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WO2018145009A1 - Méthodes et compositions à base d'adnzyme pour le traitement de la maladie de huntington - Google Patents

Méthodes et compositions à base d'adnzyme pour le traitement de la maladie de huntington Download PDF

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WO2018145009A1
WO2018145009A1 PCT/US2018/016874 US2018016874W WO2018145009A1 WO 2018145009 A1 WO2018145009 A1 WO 2018145009A1 US 2018016874 W US2018016874 W US 2018016874W WO 2018145009 A1 WO2018145009 A1 WO 2018145009A1
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dna
mutant huntingtin
enzymatic
mrna
dnz6
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PCT/US2018/016874
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English (en)
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Tayebeh Pourmotabbed
Anton REINER
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University Of Tennessee Research Foundation
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/711—Natural deoxyribonucleic acids, i.e. containing only 2'-deoxyriboses attached to adenine, guanine, cytosine or thymine and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7115—Nucleic acids or oligonucleotides having modified bases, i.e. other than adenine, guanine, cytosine, uracil or thymine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/12—Type of nucleic acid catalytic nucleic acids, e.g. ribozymes
    • C12N2310/127—DNAzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/34—Allele or polymorphism specific uses

Definitions

  • 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.
  • HD Huntington's disease
  • 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.
  • HD 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.
  • 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.
  • a method of modulating cellular expression of mutant huntingtin protein 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.
  • mRNA messenger ribonucleotide
  • 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.
  • 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.
  • the method for modulating the cellular expression of the mutant huntingtin protein includes reducing or inhibiting the expression of the mutant huntingtin protein.
  • the DNA oligonucleotide is a catalytic antisense polynucleotide.
  • the DNA oligonucleotide is a DNAzyme.
  • the DNA oligonucleotide can also be modified, for example, to increase the stability of the DNA oligonucleotide.
  • contacting the cell with the DNA oligonucleotide results in introduction of the DNA oligonucleotide into the contacted cell.
  • the target region of the mRNA includes a mutant huntingtin exon 1 mRNA.
  • contacting the cell with the DNA oligonucleotide results in a reduction in mutant huntingtin protein and mRNA.
  • a method for treating Huntington's disease in a subject can be selected.
  • 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.
  • mRNA messenger ribonucleotide
  • a therapeutically effective amount of one or more DNA oligonucleotides are administered to the subject.
  • the DNA oligonucleotide can include a nucleotide sequence that is identical to any one of the nucleotide sequences set forth as SEQ ID NOS: l-4.
  • 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.
  • the method of treatment includes reducing or inhibiting the expression of the mutant huntingtin protein in the subject.
  • the administered DNA oligonucleotide is a catalytic antisense polynucleotide.
  • 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.
  • the target region of the mRNA includes a mutant huntingtin exon 1 mRNA.
  • 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.
  • 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.
  • 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.
  • the enzymatic DNA molecule includes a modification that increases the stability of the DNA molecule.
  • the enzymatic DNA molecule can include an inverted deoxythymidine at the 3' end of the DNA molecule.
  • the target region of the mRNA includes a mutant huntingtin exon 1 mRNA.
  • 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.
  • 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.
  • 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.
  • 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.
  • the target region of the mRNA includes a mutant huntingtin exon 1 mRNA.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • DNZ1 SEQ ID NO: l
  • DNZ2 SEQ ID NO:2
  • DNZ3 SEQ ID NO:5
  • DNZ6 SEQ ID NO: 4
  • Lane 2 shows the band for mutant Htt exon 1 signal.
  • DNZ3 and DNZ4 did not cleave exon 1 RNA, as there were no cleavage products evident.
  • DNZ1 and DNZ2 did cleave exon 1 RNA, the efficiency was low.
  • 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.
  • 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.
  • 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.
  • 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 ⁇ 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.
  • 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.
  • 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.
  • 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. 11 is a graph showing the effect of DNZ6 administration on rotarod open field performance, in accordance with certain example embodiments.
  • 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).
  • 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- ⁇ , IFNy, MCP1, TNFa and IgG2a in the DNZ6-treated mice compared to PBS mice.
  • 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.
  • 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.
  • [ 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.
  • FIG. 15 is a graph showing the elimination profile of DNZ6 in healthy mice, in accordance with certain example embodiments.
  • the rate of clearance of DNZ6 was also determined as a function of time.
  • 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.
  • compositions that include DNA oligonucleotides that have targeting specificity for RNA encoding mutant huntingtin protein.
  • DNA oligonucleotides that have targeting specificity for RNA encoding mutant huntingtin protein.
  • DNA oligonucleotides When introduced into a cell, such DNA oligonucleotides reduce expression of the mutant huntingtin protein in a cell.
  • 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.
  • mutant huntingtin protein is selectively reduced or inhibited, in accordance with the methods and compositions described herein.
  • the methods and compositions describe herein can be used to treat Huntington's disease, including adult and juvenile onset Huntington's disease.
  • 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).
  • 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).
  • 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.
  • a relatively low concentration of DNAzymes is sufficient to efficiently reduce levels of specific disease- causing proteins and thereby abate disease.
  • DNAzymes that cleave RNA coding for specific protein products related to Huntington's disease.
  • 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.
  • DNAzyme technology to reduce production of mutant huntingtin protein, the causal agent in Huntington's disease (HD).
  • 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.
  • systemic DNAzyme therapy can be discontinued at any time, adverse side effects can be more readily curtailed than with other gene therapy approaches.
  • viral delivery of knockdown constructs as is the case for shRNA therapy, is permanent and any adverse side effects cannot be easily halted.
  • systemic DNAzyme treatment as described herein, may improve overall health in HD victims.
  • 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.
  • 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.
  • signaling 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.
  • zymatic 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a target cell can comprise a cell that expresses mutant huntingtin protein gene.
  • cellular 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.
  • 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.
  • 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.
  • modulate can mean “inhibit”, “suppress,” or “activate”, but the use of the word “modulate” is not limited to this definition.
  • 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.
  • inhibition with a DNA oligonucleotide described herein results in a decrease in the steady state level of a target RNA, such as an mRNA.
  • inhibition with the DNA oligonucleotide described herein results in a decrease in the steady state of mRNA transcripts encoding mutant huntingtin protein.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • a gene comprises a coding strand and a non-coding strand.
  • 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.
  • the coding/sense strand includes thymidine residues instead of the uridine residues found in the corresponding mRNA.
  • the coding/sense strand can also include additional elements not found in the mRNA including, but not limited to promoters, enhancers, and introns.
  • 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.
  • 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.
  • 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.
  • 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.
  • sequence identity refers 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.
  • 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.
  • NCBI National Center for Biotechnology Information
  • 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).
  • 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.
  • 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.
  • 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.
  • nucleic acid bases 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.
  • modified bases in this aspect is meant nucleotide bases other than adenine, guanine, cytosine, and uracil at position or their equivalents.
  • 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 ⁇ -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.
  • 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.
  • 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.
  • DNA molecule is a polymeric chain of single- or double-stranded nucleotides and also referred to herein as “oligonucleotide” and “polynucleotide.”
  • oligonucleotide and polynucleotide.
  • 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.
  • 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 ⁇ -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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • administering refers to the introduction of a composition into a subject by a chosen route.
  • 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.
  • 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.
  • infusion pump e.g. an osmotic infusion pump
  • stent e.g. an osmotic infusion pump
  • 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.
  • 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).
  • polypeptide refers to any polymeric chain of amino acids.
  • peptide and protein are used interchangeably with the term polypeptide and also refer to a polymeric chain of amino acids.
  • 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.
  • biodegradable linker 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.
  • biodegradable refers to degradation in a biological system, for example enzymatic degradation or chemical degradation.
  • biologically active molecule refers to compounds or molecules that are capable of eliciting or modifying a biological response in a system.
  • biologically active molecules 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.
  • carrier refers to conventional pharmaceutically acceptable carriers.
  • the nature of the carrier will depend on the particular mode of administration being employed.
  • 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.
  • non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate.
  • 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.
  • 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.
  • 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
  • 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.
  • an 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.
  • 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.
  • the therapeutically effective amount of the composition may inhibit or decrease the expression of mutant huntingtin protein.
  • the therapeutically effective amount of the composition may reduce the appearance of mutant huntingtin protein aggregates in a tissue.
  • 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.
  • a label includes, for example, a moiety via which an oligonucleotide can be detected or purified.
  • purified refers 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.
  • purified does not require absolute purity. Rather, this term is intended as a relative term.
  • 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).
  • 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.
  • the nucleic acid can be purified or isolated, for example, and still include components or impurities generated from the synthesis reaction.
  • 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.
  • a subject includes a subject suffering from Huntington's disease.
  • treating refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop.
  • ameliorating 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.
  • 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.
  • 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.
  • vector 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • promoter refers 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.
  • 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.
  • promoters are often classified as “constitutive”, “tissue-specific”, “cell-type-specific”, or “inducible”, depending on their functional activities in vivo or in vitro.
  • 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 ⁇ -actin promoter (see, e.g. Williams et al., 1993), and other constitutive promoters known to those of skill in the art.
  • HPRT hypoxanthine phosphoribosyl transferase
  • DHFR dihydrofolate reductase
  • PGK phosphoglycerate kinase
  • pyruvate kinase phosphoglycerate mutase
  • ⁇ -actin promoter see, e.
  • tissue-specific or “cell-type-specific” promoters 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.
  • 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
  • host cell 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.
  • 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.
  • 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.
  • the substrate binding domain 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.
  • the enzymatic DNA molecule first recognizes the target RNA and then binds a target RNA through complementary base-pairing.
  • 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.
  • 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.
  • 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.
  • 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.
  • the DNA oligonucleotide sequence is a DNAzyme.
  • 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.
  • the enzymatic DNA molecule has a nucleotide sequence comprising the sequence set forth as any one of SEQ ID NOS: 1-4.
  • 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.
  • 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.
  • the conserved core comprises one or more conserved sequences.
  • 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.
  • 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.
  • the enzymatic DNA molecule comprises a catalytic core having the sequence set forth as SEQ ID NO: 7 (i.e., ggctagctacaacga).
  • 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.
  • the conserved core can comprise a substitution in a conserved sequence, but wherein intramolecular interactions are preserved by the substitution.
  • 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.
  • 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.
  • 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.
  • 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.
  • the substrate binding domain includes 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 nucleotides.
  • 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).
  • the catalytic domain may optionally contain stem-loop structures in addition to the nucleotides required for catalytic activity.
  • the enzymatic nucleic acid molecules described herein can have substrate binding domains that are contiguous or noncontiguous and can be varying lengths.
  • the length of each substrate binding domain may be greater than or equal to four nucleotides, such as 5-30 nucleotides.
  • 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.
  • 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).
  • the enzymatic DNA molecules disclosed herein may also include those with altered substrate binding domains.
  • 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
  • binding specificity refers to the substrate binding domain being identical to or complementary to (i.e., able to base-pair with) a portion of its substrate.
  • identity or complementarity can be 100%, but can be less if desired.
  • 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.
  • 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.
  • 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.
  • 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.
  • the enzymatic DNA molecules provided herein 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.
  • 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.
  • nucleic acid motifs 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.
  • 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.
  • a vector for producing the enzymatic DNA molecules described herein includes a sequence encoding one more of the DNA molecules described herein.
  • 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.
  • 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.
  • 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.
  • small-scale syntheses can be conducted on an Applied BiosystemsTM 3400 DNA Synthesizer (Applied Biosystems Inc., Foster City, Calif., United States of America) along with standard protocols associated therewith.
  • deprotection of the DNA-based oligonucleotides is performed in accordance with methods generally known to those skilled in the art.
  • 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.
  • 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.
  • a target cell 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.
  • 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.
  • 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.
  • 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.
  • enzymatically active portions e.g. catalytic domains
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the enzymatic DNA molecules described herein can be modified extensively to enhance stability.
  • 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).
  • 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.
  • 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.
  • nucleic acid molecules incorporating various modifications 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.
  • 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).
  • 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.
  • 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).
  • conjugates and/or complexes of enzymatic DNA molecules 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.
  • 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.
  • the transporters described are designed to be used either individually or as part of a multi-component system, with or without degradable linkers.
  • 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.
  • 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.
  • 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.
  • the enzymatic DNA molecules described herein are modified to comprise one or more 5' and/or 3 '-cap structures.
  • 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.
  • 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 nu
  • 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;
  • 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.
  • oligonucleotide backbone modifications see Hunziker & Leumann, 1995 and De Mesmaeker et al., 1994.
  • amino 2'— NH2 or 2'-0— NH2, which can be modified or unmodified.
  • 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.
  • 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).
  • compositions that are useful for treating Huntington's disease.
  • the methods for treating Huntington's disease make use of the compositions and formulations described herein.
  • methods for modulating the cellular expression of mutant huntingtin protein 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 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.
  • 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.
  • 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.
  • the DNA oligonucleotide can exert its catalytic activity on the mRNA encoding the mutant huntingtin protein, thereby modulating the expression the mutant huntingtin protein.
  • the enzymatic DNA oligonucleotides can thus be used to treat Huntington's disease.
  • the methods include, for example, selecting a subject having Huntington's disease.
  • 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.
  • 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.
  • 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.
  • 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.
  • mRNA messenger ribonucleotide
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • mutant huntingtin protein or reduction of mutant huntingtin protein as described herein can be determined by various mean known in the art.
  • 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.
  • 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.
  • compositions 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.
  • 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 can be used in conjunction with the methods described herein, such as in the treatment of Huntington's disease.
  • 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.
  • 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.
  • the DNA oligonucleotides of the compositions can be an enzymatic DNA oligonucleotide, such as a DNAzyme.
  • 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.
  • the DNAzyme of the composition has a nucleotide sequence including any one of the sequences set forth as SEQ ID NOS. 1-4.
  • 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.
  • the DNA oligonucleotide of the composition is a catalytic antisense polynucleotide.
  • 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.
  • the DNA oligonucleotide can include an inverted deoxythymidine at the 3' end of the DNA oligonucleotide.
  • 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.
  • 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
  • another DNA oligonucleotide of the composition can have binding specificity for a different region of the mRNA encoding the mutant huntingtin protein.
  • the composition can include a carrier, which can be a pharmaceutically acceptable carrier thereby providing a composition suitable for administration to a subject.
  • a suitable pharmaceutical formulation can be used to prepare the composition for administration to a subject.
  • 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.
  • 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., GibcoTM PBS or LonzaTMPBS).
  • compositions can include aqueous and nonaqueous sterile suspensions, such as suspending agents and thickening agents.
  • 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.
  • 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).
  • 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 of the compositions and formulations described herein can be by any method known to those skilled in the art.
  • 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.
  • 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.
  • 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.
  • the method of administration encompasses features for steady-state regionalized delivery or accumulation at the site in need of treatment.
  • 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.)).
  • ALZET® mini-osmotic pump DURECT Corporation, Cupertino, Calif., U.S.A.
  • 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.
  • a therapeutically effective amount of a composition described herein is delivered to a subject in need thereof.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the DNA oligonucleotides as described herein can be distributed to the heart, spleen, lung, kidney, liver, brain, or other tissues, organs, of fluids.
  • 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.
  • the DNA oligonucleotides described herein can be rapidly cleared.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Htt huntingtin
  • DNZ1 SEQ ID NO: l
  • DNZ2 SEQ ID NO:2
  • DNZ3 SEQ ID NO:5
  • DNZ4 SEQ ID NO:6
  • 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.
  • 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).
  • 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.
  • HD Huntington disease
  • DNZ3 SEQ ID NO:5
  • DNZ4 SEQ ID NO:6
  • 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.
  • mice were obtained from The Jackson LaboratoryTM. 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.
  • Example 2A - Systemic DNAzyme Therapy Showed Strong Benefit with Behavioral Testing in R6/2 Mice.
  • the high dose DNZ6 mice showed a yet stronger clasping benefit than did the low dose DNZ6 mice, as shown in FIG. 4B.
  • 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).
  • 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.
  • Example 2B - Systemic DNAzyme Therapy Reduced Mutant Huntingtin mRNA, Reduced Protein Aggregates, and Improved Survival in R6/2 Mice.
  • mutant protein aggregates 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.
  • DNZ6 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).
  • mice 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.
  • 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).
  • FIG. 11 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 InstrumentsTM (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).
  • 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).
  • 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 ⁇ , 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.
  • IgG anti-DNA antibody immunoglobin G
  • IFN interferon
  • MCP monocyte chemotactic protein
  • TNF tumor necrosis factor
  • 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.
  • 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'-actcgaaggggctagctacaac

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Abstract

L'invention concerne des méthodes et des compositions qui permettent de traiter la maladie de Huntington. Par exemple, l'invention concerne des compositions qui comprennent des oligonucléotides d'ADN qui présentent une spécificité de ciblage pour l'ARN codant pour la protéine huntingtine mutante. Lorsqu'ils sont introduits dans une cellule, les oligonucléotides d'ADN diminuent l'expression de la protéine huntingtine mutante dans la cellule. Par exemple, l'oligonucléotide d'ADN, fonctionnant comme une molécule d'ADN à activité enzymatique (ou ADNzyme), clive l'ARN codant pour la protéine mutante liée à l'ARNm, rendant ainsi l'ARNm incapable d'expression. Ceci signifie que l'événement de clivage rend l'ARN non fonctionnel et diminue ou supprime l'expression de la protéine provenant de cet ARN. Par conséquent, la synthèse de la protéine huntingtine mutante est sélectivement diminuée ou inhibée, conformément aux méthodes et aux compositions décrites dans la description. Du fait qu'elles suppriment ou diminuent l'expression de la protéine huntingtine mutante, les méthodes et les compositions de l'invention peuvent être utilisées pour traiter la maladie de Huntington, y compris les formes adulte et juvénile de la maladie de Huntington.
PCT/US2018/016874 2017-02-06 2018-02-05 Méthodes et compositions à base d'adnzyme pour le traitement de la maladie de huntington WO2018145009A1 (fr)

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