Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
  • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P13/00—Drugs for disorders of the urinary system
  • A61P13/12—Drugs for disorders of the urinary system of the kidneys
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
  • A61P21/02—Muscle relaxants, e.g. for tetanus or cramps
• • 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
• • 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
  • A61P25/16—Anti-Parkinson drugs
• • 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
  • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
  • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/04—Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

• the present invention is in the field of cell biology, and relates to compounds, compositions, and pharmaceutical compositions capable of disrupting pathological protein aggregates, to methods for identifying such compounds, compositions, and pharmaceutical compositions, and to methods for treating diseases that are characterized by protein misassembly and aggregation.
• the misassembly and aggregation of proteins that are normally soluble appears to be responsible for a wide variety of diseases. These include inherited neurodegenerative diseases, such as Huntington's disease (“HD”), Alzheimer's disease, cystic fibrosis, amyotrophic lateral sclerosis and Parkinson's disease; inherited blood disorders; infectious prion diseases, such as scrapie of sheep and goats, bovine spongiform encephalopathy of cattle, and kuru, Creutzfeldt-Jakob Disease (CJD), Gerstmann-Straussler-Sheinker Disease, and fatal familial insomnia of humans.
• HD Huntington's disease
• CJD Creutzfeldt-Jakob Disease
• Gerstmann-Straussler-Sheinker Disease and fatal familial insomnia of humans.
• Huntington's disease belongs to a family of neurodegenerative diseases, including spinobulbar muscular atrophy, spinocerebellar ataxia types 1 , 2, 3, 6, and 7, and dentatorubral-pallidoluysian atrophy that in most cases are dominantly inherited and that are characterized by mutations in which CAG trinucleotide sequences are expanded, causing the translation of proteins with abnormally long polyglutamine tracts. See, e.g., Carmichael ef a/., Proc. Nat'l Acad. Sci. USA, 97:9701-9705 (2000). Although the diseases result in neurodegeneration at different locations, they all share the same basic pathological cause: the formation of intracellular protein aggregates within the vulnerable neurons and the consequent loss of function of those neurons.
• the protein aggregates are composed of ubiquitinated, N-terminal fragments of "Huntingtin", the protein encoded by the gene associated with the disease. DiFiglia et al., Science, 277:1990-93 (1997). These fragments include the region of polyglutamine expansion that is associated with the disease-causing form of the protein. Both formation of these aggregates in mammalian cell models and cell death have been found to be reduced in the presence of a bacterial chaperone, GroEL, and HSP104, a yeast heat shock protein, suggesting that there is a causal link between aggregation of the protein and disease pathology. Carmichael ef al., Proc. Nat'l Acad. Sci. USA, 97:9701-9705 (2000),
• Alzheimer's disease is another disorder in which protein aggregation appears to be responsible, at least in part, for the pathology of the disease.
• Amyloid isolated from the brain tissue of Alzheimer's disease patients consists mainly of proteins from the family designated "A ⁇ " (for Amyloid plaque of ⁇ _ secondary structure). Koo et al., Proc. Nat'l Acad. Sci. USA, 96:9989-90 (1999).
• a minor component of the amyloid plaques, A ⁇ 4 2 contains a sequence that can form unusually stable and ordered fibrils.
• This component may be responsible for "nucleating" fibril formation, Although A ⁇ 2 levels are not elevated in the most common, sporadic forms of Alzheimer's disease, they do increase in patients with mutations in presenilin 1 , presenilin 2 and amyloid beta-protein precursor that are linked to familial Alzheimer's disease. Scheuner et al., Nature Med., 2:864-70 (1996). Increased levels of A ⁇ 2 may arise from changes in the cleavage pattern of the ⁇ -amyloid precursor proteins ("APP") (Sisodia, Science, 289:2296-97 (2000)) or by decreased levels of degradation of A ⁇ 42 itself. Iwata et al., Science, 292:1550-52 (2001).
• APP ⁇ -amyloid precursor proteins
• Parkinson's disease is another common neurodegenerative motor disorder that is believed to result from improper protein interactions. Although environmental factors had long been thought to be responsible for the condition, genetic factors have now been implicated as well. See Cole et al, Neuromolecular Med., 1 :95-109 (2002). In particular, mutations in the gene encoding the presynaptic protein, ⁇ -synuclein, may be responsible for the accumulation of this protein in Lewy bodies, the neuropathological hallmark of Parkinson's disease. [0009] Prion diseases also appear to result from the misfolding and aggregation of specific proteins. PrP c is the normal form of the prion protein and is encoded by a single- copy gene in mammals.
• PrP Sc Basler ef al., Cell, 46:417-28 (1986). When expressed, the protein is generally found on the surface of neuronal cells. Prion diseases are believed to result from the conversion of the normal, PrP c -form of the prion protein to an insoluble, disease- causing form, PrP Sc . Although the amino acid sequences of the two forms are identical, the proteins appear to differ in conformation. Pan et al., Proc. Nat'l Acad. Sci. USA, 90:10962- 66 (1993). PrP Sc appears to be involved both in the transmission of prion diseases and in their pathogenesis.
• prion diseases include scrapie of sheep and goats; bovine spongiform encephalopathy of cattle; and kuru, Creutzfeldt-Jakob Disease, Gerstmann-Straussler-Sheinker Disease, and fatal familial insomnia of humans. See Prusiner et al., U.S. Patent No. 6,214,366.
• Protein misassembly and aggregation have also been implicated in the syndromes known as familial amyloid polyneuropathy and senile systemic amyloidosis. Kelly, Curr. Opin, Struct. Biol., 6:11-17 (1996).
• Amyloid fibrils form in various tissues from mutated forms of transthyretin, a tetrameric protein involved in the transport of thyroxine and retinol. Jacobson et al, Adv. Hum. Genet, 20:69-123 (1991).
• the mutations appear to destabilize the tetrameric structure and allow an amyloidogenic intermediate to form under the low pH conditions found in the lysosome. Miroy et al, Proc. Nat'l Acad. Sci. USA 93:15051-56 (1996).
• the wild-type form of the protein remains tetrameric and nonamyloidogenic under these conditions.
• diseases are thought to involve protein misfolding, misassembly, and/or aggregation.
• diseases include: amyotrophic lateral sclerosis (superoxide dismutase), Pick's disease (tau protein in Pick bodies), diabetes type II (amylin), multiple myeloma-plasma cell dyscrasias (IgG light chain), medullary carcinoma of the thyroid (procalcitonin), chronic renal failure ( ⁇ 2-microglobulin), congestive heart failure (atrial natriuretic factor), chronic inflammation (serum amyloid A), atherosclerosis (apoA1), familial amyloidosis (gelsolin). See Prusiner et al., U.S. Patent No. 6,214,366.
• a small-molecule inhibitor of the binding of serum amyloid P component ("SAP") to fibrils has recently been identified and suggested for use in the treatment of human amyloidosis.
• SAP serum amyloid P component
• Pepys ef al. Nature, 417:254-59 (2002).
• Treatment of seven human systemic amyloidosis patients with a small-molecule inhibitor of SAP binding to amyloid fibrils resulted in a substantial decrease in levels of circulating SAP.
• Pepys et al. Nature, 417:254-59 (2002).
• Methods to screen for additional inhibitors of SAP binding to amyloid fibrils have been disclosed.
• Transglutaminase inhibitors such as cystamine and monodansyl cadaverine, have been used to suppress aggregate formation and apoptotic cell death in cultured cells that express proteins containing expanded polyglutamine segments.
• Tsuji U.S. Patent No. 6,355,690.
• the present invention is based upon the unexpected discovery that oligonucleotides unrelated in sequence to that of the nucleic acid which encodes the protein aggregant can be effective in disrupting or preventing aggregation in disorders of protein assembly.
• the invention provides a method for identifying oligonucleotides that are effective to prevent, reduce or disrupt aggregation of a protein aggregant in a cell.
• the method comprises identifying, from a plurality of oligonucleotide species differing in sequence, those oligonucleotide species that are effective to prevent, reduce, or disrupt aggregation of a protein aggregant in a cell, by introducing each of a plurality of oligonucleotide species of disparate sequence separately into cells that have or are likely to develop protein aggregates, and identifying oligonucleotide species that are effective at preventing, reducing, or disrupting aggregation and/or increasing cell survival.
• the protein aggregant may be selected, among others, from the group consisting of huntingtin (htt), A ⁇ , tau, ⁇ -synuclein, atropin-1 , ataxin-1 , ataxin-2, ataxin-3, ataxin-7, alpha 1A, PrP Sc , transthyretin, superoxide dismutase, amylin, IgG light chain, procalcitonin, ⁇ a-microglobulin, atrial natriuretic factor, serum amyloid A, apoA1 , and gelsolin.
• htt huntingtin
• a ⁇ A ⁇
• tau ⁇ -synuclein
• atropin-1 ataxin-1
• ataxin-2 ataxin-2
• ataxin-3 ataxin-7
• alpha 1A PrP Sc
• transthyretin superoxide dismutase
• amylin IgG light chain
• procalcitonin ⁇ a-microglobulin
• the oligonucleotides can be at least 4 nt in length, typically at least 6 nt in length, and may usefully be at least 9 nt, 25 nt, even 30 nt or more in length; in some embodiments, the oligonucleotides can be at laest 35 nt, 40 nt, 45 nt, even 50 nt in length or more.
• the oligonucleotides may usefully and typically have a modification, such as one or more phosphorothioate linkages or 2'-OMe analogues.
• the oligonucleotides are nonidentical and noncomplementary in sequence to any portion of 10 or more contiguous nucleotides of the nucleic acid that encodes the protein aggregant. In other embodiments, the oligonucleotides may bear sequence complementarity or identity, at least in part, to a portion of the nucleic acid sequence that encodes the protein aggregant. In screening embodiments, the plurality of oligonucleotide species are typically nonidentical to one another. [0026] In one series of embodiments, the oligonucleotides are introduced into the cells in vitro, typically by a transfection method selected from the group consisting of passive transfection, chemical transfection and mechanical transfection.
• Either or both of the oligonucleotide and protein aggregant can be detectably labeled.
• the label is recombinantly fused to the aggregant.
• Such labels may, for example, be a polypeptide comprising a GFP-like chromophore.
• the oligonucleotides are introduced into the cells in vivo.
• the invention provides a method of treating a subject having a disorder of protein assembly.
• the method comprises administering an effective amount of a composition that comprises at least one oligonucleotide species that prevents, reduces, or disrupts protein aggregation, optionally in admixture with a pharmaceutically acceptable carrier or excipient.
• the oligonucleotide is at least 4 nt in length, typically at least 6 nt in length, and may usefully be at least 9 nt, 25 nt, even 30 nt or more in length; in some embodiments, the oligonucleotides can be at least 35 nt, 40 nt, 45 nt, even 50 nt in length or more.
• the oligonucleotides may usefully and typically have a modification, such as one or more phosphorothioate linkages or 2'-OMe analogues.
• the oligonucleotides are nonidentical and noncomplementary in sequence to any portion of 10 or more contiguous nucleotides of the nucleic acid that encodes the protein aggregant. In other embodiments, the oligonucleotides may bear sequence complementarity or identity, at least in part, to a portion of the nucleic acid sequence that encodes the protein aggregant.
• At least one of the oligonucleotide species in the pharmaceutical composition comprises at least one terminal modification, such as a terminal phosphorothioate linkage or 2'-OMe analogue.
• the composition comprises at least two oligonucleotide species differing in one or more of sequence, length, or composition, often as many as 3, 4, 5, or even as many as 10 - 50 different oligonucleotide species that differ in any one or more of sequence, length, or composition.
• the therapeutic method may be used to treat disorders having a protein aggregation or misassembly etiology, such as Alzheimer's disease, Huntington's disease, cystic fibrosis, amyotrophic lateral sclerosis, Parkinson's disease, spinobulbar muscular atrophy, spinocerebellar ataxia types 1 , 2, 3, 6, and 7, dentatorubral-pallidoluysian atrophy, prion diseases, scrapie, bovine spongiform encephalopathy, CJD, new variant CJD, Pick's disease, diabetes type II, multiple myeloma-plasma cell dyscrasias, medullary carcinoma of the thyroid, chronic renal failure, congestive heart failure, chronic inflammation, atherosclerosis (apoA1 ), and familial amyloidosis.
• a protein aggregation or misassembly etiology such as Alzheimer's disease, Huntington's disease, cystic fibrosis, amyotrophic
• FIG. 1 A is a flow chart displaying an experimental protocol for assaying oligonucleotides in cells cultured in vitro for their ability to disrupt or prevent aggregation of a protein aggregant, according to the present invention
• FIG. 1 B is a flow chart displaying in greater detail an experimental protocol for assaying oligonucleotides in cells cultured in vitro for their ability to disrupt or prevent aggregation of a protein aggregant, according to the present invention
• FIG. 1 C is a flow chart schematizing an experimental protocol for assaying olignucleotides in cells cultured in vitro for their ability to disrupt or prevent aggregation of a protein aggregant, based upon cellular survival, according to the present invention
• FIGS. 2A - 2C are fluorescence micrographs of PC12 cell cultures in which aggregates of huntingtin-GFP fusion protein appear light against a dark background, with FIG.
• FIG. 2A showing a control culture
• FIG. 2B showing a diminution in aggregate formation after transfection with oligonucleotide Kan UD3T/25G according to the present invention
• FIG. 2C showing a similar degree of diminution of aggregate formation after transfection with oligonucleotide Kan uD12T/25G according to the present invention (micrographs not to same scale);
• FIGS. 3A - 3C are fluorescence micrographs of PC12 cell cultures in which aggregates of huntingtin-GFP fusion protein appear light against a dark background, with FIG. 3A showing a control culture, FIG. 3B showing a diminution in aggregate formation after transfection with oligonucleotide Kan URD3/25G according to the present invention, and FIG. 3C showing the effect of transfecting with oligonucleotide Kan uR/25G according to the present invention;
• FIGS. 4A - 4E show fluorescence micrographs of PC12 cultures, with aggregates of huntingtin-GFP fusion proteins appearing light against a dark background, with FIGS. 4D and 4E showing untransfected control cultures, and FIGS. 4A - 4C showing cultures transfected with three different oligonucleotides according to the present invention, as indicated;
• FIG. 5 is a chart quantifying aggregate formation in a cell-based assay performed with the indicated oligonucleotides essentially in accordance with the protocol of FIG. 1 B, with the visible aggregates scored according to an odds ratio, the average fraction of cells containing aggregates reported in parentheses, and the error bar indicating the standard deviation calculated from averages of four independent experiments;
• FIG. 6 is a chart quantifying aggregate formation in a cell-based assay performed with the indicated oligonucleotides essentially in accordance with the protocol of FIG. 1B, with the visible aggregates scored according to an odds ratio, the average fraction of cells containing aggregates reported in parentheses, and the error bar indicating the standard deviation calculated from averages of four independent experiments;
• FIG. 7 charts cell survival after induction of mutant huntingtin production in the absence and in the presence of oligonucleotide HDS-9, according to the protocol schematized in FIG. 1C;
• FIG. 8 is a photomicrograph demonstrating the ready visualization of live cells attached to the flask surface in the cell survival assay of the present invention.
• FIG. 9A shows recombinant mutant huntingtin N-terminal aggregates captured on a cellulose acetate filter after incubation with the indicated oligonucleotides or compounds, according to the present invention, with FIG. 9B tabulating the quantity of aggregates as a percentage of aggregates observed in the negative control,
• compositions comprising oligonucleotides as short as about 4 nucleotides in length, and as long as about 25 nt in length, can effect the disruption of proteins that are pathologically aggregated within cells (hereinafter also called “protein aggregants” or “aggregants”).
• protein aggregants or "aggregants”
• the effect can be observed with oligonucleotides that bear no identifiable sequence relationship to the sequence of the gene encoding the protein aggregant.
• oligonucleotides Given the ease with which oligonucleotides can be synthesized, the ease with which they can be delivered to the interior of cells, the lack of systemic toxicity, and the wealth of dosing experience derived from a decade or more of antisense approaches, the use of short oligonucleotides to disrupt protein aggregations provides significant advantages over approaches currently being contemplated to treat these diseases.
• the oligonucleotides used in the compositions and methods of the present invention can be as short as 4 nucleotides in length, and as long as 25 nucleotides in length, and thus can be 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, exclusive of optional terminal blocking groups.
• the oligonucleotides can comprise nucleobases naturally found in nature in native 5'-3' phosphodiester internucleoside linkage — e.g., DNA, RNA, or chimeras thereof — or can contain any or all of nucleobases not found in nature (non-native nucleobases), nonnative internucleobase bonds, or post-synthesis modifications, either throughout the length of the oligonucleotide or localized to one or more portions thereof.
• nucleobases naturally found in nature in native 5'-3' phosphodiester internucleoside linkage — e.g., DNA, RNA, or chimeras thereof — or can contain any or all of nucleobases not found in nature (non-native nucleobases), nonnative internucleobase bonds, or post-synthesis modifications, either throughout the length of the oligonucleotide or localized to one or more portions thereof.
• the oligonucleotides of the present invention may usefully comprise altered, often nuclease-resistant, internucleoside bonds, as are typically used in antisense applications, See, e.g., Hartmann ef al. (eds.), Manual of Antisense Methodology (Perspectives in Antisense Science), Kluwer Law International (1999) (ISBN:079238539X); Stein ef al. (eds.), Applied Antisense Oligonucleotide Technology, Wiley-Liss (cover (1998) (ISBN: 0471172790); Chadwick et al. (eds.), Oligonucleotides as Therapeutic Agents - Symposium No.
• Modified oligonucleotide backbones may include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'- amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonat.es, thionoalkylphosphotriesters, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'
• modified oligonucleotide backbones useful in the oligonucleotides of the present invention include those that lack a phosphorus atom, such as backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages
• siloxane backbones (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, 0, S and CH2 component parts.
• Representative U.S. patents that teach the preparation of the above backbones include, but are not limited to, U.S. Pat. Nos.
• the oligonucleotides of the present invention may also include nonnaturally occurring nucleobases, either in standard phosphodiester linkage, where the chemistry allows, or with other types of linkage not found in naturally occurring nucleic acids (as would be clear to the person skilled in the art, various nucleobases which previously have been considered nonnaturally occurring have subsequently been found in nature).
• the oligonucleotides of the present invention may thus include nucleobases such as the known purine and pyrimidine heterocycles, and also heterocyclic analogues and tautomers thereof.
• nucleobases are adenine, guanine, thymine, cytosine, uracil, purine, xanthine, diaminopurine, 8-oxo-N 6 -methyladenine, 7-deazaxanthine, 7-deazaguanine, N 4 ,N 4 -ethanocytosine, N 6 ,N 6 -ethano-2,6-diaminopurine, 5-methylcytosine, 5-(C 3 -C 6 )-alkynylcytosine, 5-fluorouracil, 5-bromouracil, pseudoisocytosine, 2-hydroxy-S- methyl-4-triazolopyridine, isocytosine, isoguanine, inosine and the "non-naturally occurring" nucleobases described in U.S.
• locked nucleic acid (LNA) analogues may have utility for particular protein aggregants, although LNA-containing oligonucleotides tested to date have proven poorly effective in disaggregating huntingtin aggregates, as further described in the Examples below.
• LNAs are bicyclic and tricyclic nucleoside and nucleotide analogues and the oligonucleotides that contain such analogues.
• the basic structural and functional characteristics of LNAs and related analogues are disclosed in various publications and patents, including WO 99/14226, WO 00/56748, WO 00/66604, WO 98/39352, US Pat. No. 6,043,060, and US Pat. No. 6,268,490, all of which are incorporated herein by reference in their entireties.
• the oligonucleotides of the present invention may also usefully include Z-O-alkyl analogues, such as 2'-OMe analogues; when linked to deoxyribonucleotides in 5'-3' phosphodiester bonds, the resulting oligonucleotide is a chimera of RNA and DNA.
• Z-O-alkyl analogues such as 2'-OMe analogues
• the oligonucleotides useful in the present invention can also optionally include end-groups, at either or both of the 5' and 3' termini; such end-groups may usefully reduce degradation or, in addition or in the alternative, provide other functionalities.
• the 5' terminus may be phosphorylated, either chemically or enzymatically, thus increasing the oligonucleotide's negative charge.
• the 5' end may, in the alternative, be modified to include a primary amine group, typically appended during solid phase synthesis through use of an amino modifying phosphoramidite, such as a ⁇ -cyanoethyl (CE) phosphoramidite (Glen Research, Inc., Sterling, VA).
• an amino modifying phosphoramidite such as a ⁇ -cyanoethyl (CE) phosphoramidite (Glen Research, Inc., Sterling, VA).
• the 5' end may instead be modified to display a reactive thiol group, which can be appended during solid phase synthesis through use of a thiol modified phosphoramidite, such as (S-Trityl-6-mercaptohexyl)-(2-cyanoethyl)-(N,N-diisopropyl)- phosphoramidite (Glen Research, Inc., Sterling, VA).
• a thiol modified phosphoramidite such as (S-Trityl-6-mercaptohexyl)-(2-cyanoethyl)-(N,N-diisopropyl)- phosphoramidite
• Amine and thiol-modified oligonucleotides can be readily conjugated to other moieties, such as proteins, lipids, or carbohydrates. [0063] Among such moieties are usefully those that serve to target the oligonucleotide to the cell type of therapeutic interest.
• WO 02/47730 and WO 00/37103 describe compounds for intracellular delivery of therapeutic moieties to nerve cells.
• the targeting moieties are neurotrophins - such as NGF, BDNF, NT-3, NT-4, NT-6, and fragments thereof — that effect the targeted internalization of the compound by nerve cells of various classes.
• Such moieties may usefully be appended to the oligonucleotides of the present invention that are intended to disrupt protein aggregations characteristic of neurological disorders, such as spinobulbar muscular atrophy, spinocerebellar ataxia types 1 , 2, 3, 6, and 7, dentatorubral-pallidoluysian atrophy, Parkinson's disease, and the prion-based encephalopathies.
• oligonucleotide of the present invention facilitate passage across the blood brain barrier, such as the 0X26 monoclonal antibody (reviewed in Pardridge, "Brain drug delivery and blood-brain barrier transport", Drug Delivery 3:99-115 (1996), incorporated herein by reference in its entirety; see also U.S. Patent Nos. 5,154,924 and 5,977,307, incorporated herein by reference in their entireties), or target liver cells, such as lactosaminated albumin (Ponzetto ef al., Hepatology 14(1):16-24 (1991), incorporated herein by reference in its entirety).
• the 3' end of the oligonucleotide of the present invention may similarly be amine or thiol modified to permit the ready conjugation of the oligonucleotide to, among others, proteins, carbohydrates, and lipids.
• Fluorescent labels useful for end-modification include, for example, fluorescein isothiocyanate (FITC), allophycocyanin (APC), R-phycoerythrin (PE), peridinin chlorophyll protein (PerCP), Texas Red, Cy3, Cy5, Cy7, and fluorescence resonance energy tandem fluorophores such as PerCP-Cy5.5, PE-Cy5, PE-Cy5.5, PE-Cy7, PE-Texas Red, and APC-Cy7.
• FITC fluorescein isothiocyanate
• APC allophycocyanin
• PE R-phycoerythrin
• PerCP peridinin chlorophyll protein
• Texas Red Cy3, Cy5, Cy7
• fluorescence resonance energy tandem fluorophores such as PerCP-Cy5.5, PE-Cy5, PE-Cy5.5, PE-Cy7, PE-Texas Red, and APC-Cy7.
• fluorophores usefully appended to the 5' or 3' ends of the oligonucleotides of the present invention include, inter alia, Alexa Fluor® 350, Alexa Fluor® 488, Alexa Fluor® 532, Alexa Fluor® 546, Alexa Fluor® 568, Alexa Fluor® 594, Alexa Fluor® 647 (monoclonal antibody labeling kits available from Molecular Probes, Inc., Eugene, OR, USA), BODIPY dyes, such as BODIPY 493/503, BODIPY FL, BODIPY R6G, BODIPY 530/550, BODIPY TMR, BODIPY 558/568, BODIPY 558/568, BODIPY 564/570, BODIPY 576/589, BODIPY 581/591 , BODIPY TR, BODIPY 630/650, BODIPY 650/665, Cascade Blue, Cascade Yellow, Dansyl, l
• the oligonucleotides may also include a 3' and/or 5' group useful for secondary labeling or purification, such as biotin, dinitrophenyl, or digoxigenin.
• a 3' and/or 5' group useful for secondary labeling or purification such as biotin, dinitrophenyl, or digoxigenin.
• the oligonucleotides of the present invention can usefully be synthesized using standard solid phase chemistries appropriate to the nucleobases and linkages desired.
• the oligonucleotides of the present invention can usefully be synthesized combinatorially, providing oligonucleotides of all possible sequences for any desired length of oligonucleotide, from which desired sequences can thereafter be selected.
• the invention provides a method for identifying, from a plurality of oligonucleotides differing in sequence, those oligonucleotides that are effective to disrupt aggregation of a protein aggregant in a cell.
• the method comprises introducing each of a plurality of oligonucleotides of disparate sequence separately into cells that have or are likely to develop protein aggregates, and identifying the oligonucleotide that is most effective at disrupting or preventing aggregation.
• the cells are typically cultured cells, and the oligonucleotides are thus introduced into the cells in vitro. In other embodiments, however, the cells are present within a laboratory animal, and the oligonucleotides are introduced by administration to the animal, [0078]
• the cells chosen for use in this method exhibit or develop aggregation of proteins that are desired to disrupted.
• the cells chosen for use in the assay exhibit or develop huntingtin aggregation; one such cell line is described in Example 1 , below.
• a ⁇ Amyloid plaque of ⁇ secondary structure
• the cells chosen exhibit or develop A ⁇ aggregation.
• the oligonucleotides are desired to reduce or prevent aggregation of ⁇ - synuclein
• the cells chosen exhibit or develop ⁇ -synuclein aggregation.
• protein aggregants useful in the assays of the present invention include, e.g., atropin-1 , ataxin-1 , ataxin-2, ataxin-3, ataxin-7, alpha 1A, a tau protein, PrP Sc , and transthyretin.
• the cells can be naturally occurring, e.g. derived from a patient having the disorder desired to be treated, or can be engineered. Accordingly, the protein aggregation can comprise a naturally-occurring, albeit pathologically aggregated, protein aggregant, or can comprise a non-naturally occurring protein aggregant.
• fusions that comprise the protein aggregant, or an aggregation-competent portion thereof, and a detectable marker are particularly useful.
• GFP-like chromophore means an intrinsically fluorescent protein moiety comprising an 11 -stranded ⁇ -barrel ( ⁇ -can) with a central ⁇ -helix, the central ⁇ -helix having a conjugated ⁇ -resonance system that includes two aromatic ring systems and the bridge between them.
• intrinsically fluorescent is meant that the GFP-like chromophore is entirely encoded by its amino acid sequence and can fluoresce without requirement for cofactor or substrate.
• the PC12 neuronal cell lines described in Example 1 contain an engineered HD gene exon 1 containing alternating, repeating codons ... CAA CAG CAG CAA CAG CAA ... fused to an enhanced GFP (green fluorescent protein) gene.
• an engineered HD gene exon 1 containing alternating, repeating codons ... CAA CAG CAG CAA CAG CAA ... fused to an enhanced GFP (green fluorescent protein) gene.
• the GFP-like chromophore can be selected from GFP-like chromophores found in naturally occurring proteins, such as A. victoria GFP (GenBank accession number AAA27721), Renilla reniformis GFP, FP583 (GenBank accession no. AF168419) (DsRed),
• FP593 (AF272711), FP483 (AF168420), FP484 (AF168424), FP595 (AF246709), FP486 (AF168421), FP538 (AF168423), and FP506 (AF168422)
• Methods for determining the minimal domain required for fluorescence are known in the art. Li et al., "Deletions of the Aequorea victoria Green Fluorescent Protein Define the Minimal Domain Required for Fluorescence," J. Biol. Chem. 272:28545-28549 (1997).
• the GFP-like chromophore can be selected from GFP-like chromophores modified from those found in nature. Typically, such modifications are made to improve recombinant production in heterologous expression systems (with or without change in protein sequence), to alter the excitation and/or emission spectra of the native protein, to facilitate purification, to facilitate or as a consequence of cloning, or are a fortuitous consequence of research investigation.
• U.S. Pat. Nos. 6,090,919 and 5,804,387 is a red-shifted, human codon-optimized variant of GFP that has been engineered for brighter fluorescence, higher expression in mammalian cells, and for an excitation spectrum optimized for use in flow cytometers.
• EGFP can usefully contribute a GFP-like chromophore to the fusion proteins of the present invention.
• a variety of EGFP vectors, both plasmid and viral, are available commercially (Clontech Labs, Palo Alto, CA, USA), including vectors for bacterial expression, vectors for N-terminal protein fusion expression, vectors for expression of C-terminal protein fusions, and for bicistronic expression.
• EBFP enhanced blue fluorescent protein
• BFP2 contain four amino acid substitutions that shift the emission from green to blue, enhance the brightness of fluorescence and improve solubility of the protein, Heim ef al., Curr. Biol. 6:178-182 (1996); Cormack et al., Gene 173:33-38 (1996).
• EBFP is optimized for expression in mammalian cells whereas BFP2, which retains the original jellyfish codons, can be expressed in bacteria; as is further discussed below, the host cell of production does not affect the utility of the resulting fusion protein.
• the GFP-like chromophores from EBFP and BFP2 can usefully be included in the fusion proteins of the present invention, and vectors containing these blue-shifted variants are available from Clontech Labs (Palo Alto, CA, USA).
• EYFP enhanced yellow fluorescent protein
• Clontech Labs contains four amino acid substitutions, different from EBFP, Ormo et al., Science 273:1392-1395 (1996), that shift the emission from green to yellowish-green. Citrine, an improved yellow fluorescent protein mutant, is described in Heikal ef al., Proc. Natl. Acad. Sci. USA 97:11996-12001 (2000).
• ECFP enhanced cyan fluorescent protein
• Clontech Labs, Palo Alto, CA, USA contains six amino acid substitutions, one of which shifts the emission spectrum from green to cyan. Heim ef al., Curr. Biol.
• the GFP-like chromophore of each of these GFP variants can usefully be included in fusion protein aggregants of the present invention.
• the GFP-like chromophore can also be drawn from other modified GFPs, including those described in U.S. Pat. Nos.
• the fused marker is fluorescent, e.g. a protein moiety having a GFP-like chromophore
• aggregation can be observed visually, typically using a fluorescence microscope.
• High throughput apparatus such as the Amersham Biosciences IN Cell Analysis System and Cellomics® ArrayScan HCS System permit the subcellular location and concentration of fluorescently tagged moieties to be detected and quantified, both statically and kinetically. See also, U.S. Patent No. 5,989,835, incorporated herein by reference in its entirety.
• Markers other than fluorescent markers may be used, and markers need not be fused recombinantly to the aggregating protein.
• oligonucleotides having an inhibitory effect on protein aggregation may be found to associate physically with the misassembled proteins. Isolating the protein aggregant under conditions suitable for continued binding of the oligonucleotide to the protein aggregant may thus permit enrichment for those oligonucleotides that have greatest affinity for the protein aggregant. See Kazantsev ef al, Nature Genetics 30:367-76 (2002), incorporated herein by reference in its entirety. [0100] Markers need not be fused recombinantly to the protein aggregant. For example, the protein aggregant can be marked by subsequent staining.
• the oligonucleotide may be labeled.
• Labeling the oligonucleotide is particularly useful for purposes of measuring, and normalizing to, the amount of oligonucleotide that enters the cells being assayed. Labeling of the oligonucleotides also permits the intracellular and extracellular distributions of the oligonucleotides to be assayed.
• the protein aggregant is also labeled, since the subcellular distribution of oligonucleotide and protein aggregant may differ and provide complementary information.
• the oligonucleotides may, for example, be labeled with a radionuclide, a fluorophore, or a visualizable hapten.
• a radionuclide When labeled with a radionuclide, the oligonucleotide's subcellular localization may be detected, e.g., using x-ray film or a phosphorimager.
• the oligonucleotide is typically labeled with a fluorophore having excitation and/or emission spectrum distinguishable from that optionally used to label the protein aggregant, and the oligonucleotide position and concentration is monitored using appropriate fluorescence detection devices.
• the oligonucleotides may be labeled during or after synthesis. As described above, the label can be localized to the 5' and/or 3' terminus, In addition or in the alternative, the label can be positioned within the oligonucleotide, [0106]
• the cells used in the methods of this aspect of the invention are typically clonal lines that identically express the protein aggregant.
• the protein aggregant can be expressed from the cell's chromosome, either from its native locus or from another location into which an engineered construct has been integrated, or from an episomal construct.
• the oligonucleotides to be tested for their ability to disrupt protein aggregation can be introduced into the cells by well-known transfection techniques.
• kits are available for calcium phosphate transfection (CalPhosTM Mammalian Transfection Kit, Clontech Laboratories, Palo Alto, CA, USA), and lipid-mediated transfection can be practiced using commercial reagents, such as LIPOFECTAMINETM 2000, LIPOFECTAMINETM Reagent, CELLFECTIN® Reagent, and LIPOFECTIN® Reagent
• Mechanical means may also be used, such as electroporation, biolistics, and microinjection. Protocols for electroporating mammalian cells can be found online in
• Each oligonucleotide of distinct sequence and/or composition may be assayed individually, and its effectiveness in disrupting or preventing protein aggregation compared to that of other oligonucleotides.
• pools of oligonucleotides may be tested, either to facilitate initial screening or to identify combinations of oligonucleotides with additive or synergistic effect.
• the oligonucleotides typically will be included within compositions suitable for introduction into cell culture, such as buffered aqueous compositions, Depending upon the duration of the assay, which typically ranges from hours to days, the oligonucleotides may preferably be formulated as sterile aqueous compositions.
• the cells to be tested will be tested in a serum-free medium to prevent adventitious sequestration of the oligonucleotide by proteins in the medium.
• the degree of protein aggregation is assessed, and the efficacy of the oligonucleotide in disrupting or preventing protein aggregation determined.
• the efficacy may be measured statically, at any of a variety of time points, or kinetically, and various metrics of efficacy may be used.
• the degree of aggregation may measured as the total volume of protein aggregation within the cell at a particular time point after administration; as the number of separately distinguishable aggregates, such as "pinpoint aggregates"; as the greatest density of protein aggregation within the cell at a particular time point after administration; as the difference between greatest and least density of protein aggregation within the cell at a particular time point after administration,
• the effective degree of disruption may be measured as the rate at which the density, or volume, of aggregation dissipates in one or more regions of the cell. The choice among such metrics will be dictated, in part, by the cell type and aggregants selected for assay, and is well within the skill in the art.
• the assay method may, and typically will, be repeated, until one or more oligonucleotides, alone or in combination, are identified that possess the desired degree of efficacy.
• oligonucleotides effective in disrupting or preventing aggregation will typically be chosen through in vitro assays such as those set forth above and in the Examples below, in other embodiments of this aspect of the invention the oligonucleotides will be assayed in vivo using an animal model of protein aggregation. In such in vivo assays, the efficacy of the oligonucleotide can be assessed additionally by using clinical indicia of efficacy, such as diminution or delay of symptoms. In non-human animals, efficacy can also be assessed using post-mortem assays following sacrifice. A variety of such assays are described in the Examples that follow.
• the invention provides methods of treating human and animal subjects having disorders of protein assembly.
• the method comprises administering an effective amount of a composition comprising at least one oligonucleotide species that disrupts or prevents protein aggregation, optionally in admixture with a pharmaceutically acceptable carrier or excipient.
• disorders amenable to treatment in the methods of this aspect of the invention include those set forth in Table 1 :
• the administered composition will comprise at least one oligonucleotide prior- demonstrated, either in vitro or in an in vivo model, to disrupt or prevent aggregation of the pathogenic protein, and may include any of the structural modifications described above.
• the composition will comprise at least one species of oligonucleotide, and may comprise at least 2, 3, 4, 5, 10, 20, 25, 30, 40 and even as many as 50 to 60 different species, which may differ from one another in any one or more of sequence, length, or composition (such as presence, location, and number of altered internucleobase bonds).
• Pharmaceutically acceptable carriers and/or excipients are optionally, but typically, included and are chosen for suitability with the desired method of administration.
• one exemplary carrier for use with the oligonucleotides of the invention includes nucleic acids, or analogues thereof, that do not themselves possess biological activity perse but that are recognized by in vivo processes that would otherwise reduce the bioavailability of the active oligonucleotides, for example by degrading the active oligonucleotides or promoting their removal from circulation.
• the pharmaceutically acceptable carrier and/or excipient may be liquid or solid and is chosen based, at least in part, upon the desired route of administration so as to provide for the desired bulk, consistency, etc., when combined with the oligonucleotides and the other components of a given pharmaceutical composition.
• Routes of administration useful in the practice of this aspect of the invention include both enteral and parenteral routes, including oral, intravenous, intramuscular, subcutaneous, inhalation, topical, sublingual, rectal, intra-arterial, intramedullary, intrathecal, intraventricular, transmucosal, transdermal, intranasal, intraperitoneal, intrapulmonary, and intrauterine.
• a useful embodiment makes use of neutral liposomes that carry the oligonucleotides and that are decorated on the surface with several thousand strands of polyethyleneglycol (PEG) as described in Pardridge, U.S. Patent No, 6,372,250.
• PEG polyethyleneglycol
• the surface coating prevents the absorption of blood proteins to the surface of the liposome and slows the removal of the liposomes from the blood. It also provides sites for the attachment of ligands recognized by the carrier-mediated transport and receptor-mediated transcytosis systems to allow passage of the liposomes across the blood-brain barrier. In some cases, the ligands mediate the uptake of the pegylated liposomes by cells through the receptor- mediated endocytosis system.
• oligonucleotide formulation is administered intranasally, e.g., by applying a solution containing the oligonucleotides to the nasal mucosa of a patient, This method of administration can be used to facilitate retrograde transport of the oligonucleotides into the brain.
• the oligonucleotides can thus be delivered to brain cells without subjecting the patient to surgery. See, U.S. Patent Nos.
• the oligonucleotides are delivered to the brain by osmotic shock according to conventional methods for inducing osmotic shock.
• oligonucleotides of the invention are administered and dosed in accordance with standard medical practice, taking into account the clinical condition of the individual patient, the site and method of administration, scheduling of administration, patient age, sex, body weight and other factors known to medical practitioners.
• the pharmaceutically "effective amount" for purposes herein is thus determined by such considerations as are known in the art.
• the amount is effective either to achieve improvement in clinical signs and/or symptoms — including but not limited to decreased levels of misassembled or aggregated proteins, or improvement or elimination of symptoms and other clinical endpoints — or to delay onset of or progression of signs or symptoms of disease, as are selected as appropriate clinical indicia by those skilled in the art. Cure is not required, nor is it required that improvement or delay, as above described, be achievable in a single dose.
• the pharmaceutical composition is preferably administered in an amount effective to reverse protein misassembly and aggregation by at least about 10%, 20%, 30%, 40%, even at least about 50%, 60%, 70%, most preferably at least about 80-100%, although such dramatic effect is not required. It is preferred that the amount administered is an amount effective to maximize reversal of protein misassembly and aggregation while minimizing toxicity.
• the dosage can vary depending on the number of cells affected, the location of the cells, the route of administration, the delivery mode, whether treatment is localized or systemic, and whether the treatment is being used in conjunction with other treatment methodologies. Dosages can be determined using standard methodologies. Those skilled in the art can determine appropriate dosages and schedules of administration depending on the situation of the patient.
• the composition is preferably administered until reversal of protein misassembly and aggregation is obtained. Preferably the composition is administered from about 2 days up to a year, although chronic lifetime administration is not precluded.
• the time of administration can be coupled with other treatment methodologies.
• the oligonucleotide treatment may be applied before, after, or in combination with other treatments such as surgery or treatment with other agents. The length of time of administration can be varied depending on the treatment combination selected.
• PC12 neuronal cell lines provided by L. Thompson (UCI), are used. See Boado ef al, J. Pharmacol, and Experimental Therapeutics 295(1 ):239-243 (2000), the disclosure of which is hereby incorporated by reference. [0146] Each PC12 cell line has a construct encoding a fusion of HD exon 1 to GFP (see
• These cells thus contain an engineered HD gene exon 1 containing alternating, repeating codons ... CAA CAG CAG CAA CAG CAA ... fused to an enhanced GFP gene.
• Expression of the fusion gene leads to the appearance of green fluorescence co-localized to the site of protein aggregates.
• the fusion gene is under the control of an inducible promoter regulated by muristerone.
• One cell line used in these experiments has a construct with approximately 46 glutamine repeats (encoded by either CAA or CAG); another cell line used in these experiments has about 103 glutamine repeats.
• PC12 cells are grown in DMEM, 5% Horse serum (heat inactivated), 2.5% FBS and 1% Pen-Strep, and maintained in low amounts on Zeocin and G418. 24 hours prior to transfection, the cells are plated in 24-well plates coated with poly-L-lysine coverslips, at a density of 5x10 5 cells/ml in media without any selection, [0150] Single stranded DNA molecules having no sequence complementarity to the target
• HD gene are added to the PC12 cells bearing an HD gene exon 1-GFP fusion gene; lacking sequence complementarity, the oligonucleotides do not hybridize appreciably to DNA or RNA encoding Huntingtin protein or its complement.
• the oligonucleotides are modified as described below.
• LF2000 LipofectAMINE 2000
• Opti-Mem I serum-free medium
• the oligonucleotide is added and further incubated for 20 minutes at room temperature.
• the lipid/DNA mixture is applied to the cells and incubated at 37°C overnight.
• Muristerone is added after the overnight incubation to induce the expression of HD gene exon 1-GFP.
• the protocol is schematized in FIG. 1A.
• non-specific oligonucleotides are added to the PC 12 cells 48 hours after the induction of gene expression by addition of muristerone; and 48-72 hours later, the cells are visualized by confocal microscopy.
• oligonucleotide In the absence of oligonucleotide, activation of the promoter leads to high levels of Huntingtin-GFP fusion gene expression and, subsequently, the appearance of Huntingtin- GFP fusion protein aggregates (bright pinpoints), visible in FIGS, 2A and 3A. [0156] A visible reduction in the presence of Huntingtin-GFP fusion protein aggregates is observed in the presence of an oligonucleotide that does not hybridize appreciably to the HD gene ("non-specific" or "HD non-specific”).
• the oligonucleotide, denominated "Kan uD3T/25G” has the following sequence and structure:
• FIG. 2B shows that administration of Kan uD3T/25G results in a reduction in Huntingtin-GFP fusion protein aggregates, as compared to cells that are induced but not transfected (FIG. 2A).
• FIG. 2A shows that Similar results are seen with a 25mer HD non-specific single stranded oligonucleotide having all phosphorothioate linkages, denominated Kan uD12T/25G and having the following structure
• FIGS. 2B and 2C are not shot at the same magnification.
• each terminus has three 2'O-Me analogues (shown in lower case). Compare FIG. 3B to FIG. 3A.
• Kan UR/25G is a 25-mer containing all 2'-OMe analogues (shown in lower case):
• Kan uR/15G is a 15-mer containing all 2'-OMe analogues (shown in lower case): 5' gcccagtcgtagccg 3' [SEQ ID N0:6] (Kan UR/15G).
• non-specific single stranded DNA such as Kan UD3T/25G, which has three phosphorothioates at each terminus, or such as Kan UD12T/25G or Kan uD7T/15G, which are substituted with all phosphorothioates, are effective in reducing the number of HD protein aggregates formed after induction.
• a single stranded DNA with three 2'-0-methyl RNA at each terminus, such as Kan uRD3/25G is effective, but less so than Kan UD3T/25G or Kan UD12T/25G.
• Non-specific double stranded chimeric RNA/DNA oligonucleotides are also less effective in reducing the number of aggregates (not shown).
• oligonucleotide with all 2'-0-methyl RNA residues has little to no effect.
• oligonucleotides comprising different lengths, different base composition, different base modifications, and different concentrations are examined to determine optimal length, composition, sequence, and concentration to effect HD disaggregation, [0165] Similar systems are designed to identify oligonucleotides having greatest ability to disaggregate other proteins, such as ⁇ -synuclein, A ⁇ , and prions.
• oligonucleotides include LNA residues, denoted by a "+" prefix, and include the following: 5' +C+T+CA+GG+AG+T+C+AG+G+TG 3' [SEQ ID NO:7] (klo17LNA)
• PC12 cells (Boado ef al, J. Pharmacol, and Experimental Therapeutics 295(1): 239-243 (2000)) are used. These particular PC12 cells contain a CAG or CAA repeat of approximately 103 in the CAG/CAA tract, encoding the poly Q tract, in the first exon of the HD gene fused to an eGFP (enhanced GFP) fusion reporter construct.
• This PC12 cell line has a construct (see Examples 1 and 2 and Kazantsev ef al, Proc. Nat'l Acad. Sci. USA 96: 11404-09 (1999)) integrated into its genome. These cells thus contain an engineered HD gene exon 1 containing alternating, repeating codons ...
• the HD gene exon 1-GFP fusion gene in these PC12 cells is under the control of an inducible promoter regulated by muristerone. [0168] The protocol described in Example 1 for these PC12 cells (Boado ef al, J. Pharmcol Exp Ther. 295(1): 239-243 (2000)) is essentially followed.
• PC12 cells (PC12-HD103QE , a gift from Dr. L. Thompson, UCI) are maintained in DMEM, 5% horse serum (heat inactivated), 2.5% FBS, 1% Pen-Strep, 0.2mg/ml Zeocin, and 10O ⁇ g/ml G418.
• Cells are plated in Lab-Tek II vessels coated with poly-D-lysine coverslips at a density of 5x10 5 cells for 24 hours prior to transfection in media without selection.
• Transfection conditions are found to be best with 2 ⁇ g/ml LipofectAMINE 2000 (LF2000, Invitrogen Corp., Carlsbad, CA).
• the samples are incubated with Opti-Mem I reduced-serum medium (Invitrogen Corp., Carlsbad, CA) for 5 minutes, followed by the addition of the oligonucleotide; incubation continues for 20 minutes at room temperature.
• the lipid/oligonucleotide mixture is then applied and, 24 hours later, the cells induced with 5 mM muristerone (Invitrogen Corp.) for fusion gene expression.
• Protein aggregates are monitored for 72 hours post-transfection using a Zeiss inverted 100M Axioskop equipped with a Zeiss 510 LSM confocal microscope and a Coherent Krypton Argon laser and a Helium Neon laser,
• Approximately 500 cells per field of view are analyzed using a 100X objective in multiple ( ⁇ 3) randomly chosen fields, and counts are tallied from blinded samples. The percentage of cells containing aggregates is calculated by dividing the number of cells bearing inclusion bodies by the total number of cells expressing the HD-eGFP fusion protein. To be scored positive, a cell must have one or more aggregates. The number of cells with aggregates is determined by incorporating the average of four independent experiments.
• Odds ratio which is derived by the proportion of eGFP-expressing cells with inclusions divided by proportion of eGFP-expressing cells treated with liposome but no targeting vector, and the standard deviation are determined as described by Carmichael et al, Proc. Natl Acad. Sci. USA 97:9701-9705 (2000).
• oligonucleotides used in this study are shown below. These oligonucleotides variously contain modifications such as phosphorothioate (*) internucleoside linkages, Locked Nucleic Acid (LNA) residues (+), or 2-O-methyl residues (lower case).
• LNA Locked Nucleic Acid
• HD3S/53T is a 53-mer containing three phosphorothioate linkages at each terminus, and is complementary to the transcribed strand of the HD gene.
• HD3S/53 is identical in structure, but is complementary to the nontranscribed (NT) strand.
• HD3S/15 and HD3S/9 are complementary to the NT strand, but are 15 and 9 bases in length, respectively.
• HD1S/9 and HD1S/6 are also "specific NT" oligos - that is, complementary to the HD gene — but contain only one phosphorothioate linkage on each terminus, The shorter sequences encompass the central region of the HD-sequence contained in the 53-mer.
• the number of phosphorothioate linkages are reduced in the 9-mers and 6-mers from three to one in order to maintain the approximate ratio between the number of thioate and diester bonds linking oligomers differing in length (53 to 15 to 9 to 6).
• Others of the oligonucleotides, HD1 S/9-NS and HD1 S/6-NS are nonspecific (NS) oligomers (i.e., bear no significant complementarity to the HD-gene sequence), and contain a single phosphorothioate linkage on each end.
• Each of the oligonucleotides is transfected at the same molar amount into a PC 12 rat pheochromocytoma cell line containing an unknown number of integrated copies of the fusion gene HD103QE.
• This integrated gene is a truncated Htt sequence containing 103 CAG repeats, fused at the C terminus to an enhanced green fluorescent protein (eGFP) tag, and inducible by muristerone (Kazantsev et al, Proc. Natl Acad. Sci USA 96:11404-11409 (1999)).
• eGFP enhanced green fluorescent protein
• FIG. 1 B schematizes the experimental protocol used to evaluate the impact of these oligomers on the inhibition of aggregate formation.
• To initialize the reaction cells are maintained in low amounts of Zeocin and G418 and plated in Lab-Tek II chambers coated with poly-D-lysine coverslips. The oligos are transfected using LF2000 and, 24 hours later, the cells are induced with muristerone. Protein aggregates initially appear after 24 hours and reached a maximum between 48 and 72 hours post-induction.
• Samples are loaded into a Lab-Tek II chambered cover-glass element to improve image analyses and enable quantification. Inhibition of protein aggregation is measured by viewing cells in a Zeiss inverted 100M Axioskop confocal microscope (510LSM) using a Coherent Krypton Argon and Helium Neon laser. Inclusions are counted blindly in three randomly-selected fields of view, each containing approximately 500 cells.
• 510LSM Zeiss inverted 100M Axioskop confocal microscope
• results from the average of four independent experiments are shown in FIG. 5.
• the number of aggregates or inclusions is expressed as an odds ratio, derived by dividing the proportion of eGFP-expressing cells containing aggregates by the proportion of eGFP-expressing cells in a mock transfection with liposomes but with no added oligonucleotide. This is an appropriate statistical value for scoring visible aggregates because the total number of cells expressing eGFP can vary from day to day, but the relative proportion of cells with inclusions varies only slightly from experiment to experiment. Since the cell line used in our experiments is a single clonal isolate, the percentage of cells expressing eGFP is over 90%.
• FIG. 5 represents data obtained from experiments analyzed 72 hours after transfection, a time-point that enables sufficient expression of the fusion gene and adequate time for accumulation of the inclusion bodies.
• oligonucleotides HD3S/53T and HD3S/53 reduce the number of inclusions over 50% even though these oligonucleotides are designed to hybridize to either the transcribed (T) or nontranscribed strand of the HD gene. Shortening the oligonucleotides preserves this property: HD3S/15 (15-mer) and HD3S/9 (9- mer) are as effective as the 53-mers.
• HD12S/25-NS is a 25-mer, nonspecific for HD fusion gene, containing all PS linkages.
• HD3S/25-NS is a 25-mer, nonspecific for HD fusion gene, containing 3 terminal PS linkages
• HDR/25-NS is a 25-mer, nonspecific for the HD fusion gene, containing all 2 -0- methyl RNA.
• HD3R/25-NS is a 25-mer, nonspecific for the HD fusion gene, containing 3 terminal
• HD3L/25-NS is a 25-mer, nonspecific for the HD fusion gene, containing 3 LNA residues on each end.
• HD/58 is a 58-mer, double-stranded hairpin molecule, specific for the CAG repeat.
• HD12S/25-NS -- a nonspecific oligo with each linkage being phosphorothioate rather than phosphodiester - has an effect on inclusion reduction that is marginal in comparison to those data presented in FIG. 5. The same is true for HDR/25-
• NS an oligomer containing all 2'-0-methyl RNA bases, a modification that confers nuclease resistance, and for HDL/15-NS, a 15-mer composed of all locked nucleic acids, a modification that alters the structure of the DNA residue and makes it more RNA-like,
• the importance of terminal phosphorothioate linkages is emphasized by the results obtained with HD3L/25-NS and HD3R/25-NS, two 25-mers with either three locked nucleic acid residues or three -O-methyl RNA residues on each end. Little or no reduction in inclusions is observed when these oligomers are used.
• This cell line has incorporated a construct with essentially alternating CAACAG encoding for the poly Q tract (see Schweitzer ef al, J. Cell Science 96: 375-381 (1990), the disclosure of which is incorporated by reference herein).
• the promoter directing expression of the Huntingtin-eGFP fusion is regulated by ecdysone analogues.
• the cells ' are induced 24 hours after transfection by the addition of 0.1 ⁇ M tebufenozide (day 1). Confocal microscopy photos are taken on days 2, 3, 6 and 7 post- induction. [0205] On day 7 post-induction, there are about 1 % cells surviving in flasks treated with oligonucleotide; in contrast, by day 6 post-induction, untreated cells (ut) do not survive. [0206] Thus, treating these cells with single stranded DNA molecules causes disaggregation of the Huntingtin aggregates and increases survival of these cells. [0207] Accordingly, single stranded DNA molecules, non-specific for the HD gene, cause disaggregation of Huntingtin protein aggregates in these cells, which is manifested in these cells as cell survival.
• This cell system can be used to identify oligonucleotides that effect disruption of protein aggregation, extending cell survival.
• EXAMPLE 5 Cell Survival Assay Experiments
• PC12 cells/pBWN-Httex1-HD103QE (a gift from Dr. E. Schweitzer- UCLA) are maintained in DMEM (high glucose), 5% FBS, 10% horse serum (Invitrogen Corp.,
• Cell survival measurements are started 24 hours post-induction, using a Zeiss inverted 100M Axioskop equipped with a Zeiss 510 LSM confocal microscope and a Coherent Krypton Argon laser and a Helium Neon laser. Confocal pictures are taken over a period of seven days following induction, and surviving cells are distinguished from nonviable cells by the adherence to the flasks. Approximately 500 cells per field of view are counted in at least five randomly chosen fields and averaged over four independent experiments.
• Nonviable cells detach, round up, and remain suspended in the culture flask; they are easily removed by aspirating the media from the dish prior to counting, After seven days, almost 40% of the cells remain viable, and after 14 days the surviving cell population has more than doubled,
• the cell line PC12/pBWN-httex1-HD25QE which is identical to the PC12 cell line derivative bearing Q103 except that it contains a polyglutamine repeat length of 25, is used as a control to the cell survival assay.
• PC12/pBWN-httex1-HD25QE which is identical to the PC12 cell line derivative bearing Q103 except that it contains a polyglutamine repeat length of 25, is used as a control to the cell survival assay.
• oligonucleotides including two randomly selected PCR primers, are tested for their inhibitory effect in an in vitro mutant huntingtin aggregation assay.
• mutant huntingtin N-terminal fragment containing 58 glutamine residues is expressed in bacteria as a GST fusion.
• the purified fusion protein is treated with protease to release the N-terminal htt fragment, which forms aggregates completely within 24 hours,
• Experimental samples include 40 ⁇ M of oligonucleotides. After aggregate formation is complete, the aggregates are captured on a cellulose acetate membrane with suitable pore size and quantified.
• FIGS. 9A and 9B show the effects of the tested oligonucleotides on the formation of huntingtin aggregates.
• a known inhibitor, Congo Red As expected, a known inhibitor, Congo Red, completely blocks aggregate formation.
• RNA is much more effective than DNA.
• RNA oligonucleotide At the pH of the in vitro assay, approximately pH 8.0, the additional ribose hydroxyl groups are readily available to form hydrogen bonds.
• the greater effectiveness of such RNA oligonucleotides suggests that the additional hydrogen bonds are directly involved in the inhibition of aggregates, likely through the formation of hydrogen bond between the oligonucleotide and glutamine residues in the polyQ tract, with this interaction preventing interaction among huntingtin fragments themselves, in turn preventing the formation of aggregates.
• a transgenic mouse expressing human Huntingtin protein, a portion thereof, or fusion protein comprising human Huntingtin protein, or a portion thereof, with, for example, at least 36 CAG repeats (alternatively, any number of the CAG repeats may be CAA) in the CAG repeat segment of exon 1 encoding the poly-Q tract.
• transgenic mouse strain is the R6/2 line (Mangiarini ef al, Ce// 87: 493-506 (1996), the disclosure of which is hereby incorporated by reference in its entirety).
• the R6/2 mice are transgenic Huntington's disease mice, which over-express exon one of the human HD gene (under the control of the endogenous promoter).
• the exon 1 of the R6/2 human HD gene has an expanded CAG/polyglutamine repeat lengths (150 CAG repeats on average). These mice develop a progressive, ultimately fatal neurological disease with many features of human Huntington's disease.
• Abnormal aggregates constituted in part by the N-terminal part of Huntingtin (encoded by HD exon 1), are observed in R6/2 mice, both in the cytoplasms and nuclei of cells (Davies et al, Cell 90: 537-548 (1997), the disclosure of which is hereby incorporated by reference).
• the human Huntingtin protein in the transgenic animal has at least 55 CAG repeats and more preferably about 150 CAG repeats. These transgenic animals develop a Huntington's disease-like phenotype.
• abnormal aggregates containing the transgenic part of or full-length human Huntingtin protein are present in the brain tissue of these animals.
• the R6/2 strain is an example of such a transgenic mouse strain. See Mangiarini et al, Cell 87: 493-506 (1996), Davies et al, Cell 90: 537-548 (1997), Brouillet, Functional Neurology 15(4): 239-251 (2000) and Cha ef al, Proc. Nat'l Acad. Sci. USA 95: 6480-6485 (1998).
• mice are acclimated for 7 days and given LabDiet 5K52 and tap water ad libidum.
• mice After aclimatization, spontaneously breathing six-week-old R6/2 mice with body weight of approximately 20 g are randomly assigned to one of three treatment groups: mice
• mice 21 - 40 serve as a drug control
• mice 41 - 60 constitute the experimental group.
• mice 21 - 60 are anesthetized and a 2 cm sagittal incision made over the skull. A small opening is made on the cranium 1 mm right lateral to the bregma. A microcannula is inserted to a depth of 3 mm, secured with a dental acrylic, and attached to an Alzet 2004 osmotic pump (Alza Corp., Mountain View, CA).
• Alzet 2004 osmotic pump Alzet 2004 osmotic pump (Alza Corp., Mountain View, CA).
• the osmotic pump is filled with 200 ⁇ l of vehicle (animals 21 - 40) or oligonucleotide solution (animals 41 - 60). The pumps continuously deliver the drug or vehicle for four weeks. The experimenters are blinded to the identity of the drug used in the pump until the death of all the mice in the study.
• oligonucleotides are tested: HD3S/53T; HD3S/53; HD3S/15; HD3S/9; HD1S/9; HD1S/9-NS; HD1S/6; HD1S/6-NS; HD12S/25-NS; HDR/25-NS; HD3L/25- NS; HD3R/25-NS; HD/58 and HD3S/25-NS.
• mice 41 - 60 Survival of mice 41 - 60 is significantly increased, and their disease progression as assessed by motor performance significantly delayed, as compared to control animals 1 - 40, in mice treated with HD3S/53T; HD3S/53; HD3S/15; HD3S/9; HD1S/9; HD1S/9-NS; HD1S/6; HD1S/6-NS; and HD3S/25-NS.
• a Drosophila melanogaster model system for Huntington's disease is obtained. See, e.g., Steffan ef al, Nature, 413: 739-743 (2001) and Marsh ef al, Human Molecular Genetics 9: 13-25 (2000), the disclosure of each of which is hereby incorporated by reference.
• transgenic flies are engineered to express human Huntingtin protein, a portion thereof (such as exon 1), or fusion protein comprising human Huntingtin protein, or a portion thereof, in neurons.
• human Huntingtin protein a portion thereof (such as exon 1)
• fusion protein comprising human Huntingtin protein, or a portion thereof, in neurons.
• 4-mer oligonucleotides identified in Example 4 are administered to the transgenic Drosophila, for example by injecting pharmaceutical compositions comprising the oligonucleotides into the brain, by orally administering the oligonucleotides, or by administering the oligonucleotides as part of food. Administration of the oligonucleotides is performed at various stages of the Drosophila life cycle. [0251] The progression of the Huntington's disease-like symptoms is monitored to determine whether treatment with the oligonucleotides results in reduction or delay of symptoms.
• One or another of the following assays is additionally performed.
• disaggregation of the Huntingtin protein aggregates, or reduction in the formation of the Huntingtin protein aggregates, in these flies is monitored.
• lethality and/or degeneration of photoreceptor neurons are monitored .
• human Huntingtin protein a portion thereof (such as exon 1), or fusion protein comprising human Huntingtin protein, or a portion thereof, leads to a progressive loss of rhabdomeres.
• a microtiter plate assay for polyglutamine aggregate is obtained. See Berthelier ef al, Anal. Biochem. 295:227-236 (2001), the disclosure of which is hereby incorporated by reference.
• poly Q peptides of varying lengths are synthesized. Preferably, these peptides have pairs of Lys residues flanking the poly Q,
• the peptides can be biotinylated.
• the peptides can be about Q28.
• An exemplary peptide is biotinylated K2Q30K2.
• the peptides could be purified.
• the peptides are solubilized and disaggregated by essentially the methods described in Berthelier ef al, Analytical Biochemistry 295: 227-236 (2001), incorporated herein by reference in its entirety.
• Poly Q aggregates are then formed from the solubilized peptides as described in Berthelier ef al, Analytical Biochemistry 295: 227-236 (2001).
• the aggregates are collected by centrifugation, resuspended in a buffer (such as PBS, 0.01% Tween 20 and 0.05% aN3) and aliquoted into Eppendorf tubes.
• the tubes are snap- frozen in liquid nitrogen and stored at -80° C.
• Biotinylated peptides and aggregates of them are prepared essentially as described in Berthelier ef al, Analytical Biochemistry 295: 227- 236 (2001).
• 96-well microtiter plates with the aggregates in some or all the wells are prepared essentially as described in Berthelier ef al, Analytical Biochemistry 295: 227-236 (2001). In some experiments, 20 ng per well of aggregates are used. Aggregate extension assays are done essentially as described in Berthelier ef al, Analytical Biochemistry 295: 227-236 (2001). [0260] The microtiter aggregate extension assay is used to test the ability of the oligonucleotides described in the application, including in the Examples (the oligonucleotides could be different concentrations of HDA3T/53), to inhibit poly Q aggregate extension in this microtiter in vitro aggregate extension assay.
• W303-1 a (MAT a, Ade 2-1 , trp 1-1 , can 1- 100, leu 2-3, 112 his 3-11 , 15 ura 3-1) containing the first 170 codons of human HD with either 23 Q repeats (CAG (any of the CAG repeat may be CAA)) or 75 Q repeats, preferably fused to GFP.
• CAG any of the CAG repeat may be CAA
• oligonucleotides Dosage levels of the oligonucleotides are tested.
• the yeast cells are treated with hydroxyurea to reduce cell growth and extend the S phase of the cell cycle.
• Trichostatin A TSA is added prior to the addition of the oligonucleotides. TSA and oligonucleotide together could have a synergistic effect on the inhibition of protein misassembly.
• Inhibition of protein misassembly is carried out by dilution of the yeast in 96-well plates containing 10 3 cells per well. Huntingtin protein aggregate formation is monitored (See Hughes et al, Proc. Nat'l Acad. Sci. USA 98: 13201-13206 (2001)) using a Zeiss axiovert confocal microscope, and oligonucleotides having greatest efficacy in disrupting or inhibiting protein aggregation are identified.
• a method for identifying, from a plurality of oligonucleotide species differing in sequence and/or composition, those oligonucleotide species that are effective to disrupt aggregation of a protein aggregant in a cell comprising: introducing each of a plurality of oligonucleotide species of disparate sequence and/or composition separately into cells that have or are likely to develop aggregation of a protein aggregant, and identifying one or more of the plurality of oligonucleotide species that is effective at preventing, reducing, or disrupting said aggregation.
• the protein aggregant is selected from the group consisting of: huntingtin, A ⁇ , tau, ⁇ -synuclein, atropin-1 , ataxin-1 , ataxin-2, ataxin-3, ataxin-7, alpha 1 A, PrPSc, transthyretin, superoxide dismutase, amylin, IgG light chain, procalcitonin, ⁇ 2-microglobulin, atrial natriuretic factor, serum amyloid A, apoAI , and gelsolin.
• each of the oligonucleotide species is at least 6 nt in length
• each of the oligonucleotide species is at least 25 nt in length.

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