The present invention relates to oligonucleotide compositions and therapeutic uses thereof to modify protein-protein interactions. In particular, the invention relates to the use of a guanidine-rich oligonucleotides to disrupt disease-causing protein aggregates, for example, Huntington's Disease (HD) protein aggregates.

Description of the Invention

SUMMARY OF THE INVENTION

The present invention is based on the surprising and unexpected discovery that certain oligonucleotides are capable of disrupting protein aggregates that have been associated with disease pathologies. Therefore, the oligonucleotides of the invention can be used as therapeutics in the treatment and prevention of such diseases, and can also aid in the study of the diseases and their underlying physiological origins. In particular, the invention relates to guanosine-rich oligonucleotide compositions and associated methods of use to inhibit protein aggregates and their detrimental effects.

Although factors are known which lead to aggregation of proteins in the native state, for example, salting out, and isoelectric precipitation; the majority of cases of protein aggregation involve the intermolecular association of a partially-folded or "unfolded" intermediate state of the protein. The underlying reason is probably that partially-folded intermediates have hydrophobic patches, which normally pack together to yield the native state, but which can also interact in an intermolecular manner to form an aggregate. Diseases where protein aggregation is causal or an associated symptom and for which the present invention may be useful for treatment and/or prevention include Down's syndrome, Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), and prion diseases such as bovine spongiform encephalitis (BSE) and Creutzfeldt-Jakob Disease (CJD), cystic fibrosis, and the so-celled polyglutamine diseases (TABLE 1 (see Original Patent)), for example, Huntington's disease (HD), dentato-rubral and pallido-luysian atrophy (DRPLA) and several forms of spino-cerebellar ataxia (SCA), also have intracellular inclusions in regions corresponding to the regions of neuronal degeneration

In certain aspects the invention relates to isolated oligonucleotide compositions comprising from about 15 to about 50 nucleotides, and having at least 40% guanosine nucleotides. In certain embodiments the invention comprises oligonucleotides of SEQ ID NOs: 1-7. These gunosine rich oligonucleotides (GROs) have been shown to form higher order aggregates, for example, G-quartet structures, in which the GROs align in a parallel or antiparallel configuration. (See Biyani and Nisigaki, Gene 364: 130-38 (2005), incorporated herein by reference in its entirety). While not being limited to any particular theory, the inventors hypothesize that the higher-order structures of the GROs of the invention mediate their efficacy; i.e., inhibiting the aggregation of proteins, for example, the disease associated polyglutamine proteins. However, the GROs of the present invention may also be used generally to inhibit aggregation of other disease related proteins as indicated above. Therefore, in another aspect the oligonucleotide of the invention comprises a G-quartet structure. In a preferred embodiment, the isolated oligonucleotide of the invention comprises from 18-24 nucleotides, and has at least 95% guanosine nucleotides.

In other aspects the isolated GRO of the invention is disposed in a vector or plasmid nucleic acid for its convenient cloning, amplification, and/or transcription. In still other aspects the isolated GRO of the invention is operably linked to one or more transcription regulatory nucleic acid sequences. In yet another aspect, the isolated GRO is disposed in a vector or plasmid nucleic acid, and is operably linked with one or more transcription regulatory nucleic acid sequences.

In other aspects, the invention relates to a host cell comprising the isolated GRO of the invention. In certain embodiments, the host cell further comprises a vector or plasmid nucleic acid containing one or more transcription regulatory nucleic acid sequences operably linked with the GRO sequence of the invention. The vector or plasmid nucleic acids can be, for example, suitable for eukaryotic or prokaryotic cloning, amplification, or transcription. The vector or plasmid nucleic acids can also be stably integrated into the host cell's genome or maintained episomally.

In another aspect, the invention relates to method for inhibiting and/or reducing the aggregation of proteins. In other aspects, the invention relates to methods for inhibiting or reducing the aggregation of polyglutamine proteins, such as those that cause Huntington's Disease, or Spinocerebellar ataxia. In any embodiment of these aspects the invention comprises contacting an protein capable of forming a protein aggregate or a protein aggregate with an effective amount of a GRO of the invention to result in the inhibition of protein aggregate formation, the reduction of protein aggregation, and/or the dissociation of the components from a protein aggregate.

In other aspects, the invention relates to methods for treating and/or preventing a disease or condition in an individual related to the detrimental effects of protein aggregation. In certain embodiments, the methods of the invention comprise administering an effective amount of an isolated GRO in a pharmaceutically acceptable form to an individual in need thereof. In certain embodiments, the isolated GRO of the invention is administered together with a pharmaceutically acceptable carrier, excipient, adjuvant, amino acid, peptide, polypeptide, chemical compound, drug, biologically active agent or a combination thereof. As such, in another aspect the invention relates to therapeutic compositions comprising the isolated GRO of the invention in a pharmaceutically acceptable form together with at least one pharmaceutically acceptable carrier, excipient, adjuvant, amino acid, peptide, polypeptide, chemical compound, drug, biologically active agent or a combination thereof.

In certain embodiments the therapeutic GRO of the invention is complexed, bound, or conjugated to one or more chemical moieties to improve and/or modify, for example, bioavailability, half-life, efficacy, and/or targeting. In certain aspects of this embodiment, the GRO may be complexed or bound, either covalently or non-covalently with, for example, cationic molecules, salts or ions, lipids, glycerides, carbohydrates, amino acids, peptides, proteins, other chemical compounds, for example, phenolic compounds, and combinations thereof. In certain aspects the invention relates to a GRO of the invention conjugated to a polypeptide, for example, an antibody. In certain embodiments the antibody is specific for the protein or protein aggregate of interest and therefore targets the GRO to the protein and/or protein aggregate.

The therapeutic GRO of the invention can be administered by any suitable route recognized by those of skill in the art, for example, enteral, intravenous, intra-arterial, parenteral, topical, transdermal, nasal, and the like. In addition, the therapeutic may be in any pharmaceutically acceptable form such as, for example, a liquid, lyophilized powder, gel, pill, controlled release capsule, and the like, which is now known or becomes known to those of skill in the art.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the surprising an unexpected discovery that certain oligonucleotides are capable of inhibiting protein aggregation. The invention includes oligonucleotide compositions useful for research and therapeutic purposes.

In certain embodiments, the invention comprises an isolated polynucleotide sequence, for example, the isolated aptameric GROs of SEQ ID NOs: 1-12. By "isolated nucleic acid sequence" is meant a polynucleotide that is not immediately contiguous with either of the coding sequences with which it is immediately contiguous (one on the 5' end and one on the 3' end) in the naturally occurring genome of the organism from which it is derived. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector; into an automatically replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA) independent of other sequences. The nucleotides can be modified forms of DNA or RNA.

The present invention relates to the finding that guanosine (G) rich oligonucleotides (GROs) form functional aptamers, and are effective inhibitors of protein aggregation, for example, the aggregation of polyglutamine proteins such as huntingtin protein, which is associated with Huntington's Disease (HD). As such, the isolated GROs of the invention have therapeutic potential and can be used as a treatment for patients with diseases and conditions resulting from detrimental effects of protein aggregation, for example, Huntington's disease. While not being limited by any particular theory, the inventors postulate that the beneficial effect observed with the GROs of the invention may result from the inhibition or slowing of the aggregation process. The GROs of the invention that possess aptameric activity may also be beneficial in other amyloid or neurodegenerative diseases, for example, Alzheimer's Disease, Parkinson's Disease, spinocerebellar ataxia, and prion diseases. Moreover, the GROs of the present invention can be used to examine the relationship between cellular aggregates and toxicity in various model systems.

Therefore, in one embodiment the polynucleotide composition of the invention comprises an isolated aptameric oligonucleotide having from about 15 to about 50 nucleotides, and having at least 40% guanosine nucleotides. In certain embodiments the invention comprises oligonucleotides of SEQ ID NOs: 1-12. In another embodiment, the oligonucleotides of the invention are capable of forming G-quartet structures.

G-rich DNA and RNA have the ability to form inter- and intramolecular four-stranded structures, referred to as G-quartets. (See Biyani and Nisigaki, Gene 364: 130-38 (2005)). G-quartets arise from the association of four G-bases into a cyclic Hoogsteen H-bonding parallel or anti-parallel arrangement, and each G-base makes two hydrogen bonds with its neighbor G-base (N1 to O6 and N2 to N7). G-quartets stack on top of each other to give rise to tetrad-helical structures. The stability of G-quartet structures depends on several factors: the presence of the monovalent cations, the concentration of the G-rich oligonucleotides present, and the sequence of the G-rich oligonucleotides under study. Potassium with the optimal size to interact within a G-octamer greatly promotes the formation of G-quartet structures and increases their stability. G-quartet oligodeoxynucleotides (GQ-ODNs) have been suggested to play a critical role in several biological processes including modulation of telomere activity, inhibition of human thrombin, HIV infection, HIV-1 integrase activity, human nuclear topoisomerase 1 activity, and DNA replication in vitro. On the basis of the structure and mechanism of Stat3 activation, G-quartet-forming oligonucleotides were developed recently to block Stat3 activity within cancer cells.

While there is no hard rule governing what specific nucleotide sequence will result in the G-quartet structure, they can usually form with some iteration of a guanosine repeat, for example, GGTT.sub.n. Thus, as along as the guanosines can come in contact via parallel or anti paralell positioning, then the oligonucleotides can form higher-order structures such as the G-quartet structure. As such, the sequence of the aptameric oligonucleotide of the invention can be varied in any number of ways as long as the oligonucleotide comprises from about 15 to about 50 nucleotides, comprises at least 40% guanosine nucleotides. In a preferred embodiment, the aptameric oligonucleotides form a G-quartet structure. In certain embodiments, the invention comprises an aptameric oligonucleotide of SEQ ID NOs:1-12.

While not being limited to any particular theory, the inventors hypothesize that the higher-order structures of the aptameric GROs of the invention mediate their efficacy; i.e., inhibiting the aggregation of proteins, for example, the disease associated polyglutamine proteins. However, the aptameric GROs of the present invention may also be used generally to inhibit aggregation of other disease related proteins as indicated above. In a preferred embodiment, the isolated aptameric oligonucleotide of the invention comprises from 18-24 nucleotides, and has at least 95% guanosine nucleotides. In a particularly preferred embodiment, the invention comprises the GRO of SEQ ID NO:3. By utilizing a biochemical assay as an initial screen, SEQ ID NO:3 inhibited Htt aggregation. The monotonic G-ODN of the invention was also able to improve cell survival in PC12 cells overexpressing a mutant Htt fragment fusion gene.

In any of the embodiments described herein, the aptameric GRO of the invention may comprise one or more modified nucleotides or nucleotide analogs. Nucleotide modifications can be incorporated during or after oligonucleotide synthesis, and include modifications of the nucleobase, the sugar moiety, and/or the phosphate group.

Phosphodiester Moiety Analogs. Numerous analogs to the naturally occurring phosphodiester backbone have been used in oligonucleotide design. Phosphorothioate, phosphorodithioate, and methylphosphonate are readily synthesized using known chemical methods. Because novel nucleotide linkages can be synthesized manually to form a dimer and the dimer later introduced into the oligonucleotide via automated synthesis, the range of potential backbone modifications is as broad as the scope of synthetic chemistry. For example, the oligonucleotide may be substituted or modified in its internucleotide phosphate residue with a thioether, carbamate, carbonate, acetamidate or carboxymethyl ester.

Unlike the naturally occurring phosphodiester moieties, many phosphodiester analogs have chiral centers. For example, phosphorothioates, methylphosphonates, phosphoramidates, and alkyl phosphotriesters all have chiral centers. One skilled in the art would recognize numerous other phosphodiester analogs that possess chiral centers. Because of the importance of stereochemistry in hybridization, the stereochemistry of phosphodiester analogs can influence the affinity of the oligonucleotide for its target.

Most phosphodiester backbone analogs exhibit increased resistance to nuclease degradation. In an embodiment, phosphorothioates, methyl phosphonates, phosphorimidates, and/or phosphotriesters are used to achieve enhanced nuclease resistance. Increased resistance to degradation may also be achieved by capping the 5' and/or 3' end of the oligonucleotide. In an embodiment, the 5' and/or 3' end capping of the oligonucleotide is via a 5'-5' and/or 3'-3' terminal inverted linkage.

Phosphorothioate oligodeoxynucleotides are relatively nuclease resistant, water soluble analogs of phosphodiester oligodeoxynucleotides. These molecules are racemic, but still hybridize well to their RNA targets. Stein, C., et al. (1991) Pharmac. Ther. 52:365 384. Phosphorothioate oligonucleotides may be stereo regular, stereo non-regular or stereo random. A stereo regular phosphorothioate oligonucleotide is a phosphorothioate oligonucleotide in which all of the phosphodiester linkages or phosphorothiodiester linkages polarize light in the same direction. Each phosphorous in each linkage may be either an S.sub.p or R.sub.p diastereomer.

Sugar Moiety Analogs. Oligonucleotide analogs may be created by modifying and/or replacing a sugar moiety. The sugar moiety of the oligonucleotide may be modified by the addition of one or more substituents. For example, one or more of the sugar moieties may contain one or more of the following substituents: amino-alkylamino, araalkyl, heteroalkyl, heterocycloalkyl, aminoalkylamino, O, H, an alkyl, polyalkylamino, substituted silyl, F, Cl, Br, CN, CF.sub.3, OCF.sub.3, OCN, O-alkyl, S-alkyl, SOMe, SO.sub.2Me, ONO.sub.2, NH-alkyl, OCH.sub.2CH.dbd.CH.sub.2, OCH.sub.2CCH, OCCHO, allyl, O-allyl, NO.sub.2, N.sub.3, and NH.sub.2.

Modification of the 2' position of the ribose sugar has been shown in many instances to increase the oligonucleotide's resistance to degradation. For example, the 2' position of the sugar may be modified to contain one of the following groups: H, OH, OCN, O-alkyl, F, CN, CF.sub.3, allyl, O-allyl, OCF.sub.3, S-alkyl, SOMe, SO.sub.2Me, ONO.sub.2, NO.sub.2, N.sub.3, NH.sub.2, NH-alkyl, or OCH.dbd.CH.sub.2, OCCH, wherein the alkyl may be straight, branched, saturated, or unsaturated.

In addition, the oligonucleotide may have one or more of its sugars modified and/or replaced so as to be a ribose or hexose (i.e. glucose, galactose). Further, the oligonucleotide may have one or more modified sugars. The sugar may be modified to contain one or more linkers for attachment to other chemicals such as fluorescent labels. In an embodiment, the sugar is linked to one or more aminoalkyloxy linkers. In another embodiment, the sugar contains one or more alkylamino linkers. Aminoalkyloxy and alkylamino linkers may be attached to biotin, cholic acid, fluorescein, or other chemical moieties through their amino group.

Base Moiety Analogs. In addition, the oligonucleotide may have one or more of its nucleotide bases substituted or modified. In addition to adenine, guanine, cytosine, thymine, and uracil, other bases such as inosine, deoxyinosine, hypoxanthine may be used. In addition, isoteric purine 2'deoxy-furanoside analogs, 2'-deoxynebularine or 2'deoxyxanthosine, or other purine or pyrimidine analogs may also be used. By carefully selecting the bases and base analogs, one may fine tune the binding properties of the oligonucleotide. For example, inosine nay be used to reduce hybridization specificity, while diaminopurines may be used to increase hybridization specificity.

Adenine and guanine may be modified at positions N3, N7, N9, C2, C4, C5, C6, or C8 and still maintain their hydrogen bonding abilities. Cytosine, thymine and uracil may be modified at positions N1, C2, C4, C5, or C6 and still maintain their hydrogen bonding abilities.

Some base analogs have different hydrogen bonding attributes than the naturally occurring bases. For example, 2-amino-2'-dA forms three, instead of the usual two, hydrogen bonds to thymine (T). Examples of base analogs that have been shown to increase duplex stability include, but are not limited to, 5-fluoro-2'-dU, 5-bromo-2'-dU, 5-methyl-2'-dc, 5-propynyl-2'-dC, 5-propynyl-2'-dU, 2-amino-2'-dA, 7-deazaguanosine, 7-deazadenosine, and N2-Imidazoylpropyl-2'-dG.

Pendant Groups. A "pendant group" may be linked to the oligonucleotide. Pendant groups serve a variety of purposes which include, but are not limited to, increasing cellular uptake of the oligonucleotide, enhancing degradation of the target nucleic acid, and increasing hybridization affinity. Pendant groups can be linked to any portion of the oligonucleotide but are commonly linked to the end(s) of the oligonucleotide chain. Examples of pendant groups include, but are not limited to: acridine derivatives (i.e. 2-methoxy-6-chloro-9-aminoacridine); cross-linkers such as psoralen derivatives, azidophenacyl, proflavin, and azidoproflavin; artificial endonucleases; metal complexes such as EDTA-Fe(II), o-phenanthroline-Cu(I), and porphyrin-Fe(II); alkylating moieties; nucleases such as amino-1-hexanolstaphylococcal nuclease and alkaline phosphatase; terminal transferases; abzymes; cholesteryl moieties; lipophilic carriers; peptide conjugates; long chain alcohols; phosphate esters; amino; mercapto groups; phenolic groups, radioactive markers; nonradioactive markers such as dyes; and polylysine or other polyamines.

In one embodiment, the aptameric oligonucleotide of the invention contains at least one nucleotide conjugated to a carbohydrate, sulfated carbohydrate, or gylcan. Conjugates may be regarded as a way as to introduce a specificity into otherwise unspecific DNA binding molecules by covalently linking them to a selective oligonucleotide or polypeptide.

Cellular Uptake. To enhance cellular uptake, the oligonucleotide may be administered in combination with a carrier or lipid. For example, the oligonucleotide may be administered in combination with a cationic lipid. Examples of cationic lipids include, but are not limited to, lipofectin, dotma, dope, DMRIE and DPPES. The oligonucleotide may also be administered in combination with a cationic amine such as poly (L-lysine). Oligonucleotide uptake may also be increased by conjugating the oligonucleotide to chemical moieties such as transferrin and cholesteryls. In addition, oligonucleotides may be targeted to certain organelles by linking specific chemical groups to the oligonucleotide. For example, linking the oligonucleotide to a suitable array of mannose residues will target the oligonucleotide to the liver.

The cellular uptake and localization of oligonucleotides may be monitored by using labeled oligonucleotides. Methods of labeling include, but are not limited to, radioactive and fluorescent labeling. Fluorescently labeled oligonucleotides may be monitored using fluorescence microscopy and flow cytometry.

The efficient cellular uptake of oligonucleotides is well established. For example, when a 20 base sequence phosphorothioate (PS) oligonucleotide was Injected into the abdomens of mice, either intraperitoneally (IP) or intravenously (IV). The oligonucleotide accumulated in the kidney liver, and brain. Chain-extended oligonucleotides were also observed. Argrawal, S., et al. (1988) Proc. Natl. Acad. Sci. U.S.A. 85:7079 7083. When the PS 27-oligonucleotide was given by IV to rats, the initial T.sub.1/2 (transit out of the plasma) was 23 min, while the T.sub.1/2beta of total body clearance was 33.9 hours. The long beta half-life of elimination demonstrates that dosing could be infrequent and still maintain effective, therapeutic tissue concentrations. Iverson, P. (1991) Anti-Cancer Drug Des. 6:531.

Another aspect of the invention pertains to vectors, containing an aptameric GRO of the invention, for example, nucleic acid encoding SEQ ID NOs: 1-12, or derivatives thereof for its convenient cloning, amplification, and/or transcription. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been "operably linked." One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the transcription of sequences to which they are operatively-linked. Such vectors are referred to herein as "expression vectors". In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" can be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), and artificial chromosomes, which serve equivalent functions.

The recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, that is operatively-linked to the nucleic acid sequence to be transcribed. Within a recombinant expression vector, "operably-linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner that allows for transcription and/or expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).

The term "regulatory sequence" is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of transcription, and/or expression of protein desired, etc. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein. The recombinant expression vectors of the invention can be designed for transcription and/or expression in prokaryotic or eukaryotic cells. For example, transcription and/or expression in bacterial cells such as Escherichia coli, insect cells (using baculovirus expression vectors) yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Alternatively, the recombinant expression vector can be transcribed and/or translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.

In another embodiment, the recombinant vector is capable of directing transcription of the aptameric GRO preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert, et al., 1987. Genes Dev. 1: 268-277), lymphoid-specific promoters (Calame and Eaton, 1988. Adv. Immunol. 43: 235-275), in particular promoters of T cell receptors (Winoto and Baltimore, 1989. EMBO J. 8: 729-733) and immunoglobulins (Banedji, et al., 1983. Cell 33: 729-740; Queen and Baltimore, 1983. Cell 33: 741-748), neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle, 1989. Proc. Natl. Acad. Sci. USA 86: 5473-5477), pancreas-specific promoters (Edlund, et al., 1985. Science 230: 912-916), and mammary gland-specific promoters (e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166). Developmentally-regulated promoters are also encompassed, e.g., the murine hox promoters (Kessel and Gruss, 1990. Science 249: 374-379) and the alpha-fetoprotein promoter (Campes and Tilghman, 1989. Genes Dev. 3: 537-546).

In other aspects, the invention relates to a host cell comprising the isolated aptameric GRO of the invention. In certain embodiments, the host cell comprises a vector, plasmid or artificial chromosome nucleic acid containing one or more transcription regulatory nucleic acid sequences operably linked with the aptameric GRO sequence of the invention. The vector or plasmid nucleic acids can be, for example, suitable for eukaryotic or prokaryotic cloning, amplification, or transcription. In other embodiments, the invention comprises a plurality of aptameric GRO sequences linked contiguously as a single polynucleotide chain. In still other embodiments, the invention comprises a nucleic acid vector containing a plurality of aptameric GRO sequences linked contiguously and operably linked with the nucleic acid sequence of the vector.

The term "host cell" includes a cell that might be used to carry a heterologous nucleic acid, or expresses a peptide or protein encoded by a heterologous nucleic acid. A host cell can contain genes that are not found within the native (non-recombinant) form of the cell, genes found in the native form of the cell where the genes are modified and re-introduced into the cell by artificial means, or a nucleic acid endogenous to the cell that has been artificially modified without removing the nucleic acid from the cell. A host cell may be eukaryotic or prokaryotic. For example, bacteria cells may be used to carry or clone nucleic acid sequences or express polypeptides. General growth conditions necessary for the culture of bacteria can be found in texts such as BERGEY'S MANUAL OF SYSTEMATIC BACTERIOLOGY, Vol. 1, N. R. Krieg, ed., Williams and Wilkins, Baltimore/London (1984). A "host cell" can also be one in which the endogenous genes or promoters or both have been modified to produce the aptameric GRO of the invention.

In another aspect, the invention relates to method for inhibiting and/or reducing the aggregation of proteins. In other aspects, the invention relates to methods for inhibiting or reducing the aggregation of polyglutamine proteins, such as those that cause Huntington's Disease, or Spinocerebellar ataxia. In any embodiment of these aspects the invention comprises contacting an protein capable of forming a protein aggregate or a protein aggregate with an effective amount of a GRO of the invention to result in the inhibition of protein aggregate formation, the reduction of protein aggregation, and/or the dissociation of the components from a protein aggregate.

Plasmids disclosed herein are either commercially available, publicly available on an unrestricted basis, or can be constructed from available plasmids by routine application of well-known, published procedures. Many plasmids and other cloning and expression vectors are well known and readily available, or those of ordinary skill in the art may readily construct any number of other plasmids suitable for use. These vectors may be transformed into a suitable host cell to form a host cell vector system. Suitable hosts include microbes such as bacteria, yeast, insect or mammalian organisms or cell lines. Examples of suitable bacteria are E. coli and B. subtilis. A preferred yeast vector is pRS426-Gal. Examples of suitable yeast are Saccharomyces and Pichia. Suitable amphibian cells are Xenopus cells. Suitable vectors for insect cell lines include baculovirus vectors. Mouse, rat or human cells are preferred mammalian cells.

Transformation of a host cell with recombinant DNA may be carried out by conventional techniques as are well known to those skilled in the art. By "transformation" is meant a permanent or transient genetic change induced in a cell following incorporation of new DNA (i.e., DNA exogenous to the cell). Where the cell is a mammalian cell, a permanent genetic change is generally achieved by introduction of the DNA into the genome of the cell. By "transformed cell" or "host cell" is meant a cell (e.g., prokaryotic or eukaryotic) into which (or into an ancestor of which) has been introduced, by means of recombinant DNA techniques, a DNA molecule encoding a polypeptide of the invention (i.e., an INDY polypeptide), or fragment thereof.

Where the host is prokaryotic, such as E. coli, competent cells which are capable of DNA uptake can be prepared from cells harvested after exponential growth phase and subsequently treated by the CaCl.sub.2 method by procedures well known in the art. Alternatively, MgCl.sub.2 or RbCl can be used. Transformation can also be performed after forming a protoplast of the host cell or by electroporation.

When the host is a eukaryote, such methods of transfection with DNA include calcium phosphate co-precipitates, conventional mechanical procedures such as microinjection, electroporation, insertion of a plasmid encased in liposomes, or virus vectors, as well as others known in the art, may be used. Another method is to use a eukaryotic viral vector, such as simian virus 40 (SV40) or bovine papilloma virus, to transiently infect or transform eukaryotic cells. (Eukaryotic Viral Vectors, Cold Spring Harbor Laboratory, Gluzman ed., 1982). Preferably, a eukaryotic host is utilized as the host cell as described herein. The eukaryotic cell may be a yeast cell (e.g., Saccharomyces cerevisiae) or may be a mammalian cell, including a human cell.

Mammalian cell systems that utilize recombinant viruses or viral elements to direct expression may be engineered. For example, when using adenovirus expression vectors, the nucleic acid sequences may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region E1 or E3) will result in a recombinant virus that is viable and capable of expressing the polypeptides in infected hosts (e.g., Logan & Shenk, Proc. Natl. Acad. Sci. U.S.A. 81:3655-3659, 1984).

For long-term, high-yield production of recombinant genes, stable expression is preferred. Rather than using expression vectors that contain viral origins of replication, host cells can be transformed with the cDNA encoding an aptameric GRO controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci, which in turn can be cloned and expanded into cell lines. For example, following the introduction of foreign DNA, engineered cells may be allowed to grow for 1 to 2 days in an enriched media, and then are switched to a selective media. A number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al., Cell 11: 233, 1977), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl. Sci. U.S.A. 48: 2026, 1962), and adenine phosphoribosyltransferase (Lowy et al., Cell 22: 817, 1980) genes can be employed.

In other aspects, the invention relates to methods for treating and/or preventing a disease or condition in an individual related to the detrimental effects of protein aggregation. In certain embodiments, the methods of the invention comprise administering an effective amount of an isolated GRO in a pharmaceutically acceptable form to an individual in need thereof. In certain embodiments, the isolated GRO of the invention is administered together with a pharmaceutically acceptable carrier, excipient, adjuvant, amino acid, peptide, polypeptide, chemical compound, drug, biologically active agent or a combination thereof. As such, in another aspect the invention relates to therapeutic compositions comprising the isolated GRO of the invention in a pharmaceutically acceptable form together with at least one pharmaceutically acceptable carrier, excipient, adjuvant, amino acid, peptide, polypeptide, chemical compound, drug, biologically active agent or a combination thereof.

In certain embodiments the therapeutic GRO of the invention is complexed, bound, or conjugated to one or more chemical moieties to improve and/or modify, for example, bioavailability, half-life, efficacy, and/or targeting. In certain aspects of this embodiment, the GRO may be complexed or bound, either covalently or non-covalently with, for example, cationic molecules, salts or ions, lipids, glycerides, carbohydrates, amino acids, peptides, proteins, other chemical compounds, for example, phenolic compounds, and combinations thereof. In certain aspects the invention relates to a GRO of the invention conjugated to a polypeptide, for example, an antibody. In certain embodiments the antibody is specific for the protein or protein aggregate of interest and therefore targets the GRO to the protein and/or protein aggregate.

The efficacy of oligonucleotide therapy is also well established. For example, when a 24-base sequence PS oligonucleotide targeted to human c-myb mRNA was infused, through a miniosmotic pump, into scid mice bearing the human K562 chronic myeloid leukemia cell line, mean survival times of the mice treated with the antisense oligonucleotides were six- to eightfold longer than those of mice untreated or treated with the sense controls or treated with an oligonucleotide complementary to the c-kit proto-oncogene mRNA. Ratajczak, et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89:11823.

Therapeutic uses and formulations. The nucleic acids and proteins of the invention are useful in potential prophylactic and therapeutic applications implicated in a variety of disorders including, but not limited to: metabolic disorders, diabetes, obesity, infectious disease, anorexia, cancer, neurodegenerative disorders, Huntington's Disease, Alzheimer's Disease, Parkinson's Disorder, prion diseases (e.g., BSE and CJD), spinocerebellar ataxia, immune disorders, hematopoietic disorders, and the various dyslipidemias, metabolic disturbances associated with obesity, the metabolic syndrome X and wasting disorders associated with chronic diseases and various cancers, cardiomyopathy, atherosclerosis, hypertension, congenital heart defects, aortic stenosis, atrial septal defect (ASD), atrioventricular (A-V) canal defect, ductus arteriosus, pulmonary stenosis, subaortic stenosis, ventricular septal defect (VSD), valve diseases, tuberous sclerosis, scleroderma, lupus erythematosus, obesity, transplantation, adrenoleukodystrophy, congenital adrenal hyperplasia, prostate cancer, neoplasm; adenocarcinoma, lymphoma, uterus cancer, fertility, leukemia, hemophilia, hypercoagulation, idiopathic thrombocytopenic purpura, immunodeficiencies, graft versus host disease, AIDS, bronchial asthma, rheumatoid and osteoarthritis, Crohn's disease; multiple sclerosis, treatment of Albright Hereditary Ostoeodystrophy, and other diseases, disorders and conditions of the like.

Preparations for administration of the therapeutic complex of the invention include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's intravenous vehicles including fluid and nutrient replenishers, electrolyte replenishers, and the like. Preservatives and other additives may be added such as, for example, antimicrobial agents, anti-oxidants, chelating agents and inert gases and the like.

The nucleic acid molecules, polypeptides, and antibodies (also referred to herein as "active compounds") of the invention, and derivatives, fragments, analogs and homologs thereof, can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise the nucleic acid molecule, protein, or antibody and a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, a standard reference text in the field, which is incorporated herein by reference. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, finger's solutions, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils may also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, intraperitoneal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor.TM.. (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the active compound (e.g., the therapeutic complex of the invention) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

For oral administration, the pharmaceutical compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate). The tablets may be coated by methods well known in the art. Liquid preparations for oral administration may take the form of, for example, solutions, syrups, or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, flavoring, coloring, and sweetening agents as appropriate.

Preparations for oral administration may be suitably formulated to give controlled release of the active compound. For buccal administration the compositions may take the form of tablets or lozenges formulated in conventional manner. For administration by inhalation, the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of e.g. gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch. The compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing, and/or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use. The compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides. In addition to the formulations described previously, the compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.

Principles and considerations involved in preparing such compositions, as well as guidance in the choice of components are provided, for example, in Remington: The Science And Practice Of Pharmacy 19th ed. (Alfonso R. Gennaro, et al., editors) Mack Pub. Co., Easton, Pa.: 1995; Drug Absorption Enhancement: Concepts, Possibilities, Limitations, And Trends, Harwood Academic Publishers, Langhorne, Pa., 1994; and Peptide And Protein Drug Delivery (Advances In Parenteral Sciences, Vol. 4), 1991, M. Dekker, New York.

The active ingredients can also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacrylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles, and nanocapsules) or in macroemulsions. The formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.

Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT.TM. (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods.

In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.

The nucleic acid molecules of the invention can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see, e.g., U.S. Pat. No. 5,328,470) or by stereotactic injection (see, e.g., Chen, et al., 1994. Proc. Natl. Acad. Sci. USA 91: 3054-3057). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells that produce the gene delivery system. The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

A therapeutically effective dose refers to that amount of the therapeutic complex sufficient to result in amelioration or delay of symptoms. Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds that exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects. The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

Pharmaceutical compositions may be formulated in conventional manner using one or more physiologically acceptable carriers or excipients. Thus, the compounds and their physiologically acceptable salts and solvates may be formulated for administration by inhalation or insufflation (either through the mouth or the nose) or oral, buccal, intravenous, intraperitoneal, parenteral or rectal administration.

Also disclosed according to the present invention is a kit or system utilizing any one of the methods, selection strategies, materials, or components described herein. Exemplary kits according to the present disclosure will optionally, additionally include instructions for performing methods or assays, packaging materials, one or more containers which contain an assay, a device or system components, or the like.

In an additional aspect, the present invention provides kits embodying the complex and methods of using disclosed herein. Kits of the invention optionally include one or more of the following: (1) polypeptide or nucleic acid components described herein; (2) instructions for practicing the methods described herein, and/or for operating the selection procedure herein; (3) one or more detection assay components; (4) a container for holding nucleic acids or polypeptides, other nucleic acids, transgenic plants, animals, cells, or the like and, (5) packaging materials.
 

Claim 1 of 4 Claims

1. A method of inhibiting or reducing the aggregation of polyglutamine-containing proteins associated with polyglutamine diseases comprising: providing an oligonucleotide of from 15 to 50 nucleotides, wherein at least 60% of the nucleotides are guanosine nucleotides; providing a polyglutamine-containing protein or a protein aggregate; and contacting the polyglutamine-containing protein or protein aggregate with an effective amount of the oligonucleotide sufficient to inhibit or reduce protein aggregation.
 

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