JP2006509726A5 - - Google Patents

Description

Huntington's disease is a neurodegenerative disease that includes spinal medulla muscular atrophy, spinocerebellar ataxia type 1, type 2, type 3, type 6, and type 7, and dentate nucleus red nucleus-palyukyu Rui body atrophy. And are often characterized by mutations that are dominantly inherited and that cause CAG trinucleotide sequences to extend and cause translation of proteins with unusually long polyglutamine tracts (tracts). See, for example, Carmichael et al. , Proc. Nat'l Acad. Sci. USA, 97: 9701-9705 (2000). Also, despite the fact that the disease results in neurodegeneration at different locations, all of these diseases share the same basic pathological cause. That is, intracellular protein aggregates are formed in fragile nerves, resulting in loss of nerve function.

Intracellular protein aggregation has recently been shown to directly weaken the function of the ubiquitin-proteasome system. This ubiquitin-proteasome system serves to break down misfolded, unassembled or damaged proteins that can potentially form toxic aggregates within the cell. Bence et al. , Science, 292: 1552-55 (2001). Such dysfunction can generate a positive feedback mechanism, whereby aggregated proteins at increasing concentrations inhibit the very system involved in the degradation of those proteins, and many neurodegeneration This can result in a sharp loss of cellular function characteristic of the disease. Consistent with this point, direct inhibition of protease activity results in the accumulation of mutant huntingtin fragments in aggresome-like inclusions. Waelter et al. Mol. Biol. Cell, 12: 1393-1407 (2001).

Prion diseases are also thought to result from misfolding and aggregation of certain proteins. PrP ^c is a normal form of the prion protein and is encoded by a single copy gene in mammals. Basler et at, Cell, 46: 417-28 (1986). When expressed, this protein is usually found on the surface of nerve cells. Prion disease is believed to result in the conversion of the normal PrP ^c form of prion protein to PrP ^Sc , an insoluble and disease-causing form. Despite the identical amino acid sequence order of the two shapes, the protein's higher order structure appears to be different. Pan et al, Proc. Nat'l Acad. Sci. USA, 90: 10962-66 (1993). PrP ^Sc seems to be related to infections and pathogenic prion disease. As noted above, prion diseases include sheep and goat scrapie; cattle bovine spongiform encephalopathy; and Kuru, Creutzfeldt-Jakob disease, Gerstmann-Stroisler-Schönker disease, and human lethal familial insomnia . See Prusiner et al., US Pat. No. 6,214,366.

Many other diseases are thought to involve misfolding, misassembly and / or aggregation of proteins. Such diseases (and related proteins) include the following: That is, amyotrophic lateral sclerosis (superoxide dismutase), stenotic pericarditis (pic body tau protein), type II diabetes (amylin), multiple myeloma-plasma cell dysplasia (immunoglobulin G light chain) ), Medullary carcinoma of the thyroid (procalcitonin), chronic renal failure ( β ₂ -microglobulin), congestive heart failure (atrial natriuretic factor), chronic inflammation (serum amyloid A), atherosclerosis (ApoA1) , And familial amyloidosis (gelsolin). See Prusiner et al., US Pat. No. 6,214,366.

Using transglutaminase inhibitors (eg cystamine and mono dansylcadaverine), stretch aggregates in cultured cells expressing the protein containing polyglutamine segments formed and A Pot Shisu cell death inhibition was done. Tsuji, US Pat. No. 6,355,690.

Fusaric acid and picolinic acid have been used to inhibit zinc-dependent polymerization and fibril formation of βA _1-40 amyloid peptide . Douglas et al., US Patent Publication No. US 2002/0037908 A1. These same drugs also cause the release of β-amyloid protein from brain sections of postmortem Alzheimer's disease patients. Same as above.

The above findings suggest that agents that can inhibit the formation of protein aggregates or disrupt the structure of already formed aggregates may help treat diseases caused by protein misassembly and aggregation. It shows that there is. US Pat. No. 6,420,122 describes an in vitro method for identifying additional agents useful for causing protein aggregate breakage in cells.

The method separately introduces each of a plurality of oligonucleotide species of different sequences into a cell having or possibly generating protein aggregates, from a plurality of oligonucleotide species having different sequences, into a cell. Identifying those oligonucleotide species that are effective to prevent, reduce, or disrupt the aggregation of protein aggregates, as well as prevent, reduce, or disrupt aggregation and / or increase cell survival Identifying effective oligonucleotide species above .

The protein aggregates, in particular, Hanchin Ji emissions (htt), A [beta], tau, alpha-synuclein, atropine -1, ataxin-1, ataxin -2, ataxin3, ataxin -7, alpha1A, ^{PrP Sc,} transthyretin , Superoxide dismutase, amylin, IgG light chain, procalcitonin, β ₂ -microglobulin, atrial natriuretic factor, serum amyloid A, apoA1, and gelsolin.

Modified oligonucleotide backbones such as phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkyl phosphonates (including 3' -alkylene phosphonates and chiral phosphonates), phosphinates Phosphoramidates (eg, 3′-aminophosphoramidates and aminoalkylphosphoramidates), thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and normal 3′-5 ′ linkages Boranophosphates, as well as their 2′-5 ′ linked analogs, and those with reversed polarity, where adjacent pairs of nucleoside units are linked 3′-5 ′ to 5′-3 ′ or 2′— Bind to 5 'to 5'-2' Nucleoside base pair. Representative United States patents that teach the preparation of the above Li emissions including covalent bonding, but are not limited to, U.S. Pat. No. 3,687,808, No. 4,469,863, No. 4,476, No. 301, No. 5,023,243, No. 5,177,196, No. 5,188,897, No. 5,264,423, No. 5,276,019, No. 5,278,302 5,286,717, 5,321,131, 5,399,676, 5,405,939, 5,453,496, 5,455,233, 5,466,677, 5,476,925, 5,519,126, 5,536,821, 5,541,306, 5,550,111, 5, 563,253, 5,571,799, 5,587,361 And No. 5,625,050 can be mentioned, incorporated these disclosures as herein.

Other modified oligonucleotide backbones useful in the oligonucleotides of the invention include those lacking a phosphorus atom, such as by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl nucleosides. Examples include backbone chains that are formed, or one or more short-chain heteroatoms or heterocyclic internucleoside linkages. These are morpholino linkages (partially made from the sugar moiety of the nucleoside); siloxane backbones; sulfurized, sulfoxide, and sulfone backbones; acetyl formate and thio-formate acetyl backbones; methylene groups acetyl formate and thio-formate acetyl Skeletal chain; Alkene containing skeletal chain; Sulfamide ester backbone chain; Methyleneimino and methylene hydrazino backbone chain; Sulfonic ester and sulfonamide backbone chain; Amide backbone chain; Mixed N, O, S, and CH ₂ components Others that include a moiety are included. Representative US patents that teach the preparation of the above backbone chains include, but are not limited to, US Pat. Nos. 5,034,506, 5,166,315, 5,185,444, 5,214,134, 5,216,141, 5,235,033, 5,264,562, 5,264,564, 5,405,938, , 434,257, 5,466,677, 5,470,967, 5,489,677, 5,541,307, 5,561,225, 5,596 No. 5,086, No. 5,602,240, No. 5,610,289, No. 5,602,240, No. 5,608,046, No. 5,610,289, No. 5,618,704 No. 5,623,070, No. 5,663,312, No.5 No. 633,360, No. 5,677,437, and No. 5,677,439 can be mentioned. These disclosures are incorporated herein in their entirety.

Accordingly, the oligonucleotides of the present invention may include nucleobases known as known purines and pyrimidine heterocycles, as well as their heterocyclic analogs and tautomers. Typical examples of nucleobases are adenine, guanine, thymine, cytosine, uracil, purine, xanthine, diamino purine, 8-oxo -N ⁶ - methyl adenine, 7-de azaxanthine, 7-deazaguanine, ^N 4, N ⁴ - ^{Etanoshitoshin,} N 6, ^{N 6} - ethano-2,6-diaminopurine, ^{5-methylcytosine, 5- (C 3 -C 6)} - alkynyl cytosine, 5-fluorouracil, 5-bromouracil, pseudo-isocytosine , 2-hydroxy-S-methyl-4-triazolopyridine, isocytosine, isoguanine, inosine, and “non-naturally occurring” nucleobases disclosed in US Pat. No. 5,432,272. Which are incorporated herein by reference in their entirety.

Oligonucleotides also include 3 ′ and / or 5 ′ groups useful for secondary labeling or purification , such as biotin, dinitrophenyl, or digoxigenin.

If the desired sequence has not been determined, the oligonucleotides of the present invention can be combined and usefully synthesized to provide all possible sequence oligonucleotides to any desired length of the oligonucleotide, from which The desired sequence can then be selected.

Such combination methods are known in the art. In the simplest such method, all possible nucleobase monomers are used in each synthesis cycle. The disadvantage of this approach is that oligonucleotides of heterologous sequence are present in the mixture. Other methods allow for high-throughput parallel synthesis in which oligonucleotides with different sequences are separated. For example, Cheng et al. , "High throughput parallel synthesis of oligonucleotides with 1536-channel synthesizer," Nucl. Acids Res. 30 (18): See e93 (2002).

The oligonucleotides to be tested differ in sequence. These oligonucleotides, there are differences in further combination formed optionally, for example, the length, the presence of binding between the non-natural nucleosides, position, and number, the presence of non-natural internucleoside linkages, location, and number, unnatural nucleobase Presence, position, number and chemistry, as well as the presence, position and number of terminal modifications.

Similarly, if an oligonucleotide is required to reduce or prevent aggregation of Aβ (amyloid plaque β secondary structure), the selected cells exhibit or develop Aβ aggregation. When reducing or preventing α-synuclein aggregation, the selected cells exhibit or inhibit α-synuclein aggregation . Useful other protein aggregates in the assay of the present invention, for example, atropine 1, Ataki Thin 1, ataxin 2, ataxin 2, Ataki Thin 3, ataxin 7, alpha1A, tau protein, ^{PrP Sc,} and trans Sai Retin is mentioned.

As used herein, “GFP-like chromophore” means an internal fluorescent protein moiety composed of an 11-chain β-barrel (β-can) having a central α-helix, the central α-helix. Has a coupled π resonance system that includes two aromatic ring systems and a bridge that bridges them. “Intrinsically fluorescent” means that the GFP-like chromophore is entirely encoded by the amino acid sequence and can fluoresce without the need for cofactors or substrates. For example, the PC12 neuronal cell line described in Example 1 contains the HD gene exon 1, as shown below, which exon 1 is an alternating and repeated codon, ie. . . CAA CAG CAG CAA CAG CAA . . . And is fused to an enhanced GFP (green fluorescent protein) gene.

The label need not be recombinantly fused to the protein aggregate. For example, protein aggregates can be labeled by subsequent staining.

In general, labeling oligonucleotides also labels protein aggregates. This is because the intracellular distribution of oligonucleotides and protein aggregates can differ and provide complementary information.

Given the short oligonucleotide length, passively the oligonucleotide, for example, by endocytosis mechanism, have to be further simplified rather than be introduced.

For chemical transfection, DNA is coprecipitated with calcium phosphate and introduced using liposomes or non-liposomal lipid reagents. Commercially available kits for calcium phosphate transfection are available (CalPhos ^™ Mammalian Transfection Kit, Clontech Laboratories, Palo Alto, CA, USA). Lipid- mediated transfection can also be performed using commercially available reagents (eg, LIPOFECTAMINE ^™ 2000, LIPOFECTAMINE ^™ Reagent, CELLFECTIN® Reagent, and LIPOFECTIN® Reagent (Invitrogen, Cardobad, CADOAtm, USADOAP, USADOAP, USADOAP). , FuGENE 6, X-tremeGENE Q2, DOSPER (Roche Molecular Biochemicals, Indianapolis, IN USA), Effectene (registered trademark), PolyFect (registered trademark), Superfect (registered trademark) (Qiagen, Inc.). , CA, USA)). Other types of polycations, cationic lipids , liposomes, and polyethyleneimine (PEI) are known and can be used.

A single-stranded DNA molecule that does not have sequence complementarity to the target HD gene is added to PC12 cells carrying the HD gene exon 1-GFP fusion gene. Since no sequence complementarity, oligonucleotide does not hybridize appreciably to DNA or RNA encoding the Hanchin Chi down protein or its complement. Oligonucleotides are modified as described below.

Zeiss 510 LSM confocal microscope and Coherent Krypton
Data are acquired on a Zeiss inverted 100M Axioskop equipped with an Argon laser and a Helium Neon laser. Lab-Tek for improved imaging
Place the sample in the II chamber / cover / glass system. In seven independent experiments, and counts the number of Hanchin Chi down -GFP aggregation within the field of view of the objective lens. To suppress the observer bias in order to count the "pinpoint aggregates (pinpoint aggregate)", the plurality of scientists in different regions from control and treated cell populations, Hanchin Chi down -GFP fusion You are required to perform an unbiased count of protein aggregates.

In the absence of oligonucleotide, activation of the promoter leads to Hanchin Chi emissions -GFP fusion gene expression of high levels, then Hanchin Chi emissions -GFP fusion protein aggregates that can be seen in FIGS. 2A and 3A (bright small Point) appears.

Visible reduction in the presence of Hanchin Chi emissions -GFP fusion protein aggregates, as the oligonucleotide ( "nonspecific (non-specific)," that does not hybridize appreciably to the HD gene or "HD non-specific (HD non -Specific))). The oligonucleotide named “Kan uD3T / 25G” has the following sequence and structure: That is,

Here, an asterisk indicates a phosphorothioate bond. Figure 2B, induction is being although compared to cells not transfected (FIG. 2A), the administration of Kan uD3T / 25G indicating that results in a decrease in Hanchin Chi emissions -GFP fusion protein aggregates.

Hanchin Chi emissions -GFP fusion decreased protein aggregate formation is also observed Kan uRD3 / 25G. That is,

In certain experiments, the above reduction in Hanchin Chi emissions -GFP fusion protein aggregate formation effect, oligonucleotide excess not specific to the HD gene (> 25 [mu] g) is observed only when it is transfected. The optimal amount of HD non-specific oligonucleotide required to reduce Hanchin Chi emissions -GFP fusion protein aggregate formation may be different, it can be readily determined.

Visible reduction in the appearance of Hanchin Chi emissions -GFP protein aggregates is not observed in the presence of Klo17LNA. Indeed, among the oligonucleotides comprising LNA residues shown above in this example, it is not intended to reduce the Hanchin Chi emissions -GFP protein aggregate formation. Kan uD12T / 25G has a toxic effect on these cells (ie, causes more cell death). See FIG. 4A.

Place the sample on a Lab-Tek II chamber cover glass element that improves image analysis and allows quantification . Inhibition of protein aggregation is measured by viewing cells in a Zeiss inverted 100M Axioskop confocal microscope (510 LSM) using a Coherent Krypton Argon and Helium Neon laser. Inclusion bodies are counted with just the instrument in three randomly selected fields, each containing about 500 cells.

Example 4
Cell survival assay eGFP (enhanced GFP) gene 103 amino polyglutamine repeats (each Q is fused to (Dr.Erik Schweitzer, UCLA donated material) for detecting the disaggregation of Hanchin Chi down aggregates, CAA or encoded by either CAG essentially alternating CAACAG) of Hanchin Chi emissions including the first containing exon cell line PC12 / pBWN:. use Httex. This cell line incorporated a construct with essentially alternating CAACAG encoding the polyQ region (see Schweitzer et al., J. Cell. Science 96: 375-381 (1990), the disclosure of which is hereby incorporated by reference). Incorporated herein by reference). Promoter directing the expression of Hanchin Chi emissions -GFP fusion is regulated by ecdysone analogues.

These cells, after induction to a overwhelming Hanchin Chi emissions aggregate formation, is useful for ultimately the cells die. Thus, disruption of Hanchin Chi emissions aggregate formation, as indicated by the green fluorescence sustained, ultimately prolongs cell survival and proliferation. Make meticulous measurements to prolong cell survival.

Therefore, treatment of these cells with single stranded DNA molecules causes disaggregation of Hanchin Chi down aggregates, thereby increasing the survival of these cells.

Thus, single-stranded DNA molecules in the HD gene nonspecific causes disaggregation of Hanchin Chi down protein aggregates in these cells, dehydration aggregation is expressed as cell survival in these cells.

Example 6
In vitro assays In vitro mutation Hanchin Chi down agglutination assay for the inhibition of aggregation, a plurality of oligonucleotides including PCR primers selected to two randomized tested for its et inhibitory effect.

In essence, Huang et al. , Somat.Cell Mol.Genet.2
4 (4): 217-33 (1998), the entire disclosure of which is incorporated herein by reference, the assay is performed. In summary, the expression of mutated Hanchin Chi down N-terminal fragment containing 58 amino glutamine residue in bacteria as GST fusion. The purified fusion protein is treated with protease to release an N-terminal htt fragment that forms a complete aggregate within 24 hours. The experimental sample contains 40 μM oligonucleotide. After the formation of the aggregate is completed, the aggregate is captured on a cellulose acetate film having an appropriate pore size and quantified.

9A and 9B shows the effect of oligonucleotides tested in the formation of Hanchin Chi down aggregates.

From the results, direct interaction with the oligonucleotide and mutant Hanchin Ji emissions poly Q region is suggested.

Second, RNA is more effective than DNA. At an in vitro assay pH of about 8.0, additional ribose hydroxyl groups are readily available and form hydrogen bonds. Because of the greater effect of such RNA oligonucleotides, additional hydrogen bonds are directly involved in the inhibition of aggregates, possibly through the formation of hydrogen bonds between the oligonucleotides and glutamine residues in the polyQ region, this interaction prevents interaction between Hanchin Chi emissions fragment itself, this time suggesting that prevent the formation of aggregates.

Example 7
The Hanchin Chi down aggregates effective 4 base single-stranded oligonucleotide of the identification number of oligonucleotides in order to destroy, to screen for ability to disrupt Hanchin Chi down aggregates, 256 4 conceivable bases Synthesize all single stranded oligonucleotides.

In accordance with one or both assays described respectively in Examples 1, 2, and 3, each of these 4mer oligonucleotide, for their ability to cause disaggregation of Hanchin Chi down protein aggregates across the concentration range To identify the most effective 4mer oligonucleotide.

(Example 8)
Administration of single-stranded oligonucleotide to a transgenic animal model system HD obtain an animal model system for Huntington's disease that causes a decrease in Hanchin Chi down protein aggregates.

For example, during CAG repeat segment of exon 1 encoding the poly Q region, for example, at least 36 CAG repeats (or any number may be CAA of CAG repeats) human Hanchin Chi down-protein with, a transgenic mouse which expresses a fusion protein comprising the partial or human Hanchin Chi down-protein or portion thereof.

An example of such a transgenic mouse line is the R6 / 2 line (Mangialini et al., Cell 87: 493-506 (1996), the entire disclosure of which is incorporated herein). R6 / 2 mice are transgenic Huntington's disease mice that overexpress exon 1 of the human HD gene (under the control of the endogenous promoter). Exon 1 of the R6 / 2 human HD gene has an extended CAG / polyglutamine repeat length (on average 150 CAG repeats). These mice develop a progressive, ultimately fatal neurological disease with many features of human Huntington's disease. Hanchin Chi emissions of N-terminal portion (HD exon 1 encoded) by partially constructed abnormal aggregates in R6 / 2 mice, the cytoplasm of cells and found in the nucleus both (Davies et al., Cell 90: 537-548 (1997), the disclosure of which is incorporated herein by reference). Preferably, human Hanchin Chi down proteins in transgenic animals have at least 55 CAG repeats and more preferably about 150 CAG repeats. These transgenic animals develop a Huntington's disease-like phenotype.

These transgenic mice are characterized by reduced weight gain, longevity and movement disorders characterized by abnormal gait, resting tremor, hindlimb flexion, and hyperfunction from 8-10 weeks after birth. (See, for example, R6 / 2 strain; Mangialini et al., Cell 87: 493-506 (1996)). The phenotype progressively worsens towards a decrease in movement. The brains of these transgenic mice have changes in neurotransmitter receptors (glutamine, dopaminergic), reduced levels of N-acetylaspartate (a marker of neuronal integrity), and striatum and brain size It also shows neurochemical and histological abnormalities such as reduction. Furthermore, abnormal aggregates containing the full-length or transgenic parts of human Hanchin Chi down proteins are present in the brain tissue of these animals. The R6 / 2 strain is an example of such a transgenic mouse strain. Mangiarini et al, Cell 87: 493-506 (1996), Davies et al. , Cell 90: 537-548 (1997), Broillet, Functional Neurology 15 (4): 239-251 (2000), and Cha.
et al, Proc. Nat'l Acad. Sci. USA 95: 6480-6485 (1998).

The animals are then sacrificed and brain sections are obtained. Then, to analyze The brain sections for the presence of aggregates containing the fusion protein comprising transgenic human Hanchin Chi down protein, its partial or human Hanchin Chi down-protein or portion thereof. The analysis, for example, by staining the sections of brain tissue with anti Hanchin Ji emissions antibodies, adding a secondary antibody conjugated with recognized FITC anti Hanchin Ji emissions antibodies (e.g., anti Hanchin Ji emissions Antibody is a mouse anti- human antibody, secondary antibodies are specific for human antibodies) and visualization of protein aggregates by fluorescence microscopy. Or, it can be conjugated anti Hanchin Chi emissions antibody directly with FITC. Then visualized by fluorescence microscopy the amount of Hanchin Chi down protein aggregates.

Example 10
Administration of single-stranded oligonucleotide to a Drosophila model system HD obtain a Drosophila model system for Huntington's disease that causes a decrease in Hanchin Chi down protein aggregates. See, for example, Stefan et al. , Nature, 413: 739-743 (2001) and Marsh et al. , Human Molecular Genetics 9: 13-25 (2000) (the disclosures of each of which are incorporated herein by reference). For example, in the CAG repeat segment of exon 1 encoding the poly Q region, for example, at least 36 CAG repeats (preferably 51 or more repeats) (or any number of CAG repeats may be CAA) human Hanchin Chi down protein with a transgenic Drosophila expressing a fusion protein comprising the portion (1, etc. exon), or human Hanchin Chi down-protein or portion thereof. By operating these transgenic flies, expressing human Hanchin Chi down proteins, that part (1, etc. exon), or human Hanchin Chi down protein or a fusion protein comprising a part thereof in neurons.

In a first additional assay, monitoring disaggregation or Hanchin reduced blood down protein aggregate formation Hanchin Chi down protein aggregates in these flies.

Human Hanchin Chi down proteins, neurodegeneration due to the expression of that portion (1, etc. exon), or fusion protein comprising a human Hanchin Chi down proteins or portions thereof, produced by the photoreceptor neurons of each Drosophila ommatidium It is easily observed in a fly compound eye composed of a certain trapezoidal arrangement of seven visible sensory bodies (subcellular condensing structures). Human Hanchin Chi down proteins, that portion (1, etc. exon), or expression of a fusion protein comprising a human Hanchin Chi down proteins or portions thereof, leads to progressive loss of sensitive桿分body.

(Example 11)
Administration of single-stranded oligonucleotides to HD in vitro model system to obtain a microtiter plate assay for polyglutamine aggregates cause a decrease in Hanchin Chi down protein aggregates. Berthelier et al. , Anal. Biochem, 295: 227-236 (2001), the disclosure of which is incorporated herein by reference.

Two S.C. S. cerevisiae strains are provided. That is, the first 170 codons of human HD with either 23 Q repeats (CAG (any CAG repeat may be CAA)) or 75 Q repeats preferably fused to GFP W303-1a (MAT a, Ade 2-1, trp 1-1, can 1-100, leu 2-3, 112 his 3-11, 15 ura 3-1). Each of these strains carries an insert HD gene, preferably with an NLS (nuclear localization signal), under the control of an inducible promoter (Gal 1,10) or a constitutive promoter (GPD-1). Portion of Hanchin Ji emissions are localized in the nucleus, to form protein aggregates cliff in these cells. Hughes et al. Proc. Natl. Acad. Sci. USA 98: 13201-13206 (2001), the disclosure of which is incorporated herein by reference.

Inhibition of protein misassembly is carried out by dilution of yeast in 96 well plates containing 10 ³ cells per well. Using Zeiss Axiovert confocal microscope, monitor the Hanchin Chi down protein aggregate formation (Hughes et al, Proc.Nat'l Acad.Sci.USA 98 :. See, 13201-13206 to (2001)), protein Identify the oligonucleotide with the greatest effect of disrupting or inhibiting aggregation.

FIG. 1A is a flow chart showing an experimental protocol for assaying oligonucleotides in cells cultured in vitro for the ability to disrupt or prevent aggregation of protein aggregates according to the present invention. FIG. 1B is a flow chart showing in more detail an experimental protocol for assaying oligonucleotides in cells cultured in vitro for the ability to disrupt or prevent aggregation of protein aggregates according to the present invention. . FIG. 1C schematically illustrates an experimental protocol for assaying oligonucleotides in cells cultured in vitro based on cell survival for the ability to disrupt or prevent aggregation of protein aggregates according to the present invention. It is a flowchart shown in FIG. FIG. 1-2 is a continuation of FIG. 1-1. FIGS. 2A-2C are fluorescence micrographs of PC12 cell culture in which aggregates of huntingtin GFP fusion protein appear as light against a light-dark background, FIG. 2A shows a control culture, and FIG. 2B is in accordance with the present invention. 2C shows a decrease in aggregate formation after transfection with oligonucleotide Kan uD3T / 25G , and FIG. 2C shows a similar degree of reduction in aggregate formation after transfection with oligonucleotide Kan uD12T / 25G according to the present invention. Yes (but the magnification of the micrograph is different). FIGS. 3A-3C are fluorescence micrographs of PC12 cell culture in which aggregates of huntingtin GFP fusion protein appear as light against a light-dark background, FIG. 3A shows a control culture, and FIG. 3B is in accordance with the present invention. Te exhibit reduced aggregation formation after transfection with oligonucleotide Kan uRD3 / 25G, further FIG. 3C shows a similar extent as the reduction in aggregate formation after transfection with oligonucleotide Kan uR / 25G based on the present invention Yes. 4A to 4 E are fluorescence micrographs of look PC12 cultures as light aggregates of huntingtin GFP fusion protein against dark background, FIGS. 4D and 4E show the control culture that was not transfected 4A to 4C show cultures transfected with three different oligonucleotides according to the present invention, as shown. FIG. 5 is a presentation oligonucleotide essentially based on the protocol of FIG. 1B, visible aggregates scored based on odds ratio (average percentage of cells containing aggregates reported in parentheses), Figure 6 is a chart quantifying aggregate formation in a cell-based assay with error bars indicating standard deviations calculated from the average of four independent experiments. FIG. 6 shows the displayed oligonucleotides essentially based on the protocol of FIG. 1B, visible aggregates scored based on odds ratio (average percentage of cells containing aggregates reported in brackets), FIG. 6 is a chart quantifying aggregate formation in cell-based assays with error bars indicating standard deviations calculated from the average of four independent experiments. FIG. 7 shows cell survival after introduction of a mutant huntingtin product in the presence and absence of oligonucleotide HDS-9, based on the protocol illustrated in FIG. 1C. FIG. 8 is a photomicrograph showing rapid visualization of viable cells attached to the flask surface in the cell viability assay of the present invention. FIG. 9A shows recombinant mutant huntingtin N-terminal aggregates captured on a cellulose acetate filter after incubation with an indicator oligonucleotide or compound according to the present invention. In FIG. 9B, the amount of aggregates is tabulated as a percentage of aggregates observed in the negative control.

Claims (20)

A method of identifying from a plurality of oligonucleotide species having different sequences and / or compositions, those oligonucleotide species effective to prevent aggregation of protein aggregates in a cell, comprising:
Each of a plurality of oligonucleotide species of different sequence and / or composition is introduced separately into a cell where the protein aggregates are aggregated or where such aggregation may occur to prevent, reduce or reduce the aggregation; or Identifying one or more of a plurality of oligonucleotide species effective in preventing. The protein aggregates, Hanchin Ji emissions, A [beta], tau, alpha-synuclein, atropine -1, ataxin-1, ataxin -2, ataxin3, ataxin -7, alpha1A, PrPSc, transthyretin, superoxide dismutase, amylin The method of claim 1, wherein the method is selected from the group consisting of:, IgG light chain, procalcitonin, β2-microglobulin, atrial natriuretic factor, serum amyloid A, apo A1, and gelsolin. The method of claim 1, wherein each of the oligonucleotide species is at least 6 nt long. 4. The method of claim 3, wherein each of the oligonucleotide species is at least 25 nt long. The method of claim 1, wherein at least a plurality of the oligonucleotide species comprises at least one phosphorothioate linkage. 6. The method of claim 5, wherein at least a plurality of the oligonucleotide species comprises at least one phosphorothioate linkage at each end. The method of claim 1, wherein the oligonucleotide is introduced into the cell in vitro. The method of claim 1, wherein the protein aggregate is detectably labeled. 9. The method of claim 8, wherein the label is recombinantly fused to the aggregate. 13. A method according to claim 12, wherein the label is a polypeptide comprising a GFP-like chromophore. A composition for treating a subject having a disease caused by aggregation of protein aggregates, comprising:
A composition comprising at least one oligonucleotide species, optionally mixed with a pharmaceutically acceptable carrier or excipient, to prevent, retard or prevent aggregation of the protein. The diseases are Alzheimer's disease, cystic fibrosis, amyotrophic lateral sclerosis, Parkinson's disease, spinal medullary atrophy, spinocerebellar ataxia type 1, spinocerebellar ataxia type 2, spinocerebellar ataxia Type 3, spinocerebellar ataxia type 6, spinal cerebellar ataxia type 7, dentate nucleus red nucleus pallidus atrophy, prion disease, scrapie, bovine spongiform encephalopathy, CJD, new variant CJD, The group consisting of Pick's disease, Type II diabetes, multiple myeloma-plasma cell dysplasia, medullary thyroid cancer, chronic renal failure, congestive heart failure, chronic inflammation, atherosclerosis (ApoA1), and familial amyloidosis 12. A composition according to claim 11, selected from: 12. The composition of claim 11, wherein the at least one oligonucleotide species is at least 6 nt long. 14. The composition of claim 13, wherein the at least one oligonucleotide species is at least 25 nt long. 12. The composition of claim 11, wherein the at least one oligonucleotide species comprises at least one terminal modification. 16. The composition of claim 15, wherein the at least one terminal modification is selected from the group consisting of a phosphorothioate linkage and a 2'-OMe analog. The composition of claim 16, wherein the terminal modification is at least one phosphorothioate linkage. Wherein the composition, sequence, one or more of the length, or composition comprising a different at least two oligonucleotide species composition of claim 11. Wherein the composition is suitable for administration by intravenous or intrathecal injection composition of claim 11, wherein. 12. The composition of claim 11, wherein the composition is suitable for administration by ex vivo transfection of cells from the subject and then reintroducing the transfected cells into the subject .