic DNA oligonucleotides may have binding specificity for the mutant exon 1 mRNA of the mutant huntingtin protein, whereas other enzymatic DNA oligonucleotides may have enzymatic activity for other regions of the mRNA coding mutant huntingtin protein. In certain example embodiments, the methods can include contacting the cell with a mixture of DNA oligonucleotides having a sequence set forth as any one of SEQ ID NOS. 1-4. In certain example embodiments, the methods include contacting the cell with a mixture of DNA oligonucleotides, wherein each of the DNA oligonucleotides of the mixture has a nucleotide sequence at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of the sequences set forth as SEQ ID NOS: 1-4. In certain example embodiments, the mixture includes subpopulations of DNA oligonucleotides, with each subpopulation having the same sequence. In other example embodiments, each of the DNA oligonucleotides of the mixture has the same sequence.

[00130] In certain example embodiments, the DNA oligonucleotides described herein individually, or in combination or in conjunction with other drugs, can be used to treat diseases or conditions discussed herein. For example, to treat a particular disease or condition, the DNAzymes described herein can be administered to a subject or can be introduced into other appropriate cells evident to those skilled in the art, individually or in combination with one or more drugs under conditions, suitable for the treatment.

[00131] When used in accordance with the methods described herein, enzymatic DNA oligonucleotides can inhibit or reduce the formation of mutant huntingtin protein within a cell or tissue, for example, thereby inhibiting or reducing the formation and/or aggregation of mutant huntingtin protein in the subject. In certain example embodiments, contacting a cell or tissue with one or more enzymatic DNA oligonucleotides as described herein may reduce mutant huntingtin protein in the cell or tissue by at about 10%, 15%, 20%, 25%, 30%, 40%, 50%, 55%, 60% or more as compared to a non-treated cell or tissue. In certain example embodiments, administration of one or more enzymatic DNA oligonucleotides as described herein to a subject may reduce the amount of mutant huntingtin protein in a subject by at least about 10%, 15%, 20%, 25%, 30%, 40%, 50%, 55%, 60% or more, thereby treating the subject.

[00132] As those skilled in the art will appreciate, inhibition of mutant huntingtin protein or reduction of mutant huntingtin protein as described herein can be determined by various mean known in the art. For example, levels of mutant huntingtin can be measured in the cerebrospinal fluid as obtained by lumbar puncture, or from white blood cells harvested from a blood sample.

[00133] In certain example embodiments, use of the DNA oligonucleotides in accordance with the methods described herein can increase survival rate of a subject with Huntington's disease. That is, use of the DNA oligonucleotides as described herein can increase survival rate of a subject with Huntington's disease as compared to one or more subjects with Huntington's disease not treated with the enzymatic DNA oligonucleotides. For example, use of the DNA oligonucleotides as described herein can increase survival rate beyond an average or median survival rate, such as by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65% or more.

Formulations

[00134] In certain example embodiments, compositions are provided having at least one DNA oligonucleotide as disclosed herein with binding specificity for a target region of a target nucleic acid, such as but not limited to a mRNA encoding a mutant exon 1 of the huntingtin protein. Such compositions, for example, can be used in conjunction with the methods described herein, such as in the treatment of Huntington's disease. In certain example embodiments, the DNA oligonucleotide of the compositions has a nucleotide sequence comprising one or more of the sequences set forth as SEQ ID NOS. 1-4. In certain example embodiments, the DNA oligonucleotide of the compositions has a nucleotide sequence that is about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to one or more of the sequences set forth as SEQ ID NOS. 1-4.

[00135] In certain example embodiments, the DNA oligonucleotides of the compositions can be an enzymatic DNA oligonucleotide, such as a DNAzyme. In some embodiments, the DNAzyme includes a catalytic domain flanked on each side by substrate binding domains, as described herein, each having binding specificity for a distinct nucleotide sequence of the target region. In certain example embodiments, the DNAzyme of the composition has a nucleotide sequence including any one of the sequences set forth as SEQ ID NOS. 1-4. In certain example embodiments, the DNAzyme of the composition has a nucleotide sequence has a sequence that is 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to one or more of the sequences set forth as SEQ ID NOS. 1-4.

[00136] In certain example embodiments, the DNA oligonucleotide of the composition is a catalytic antisense polynucleotide. Additionally or alternatively, the DNA oligonucleotide can include a modification as described herein, such as a modification that increases the stability of the DNA oligonucleotide and/or its catalytic activity. For example, the DNA oligonucleotide can include an inverted deoxythymidine at the 3' end of the DNA oligonucleotide.

[00137] In certain example embodiments, the composition can include more than one DNA oligonucleotide, each having binding specificity for a target region of a distinct mRNA encoding the mutant huntingtin protein. For example, one DNA oligonucleotide within the composition can have binding specificity for a target region of an mRNA encoding mutant exon 1 of the mutant huntingtin protein, whereas another DNA oligonucleotide of the composition can have binding specificity for a different region of the mRNA encoding the mutant huntingtin protein.

[00138] In certain example embodiments, the composition can include a carrier, which can be a pharmaceutically acceptable carrier thereby providing a composition suitable for administration to a subject. Any suitable pharmaceutical formulation can be used to prepare the composition for administration to a subject. For example, suitable formulations can include aqueous and non-aqueous sterile injection solutions that can contain anti-oxidants, buffers, bacteriostats, bactericidal antibiotics, and solutes that render the formulation isotonic with the bodily fluids of the intended recipient. In certain example embodiments, the pharmaceutical formulation can include one or more solvents, within which the DNA oligonucleotides as described herein can remain stable and active. Example solvents include saline and phosphate buffered saline (PBS) (e.g., Gibco™ PBS or Lonza™PBS).

[00139] Additionally or alternatively, the compositions can include aqueous and nonaqueous sterile suspensions, such as suspending agents and thickening agents. The formulations can be presented in unit-dose or multi-dose containers such as in sealed ampoules and vials, and can be stored in a frozen or freeze-dried (lyophilized) condition requiring only the addition of sterile liquid carrier, for example water for injections, immediately prior to use. Some example ingredients are SDS, in the range of about 0.1 to 10 mg/ml, about 2.0 mg/ml, and/or mannitol or another sugar, for example in the range of about 10 to 100 mg/ml, in another example about 30 mg/ml; and/or phosphate-buffered saline (PBS). As those skilled in the art will appreciate, in addition to the ingredients particularly mentioned above, the formulations can include other agents conventional in the art having regard to the type of formulation in question. For example, sterile pyrogen- free aqueous and non-aqueous solutions can be used. Administration, Dosing, & Clearance

[00140] Administration of the compositions and formulations described herein can be by any method known to those skilled in the art. For example, the administration may include intravenous administration, intrasynovial administration, transdermal administration, intramuscular administration, subcutaneous administration, topical administration, rectal administration, intravaginal administration, intratumoral administration, oral administration, buccal administration, nasal administration, parenteral administration, inhalation, and insufflation.

[00141] In certain example embodiments, suitable methods for administration of a DNA molecule of the presently disclosed subject matter include but are not limited to direct injection, pump infusion (e.g. by osmotic pump), intravenous, or intratumoral injection. Additionally or alternatively, a nucleic acid molecule can be deposited at a site in need of treatment in any other manner, for example by spraying a composition comprising a nucleic acid molecule within the pulmonary pathways. The particular mode of administering a composition of the presently disclosed subject matter depends on various factors, including the distribution and abundance of cells to be treated, the vector employed, additional tissue- or cell-targeting features of the vector, and mechanisms for metabolism or removal of the vector from its site of administration.

[00142] In certain example embodiments, the method of administration encompasses features for steady-state regionalized delivery or accumulation at the site in need of treatment. For example, a DNA molecule disclosed herein is delivered to a tumor using a mini-osmotic pump (e.g. an ALZET® mini-osmotic pump (DURECT Corporation, Cupertino, Calif., U.S.A.)). These pumps can be filled with oligonucleotides in solution, and will deliver the oligonucleotides by an osmotic displacement mechanism. Mini- osmotic pumps can have a distinct advantage over direct injection for delivery of therapeutic agents such as DNAzymes because they maintain a well-defined and consistent pattern of delivery and tissue exposure over a significant period of time. Molecular weight, physical conformation, and chemical properties do not affect the delivery rate of a given compound.

[00143] In certain example embodiments, a therapeutically effective amount of a composition described herein is delivered to a subject in need thereof. In certain example embodiments, an activity that inhibits or reduces mutant huntingtin protein aggregation is measured. Actual dosage levels of active ingredients in the compositions of the presently disclosed subject matter can be varied so as to administer an amount of the active compound(s) that is effective to achieve the desired therapeutic response for a particular subject. The selected dosage level will depend upon the activity of the therapeutic composition, the route of administration, combination with other drugs or treatments, the severity of the condition being treated, and the condition and prior medical history of the subject being treated. In certain example embodiments, the dose of DNA oligonucleotides can be 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150 mg per kg of body weight. In certain example embodiments, the dose of DNA oligonucleotides can be between closer to 100 mg/ kg of body weight, such as about 80-120 mg/kg of body weight, or even 90-110 mg/ kg of body weight.

[00144] In certain example embodiments, a skilled artisan can start doses of the compound at levels lower than required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. The potency of a composition can vary, and therefore a "therapeutically effective" amount can vary. However, using the assay methods described herein below, one skilled in the art can readily assess the potency and efficacy of a candidate modulator of this presently disclosed subject matter and adjust the therapeutic regimen accordingly.

[00145] In certain example embodiments, systemic administration of one or more of the DNA oligonucleotides described herein can result in a distribution of the one or more of the DNA oligonucleotides to a variety of organ and tissue types. For example, following intravenous injection, the DNA oligonucleotides as described herein can be distributed to the heart, spleen, lung, kidney, liver, brain, or other tissues, organs, of fluids. In certain example embodiments, the DNA oligonucleotides described herein may be distributed disproportionally amount various organs. For example, more of a given DNA oligonucleotide described herein may be distributed in the liver as compared to the heart.

[00146] Following administration of the DNA oligonucleotides to a subject, the DNA oligonucleotides described herein can be rapidly cleared. For example, the DNA oligonucleotides can be cleared via urine and feces, with the majority the DNA oligonucleotides being cleared within the first 24 hours following administration. In certain example embodiments, at least about 30%, 35%, 40%, 45%, 50%, 55%, 60% or more of the DNA oligonucleotides are cleared within the first 1, 2, 3, 4, 5, 6, or 7 hours following administration. In certain example embodiments, the majority of the remaining DNA oligonucleotides can be cleared over the next 5-72 hours, such as within about 10, 15, 20, 25, 30, 35, 40, 50, 60, or 70 hours or more.

[00147] In certain example embodiments, the DNA oligonucleotides are cleared primarily via urine excretion. That is, renal excretion can account for the major route of elimination of the DNA oligonucleotides described herein. In certain example embodiments, about 10%, 15%, 20%, 25%, 30% of the administered oligonucleotides are excreted via the urine within the first 1, 2, 3, 4, 5, 6, or 7 hours following administration. In certain example embodiments, about 35%, 40%, 45%, 50%, 55%, 60% or more of the DNA oligonucleotides are excreted in the urine within approximately the first 45, 50, 60, or 70 hours following administration.

[00148] After review of the disclosure provided herein, one of ordinary skill in the art can tailor the dosages to an individual subject, taking into account the particular formulation, method of administration to be used with the composition, and/or distribution of mutant huntingtin protein aggregates, for example. Further calculations of dose can consider subject height and weight, severity and stage of symptoms, and the presence of additional deleterious physical conditions. Such adjustments or variations, as well as evaluation of when and how to make such adjustments or variations, are well known to those of ordinary skill in the art of medicine.

EXAMPLES

[00149] The following examples further illustrate the invention but should not be construed as in any way limiting its scope. In light of the present disclosure and the general level of skill in the art, those of skill will appreciate that the following Examples are intended to be exemplary only and that numerous changes, modifications, and alterations can be employed without departing from the scope of the presently disclosed subject matter.

Example 1 - DNAzyme Design, Production and in vitro Testing.

[00150] Six different DNAzymes against the part of the huntingtin (Htt) transcript derived from human mutant exon 1 were constructed and tested in vitro for their ability to cleave mutant huntingtin mRNA. These include DNZ1 (SEQ ID NO: l), DNZ2 (SEQ ID NO:2), DNZ3 (SEQ ID NO:5), DNZ4 (SEQ ID NO:6), DNZ5 (SEQ ID NO: 3), and DNZ6 (SEQ ID NO: 4). Briefly, DNA oligonucleotides used in these experiments were synthesised by Integrated DNA Technology (Coralville, IA). DNAzymes were designed according to the rule of 10-23 DNAzyme, which contains a catalytic domain of 15 conserved deoxynucleotides flanked by two substrate-recognition domains. See Sun LQ, Cairns MJ, Saravolac EG, Baker A, Gerlach WL: Catalytic nucleic acids: from lab to applications. Pharmacol Rev 2000, 52:325-347.

[00151] As shown in FIG. 2, which shows the effects of these DNAzymes on mRNA for human mutant exon 1, two of these DNAzymes did not show in vitro ribonuclease activity against mutant exon 1 mRNA (namely, DNZ3 and DNZ4 in lanes 6 and 7), two cleaved the mRNA with low efficiency (DNZ1 and DNZ2 in lanes 4 and 5), and two cleaved with high efficiency (DNZ5 and DNZ6 in lanes 8 and 9) (FIG. 2). Note that each of the four effective DNAzymes cleaved mutant huntingtin exon 1 mRNA at a different site, as revealed by the differences in product size, because they are targeted to different parts of exon 1. A control DNAzyme with a scrambled variable sequence did not cleave mutant exon 1 mRNA, which was the expected outcome. Based on its efficacy in vitro, DNZ6 was selected for testing of its benefit in a mouse Huntington disease (HD) model expressing mutant exon 1, as described below.

[00152] While DNZ3 (SEQ ID NO:5) and DNZ4 (SEQ ID NO:6) did not cleave mutant exon 1 mRNA, these DNAzymes may very well be effective at treating Huntington disease as described herein. More particularly, DNZ3 and DNZ4 may be effective on other Htt Exon 1 RNA having different numbers of CAG repeat than those evaluated herein.

Example 2 - Testing Efficacy of Systemic DNAzyme Therapy in vivo

[00153] Testing of the effectiveness of anti-HD DNAzyme therapy was carried out in vivo using the R6/2 mouse transgenic model of Huntington's disease. This model is generally described in Mangiarini L et ah, Cell. 1996 Nov l;87(3):493-506, which is hereby incorporated herein in its entirety. Briefly, B6CBA-R6/2 (CAG 120 +/- 5) mice were obtained from The Jackson Laboratory™. These mice are transgenic for the promoter and exon 1 of a human HD gene with about 125 CAG repeats, which is ubiquitously expressed. With expression of the human HD gene these mice exhibit a progressive neurodegenerative phenotype that begins to be evident by 6-8 weeks of age, which typically culminates in death by 14-15 weeks. [00154] To test the effectiveness of DNAzyme therapy, either anti-huntingtin (Htt) DNAzyme DNZ6 or vehicle (sodium phosphate-buffered saline, PBS) was injected intraperitoneally in age-matched male R6/2 mice, and compared the results to PBS- injected wild-type (WT) males of the same age. DNZ6 was injected at either of two doses - 75 mg/kg (low dose) or 100 mg/kg (high dose). Mice were injected daily or 5 out of 7 days for different durations. Mice were then monitored for overall health (weight) and motor function (rotarod and open field), and they were afterwards sacrificed for histological and biochemical analysis.

[00155] As described in more detail below, we first examined the effects of low dose DNZ6, and found it produced slightly reduced brain levels of mutant Htt and yielded a modest functional benefit. We next examined high dose DNZ6, and found that it greatly reduced mutant Htt in the brain and in the periphery, and yielded a strong functional benefit. For many of the endpoints below, we present the effects with low dose and high dose DNZ6. Further, DNZ was found to increase survival of R6/2 mice.

Example 2A. - Systemic DNAzyme Therapy Showed Strong Benefit with Behavioral Testing in R6/2 Mice.

2.A.I. - Systemic DNZ6 Prevented Large Weight Drop in R6/2 Mice after 70 days.

[00156] The effect of low dose DNZ6 injected intraperitoneally in R6/2 mice 5 times a week was examined, beginning at the age of 32 days, compared to age-matched R6/2 mice injected according to the same schedule with PBS vehicle. Wild-type (WT) mice injected with PBS according to the same schedule also served as controls. The graph of FIG. 3 shows that WT mice steadily gain weight throughout the test period from day 32 to day 75. By contrast, weight gain in the R6/2 mice ceased around day 50. Weight thereafter remained stable in the R6/2 mice treated with DNZ6. In PBS -treated R6/2 mice, a weight decline was seen beginning at about 65 days of age, with weight gradually dropping below the initial 32-day weight by day 75. Thus, low dose DNZ6 slowed the weight loss seen in R6/2 mice, compared to PBS-treated R6/2 mice (FIG. 3).

[00157] In the cohort of R6/2 mice treated with high dose DNZ6, a similar beneficial effect on weight maintenance was seen. These mice, however, were only treated and tested until day 71 of age, before the age at which we saw maximal benefit in the low dose DNZ6 mice, and injections began somewhat later than for the low dose mice, at 48 days of age for high dose DNZ6. Nonetheless, R6/2 mice receiving high dose DNZ6 weighed nearly a gram more at 71 days of age than R6/2 mice receiving PBS over the same period. A similar outcome was seen at 71 days of age for the low dose DNZ6- treated mice, as shown in the above graph.

2.A.2. - Systemic DNZ6 Slowed Appearance of the Clasping Neurological Abnormality.

[00158] When suspended by their tail, neurologically impaired mice clasp their hindlimbs with their forelimbs, and R6/2 mice prominently show this abnormality as disease progresses. The effect of low dose DNZ6 on clasping in R6/2 mice was examined, when injected intraperitoneally with DNZ6 or PBS 5 times a week, beginning at the age of 32 days. R6/2 mice gradually developed this abnormality beginning at about 50 days of age, and it reached maximal severity in PBS-treated mice by about 75 days of age, using a 0 = normal and 3 = maximal clasping scoring system, as shown in FIG. 4A. By contrast, at these same ages, clasping was slightly less severe in DNZ6-treated mice. Note that the difference between groups was not yet evident at 71 days of age, but became evident and was sustained subsequently. WT control mice showed no clasping (as expected), and had a zero score on all test days, as shown in FIG. 4A.

[00159] The high dose DNZ6 mice showed a yet stronger clasping benefit than did the low dose DNZ6 mice, as shown in FIG. 4B. In particular, the clasping score was 40% better in DNZ6-treated mice at day 71 than in PBS-treated mice at the same time point, and was also significantly better than in low dose DNZ6 mice at the same time point (compare Fig. 4A to Fig. 4B).

2 A3. - Systemic DNZ6 Greatly Improved Rotarod and Open Field Motor Performance.

[00160] Open Field - Distance, Speed, and Anxiety. Benefit was seen in open field with high dose DNZ6 (FIG. 5). For example, WT mice injected intraperitoneally with PBS 5 times a week beginning at the age of 32 days and tested at 71 days of age traveled a distance of 26,805.7 cm in our 30-minute open field task, which is typical performance for WT mice. By contrast, R6/2 mice injected intraperitoneally with PBS at the same schedule or injected daily with PBS beginning at 48 days, and tested in both cases at 71 days of age, traveled only an average of 16,506.6 cm. R6/2 mice injected intraperitoneally with low dose DNZ6 at 5 times a week beginning at the age of 32 days and tested at 71 days of age did not show improved performance, as they traveled only an average of 15,218.1 cm. The high dose DNZ6 mice injected intraperitoneally daily beginning at the age of 48 days and tested at 71 days of age again showed a benefit that low dose DNZ6 did not. At day 71, high dose DNZ6-treated mice traveled 24,770.3 cm, which is about a 1.5-fold improvement over R6/2 control mice. Maximum speed showed a similar outcome as total distance traveled. Low DNZ did not improve a 38.5% loss seen in PBS-treated R6/2 mice, but high DNZ6 restored maximum speed to 85.0% of WT.

[00161] We also examined the effects of high dose DNZ6 on anxiety. As shown, high DNZ6 also largely rectified an anxiety increase seen in R6/2 mice at 71 days of age (FIG. 5), as shown by time spent in the middle part of the open field arena. Both PBS-treated R6/2 mice and low DNZ6-treated R6/2 mice spent about half as much time in the arena middle as WT mice (meaning approximately a doubling in anxiety). By contrast, high DNZ6 R6/2 mice spent about as much time as WT mice in the arena center (i.e. 98.8% of WT) - substantially less anxiety than seen in the PBS-treated and low DNZ6-treated R6/2 mice. These various results showing the high DNZ6 benefit in R6/2 males at 71 days for open field, expressed as a percent of WT, are shown in FIG. 5.

Example 2B. - Systemic DNAzyme Therapy Reduced Mutant Huntingtin mRNA, Reduced Protein Aggregates, and Improved Survival in R6/2 Mice.

2B.1. - Systemic DNZ6 Reduced Formation of Mutant Htt Aggregates R6/2 Brain.

[00162] Mutant huntingtin protein aggregates as 2-3 μιη wide balls within neuronal nuclei throughout the R6/2 mouse brain. These aggregates are commonly called neuronal intranuclear inclusions (Nils). The size and abundance of these mutant Htt aggregates in cortex and CA1 of hippocampus in mice from each of our R6/2 treatment groups was examined, using immunolabeling to detect the Nils. Cellular localization and development of neuronal intranuclear inclusions in striatal and cortical neurons in R6/2 transgenic mice. WT mice were not analyzed, as they do not express mutant protein, and Nils are thus completely absent in the brains of WT mice. Briefly, mutant protein aggregates (Nils) were labeled using standard indirect immunofluorescence procedures with a primary antibody against uniquitin, and a fluorophore-conjugated secondary antibody directed against the host for the primary antibody. See Mead et al., J Comp Neurol. 2002 Jul 29;449(3):241-69, which is hereby expressly incorporated herein in its entirety. Note that mutant huntingtin aggregates can be detected with anti-ubiquitin because the aggregates become highly ubiquitinated. Antigen retrieval is used to expose antigenic sites in the Nils, which otherwise largely remain cryptic. The immunofluorescent labeling is then imaged using confocal laser scanning microscopy. [00163] R6/2 mice injected intraperitoneally with PBS or low dose DNZ6 at 5 times a week beginning at the age of 32 days were examined at 76 days of age. Treatment with low dose DNZ6 reduced the abundance of mutant Htt aggregates in CA1 of the hippocampus by about 20% and the size about 12%, compared to PBS-treated R6/2 mice. In primary motor cortex, low dose DNZ6 reduced Nils in size by about 10%, but had no impact on abundance. Thus, chronic treatment with low dose Htt-DNZ appears to have only slightly reduced production of mutant protein by 10-20%, consistent with its relatively small benefit.

[00164] The R6/2 mice injected intraperitoneally daily with high dose DNZ6 or PBS beginning at the age of 48 days were examined for Nil formation at 71 days of age (FIG. 6). We found that high dose DNZ6 substantially reduced Nil abundance and size in brain. As shown in FIG. 7, the abundance of Nils in cerebral cortex and striatum was reduced to about 50% and 25% of that in PBS-treated R6/2 mice, respectively. Moreover, Nil size was reduced to 25% of that in PBS-treated R6/2 mice. As a result, the mutant Htt (mHtt) burden was reduced by about 60% for cortex and 85% for striatum. The reduction in Nil burden in cortex was significantly correlated with the improved weight maintenance and open field performance.

2B.2. - Systemic DNZ6 Greatly reduced Mutant Htt mRNA Production in R6/2 Brain and Peripheral Organs.

[00165] Knockdown efficacy using PCR measurements of brain and peripheral organ expression of mRNA for exon 1 mutant Htt was also examined. Because immunolabeling studies showed that DNZ6-mediated knockdown of mutant Htt in brain was meager with low dose DNZ6 but substantial with high dose DNZ6, we focused on samples from high dose DNZ6-treated mice. Briefly, for the knockdown efficacy study, PCR was used to measure the effect of daily high dose DNZ6 on expression of mutant Htt exon 1 in the brains of R6/2 mice treated daily with high dose DNZ6 versus those treated with PBS. PCR was also used to measure the effect of daily high dose DNZ6 on expression of endogenous mouse Htt.

[00166] As shown in the graph of FIG. 8, we found that brain message for mutant Htt exon 1 from R6/2 mice injected intraperitoneally daily with high dose DNZ6 beginning at the age of 48 days until 71 days of age was significantly reduced to about 50% of that in R6/2 mice treated with PBS according to the same schedule. When normalized to beta- actin expression, the result remained similar - the brain of high DNZ6 treated R/2 mice expressed only about 50% the amount of mutant Htt message as brain of the animals treated with PBS. Thus, the amount of mutant Htt knockdown in brain after high dose DNZ6 as detected by PCR was similar to that detected by immunolabeling for aggregated mutant in brain. Similar PCR results were obtained for peripheral organs (for example liver) - high DNZ6 yielded substantial mutant Htt message reduction compared to PBS treatment. In contrast, daily DNZ6 had no effect on message levels in brain for endogenous mouse Htt, demonstrating that DNZ6 is specific for human Htt.

2B.3. - Systemic DNZ6 Improved R6/2 Survival

[00167] Survival was compared for vehicle-treated and DNZ6-treated R6/2 mice using a mortality plot, in which the percent of original mice surviving at each given day of age out to 76 days is plotted. Note that over 80% of DNZ6-treated mice remain alive at 76 days but only about 60% of the vehicle-treated mice do. These data are presented in Figure 9.

Example 3. - Stability, Safety, & Pharmacokinetics of DNZ6

3.1. - DNZ6 is Stable and Active in a Variety of Solvents

[00168] The in vitro stability of DNZ6 was determined by incubating olionucleotides in PBS, saline and water at 37°C. An equal amount was removed at 2 and 24 hr and tested for its ability to cleave mutant exonl mRNA. Note that contrary to inactive DNZ molecules i.e. scrambled arm, scrambled active site and sense DNZ, DNZ 6 effectively cleaves mutant exon 1 mRNA, yielding different cleavage products (arrow) and solvents do not have any effect on the activity of DNZ toward cleaving Htt RNA (FIG. 10).

3.2 - Daily DNZ6 has No Adverse Effects on Behavior, Inflammation, or Liver Toxicity

[00169] To evaluate the safety of daily DNZ6 administration, the effects of daily DNZ6 administration on behavior, inflammation, and toxicity were assessed. Healthy, wild-type mice were injected i.p. with PBS or DNZ6 every day for 4 weeks. Following dosing, animals were observed daily for signs of toxicity including trouble grooming, lack of food consumption, and any other signs of lethargy; none were observed. At necropsy, liver, heart, lung, kidney, and spleen were collected and fixed in 4% paraformaldehyde, impregnated with 25% sucrose for cryoprotection, and were processed for histopathological evaluation (standard light microscopic examination of hematoxylin and eosin-stained tissue slides). [00170] To assess behavior, accelerating rotarod and open field testing were performed as shown in FIG. 11. Rotarod analysis was carried out using a San Diego Instruments™ (San Diego, CA) rodent rotarod. For the rotarod task, RPM increased from 0 to 30 over a four-minute period, and 30 RPM was then maintained for another 2 minutes. The first rotarod session was a 3 -trial training session one day, followed by a 3 -trial test session carried out the next day. Time to fall was the measure of rotarod performance. An automated 30-minute assessment of open field behavior was also conducted, using a Noldus Etho Vision video tracking system to record and digitize the mouse movements (Noldus Information Technology, The Netherlands). Software was used to dichotomize mouse movements into lingering episodes and progression segments, and calculate further parameters for these, such as speed and acceleration. Each animal was brought from its housing room, introduced into the open field arena and returned after the 30- minute session. The arena was 200 cm in diameter with a non-porous gray floor and a 50 cm high gray wall. DNZ6 had no significant effect in wild-type mice on rotarod (RR) performance or on such open field parameters as distance traveled, maximum speed, number of stops of anxiety (i.e. avoiding the arena center).

[00171] Immunogenesity and the effect of DNZ6 on innate immune system was determined by measuring the serum levels of anti-DNA antibody immunoglobin G (IgG) and the inflammatory cytokines [interleukin (IL)-6, I L 1 β , interferon (IFN)y, monocyte chemotactic protein (MCP)-l, and tumor necrosis factor (TNF)a] 4 weeks after treating the healthy mice with PBS or DNZ6, respectively using MILLIPLEX MAP Mouse Cytokine/Chemokine immunoassay kit, as described by the manufacturer (Millipore). No significant effect was seen of DNZ6 treatment in wild-type mice (FIG.

12).

[00172] Lastly, to assess the effect of DNZ6 liver toxicity, paraformaldehyde-fixed livers of animals treated with PBS or DNZ6 from day 37 to day 65 were dehydrated, embedded in paraffin, sectioned on a roatary microtome, collected on slides, and analyzed for cytotoxicity by H&E stain (FIG. 13). As shown, no differences were found between control and DNZ6 treated specimens in H&E tissue. Lobule organization is preserved with central veins in center (a). Nuclei (b) of hepatocytes are uniform in size throughout and no vacuolization is seen. Neutrophil (c) density is homogeneous and number is within normal limits. Holes (d) in section are where the sinusoids were cut along their diameter and are normal. No fibrosis, cirrhosis, or necrosis of any kind is noted. Sparse binucleation noted, within normal limits, no nuclear pleomorphism, apoptosis, steatosis, nuclear inclusions, or lymphocytosis is noted.

3.3 - DNZ6 is Broadly Distributed Among Tissue Types and is Readily Cleared

[00173] The systemic distribution of DNAzyme in mice and its rate of clearance were determined by injecting [ ^j5 S]-DNZ6 in saline i.p, to 21 healthy mice, and the amount of radioactivity present in blood and different organs was measured as a function of time. DNZ6 injected in this manner distributed to all major organs; liver, heart, spleen, kidney, and lungs (Fig. 14). Tissue distribution of [ ³⁵ S]-DNZ6 presented in average percentages revealed the order of accumulation is Liver>Kidney> Lung> Heart=Brain>Spleen.

[00174] To assess the tissue distribution profile of systemically administered DNZ6, [^Sj-D ZC-), 2()C)pL (lO mg/kg; 0.275 mCi/ kg) in saline was administered i.p. to 21 healthy mice and they were placed in metabolism cages, immediately following injection. Mice were divided into 7 groups of 3 animals each, and at 2, 4, 6, 24, 48, 72 hours, and 7 days following i.p. administration of [ ³⁵ S]-DNZ6, one group of animals was euthanized and liver, heart, lung, kidney, spleen, intestines, brain, pancreas, and stomach were removed for determination of total radioactivity. Immediately prior to sacrifice blood samples (0.5—1.0 mL) were collected from anesthetized mice via the retroorbital vein.

[00175] All samples were collected and stored at -80°C until analysis. DNZ6 injected in this manner distributed to all major organs; liver, heart, spleen, kidney, and lungs (Fig. 14). Tissue distribution of [ ⁵ S]-DNZ6 presented in average percentages revealed the order of accumulation is Liver>Kidney> Lung> Heart=Brain>Spleen

[00176] In addition to assessing the tissue distribution of DNZ6, the rate of clearance of [ ^j:, S]-DNZ6 from healthy mice was examined by determining the total radioactivity present in the urine and feces of the treated animals as a function of time. Renal excretion represented the major pathway of DNZ6 elimination (Fig. 15). The amount of DNZ6 excreted in urine was about 5 -fold greater than the amount released from feces over the 72 h period (Fig. 15). As expected, DNZ6 excretion was time-dependent with the majority being eliminated in the first 72 hours (Fig. 15). SEQUENCE LISTING FREE TEXT

[00177] The nucleic and amino acid sequences listed below use standard letter abbreviations for nucleotide base. If only one strand of each nucleic acid sequence is shown, the complementary strand is understood as included by any reference to the displayed strand.

SEQ ID NO: 1 (or DNZ ^* 1 ) 5'-tttccagggggctagctacaacgacgccatggt-3' SEQ ID NO: 2 (or DNZ 2) 5'-cttcatcagggctagctacaacgattttccagg-3' SEQ ID NO: 3 (or DNZ 5) 5 ' -cttg agg g ag g ctag ctacaacg atcg aagg cc-3 ' SEQ ID NO: 4 (or DNZ 6) 5'-ttggaaggaggctagctacaacgattgagggac-3' SEQ ID NO: 5 (or DNZ 3) 5'-aggccttcaggctagctacaacgacagcttttc-3' SEQ ID NO: 6 (or DNZ 4) 5'-actcgaaggggctagctacaacgacttcatcag-3' SEQ ID NO: 7: (core) 5'-ggctagctacaacga-3'

*DNZ = DNAzyme