WO2006130949A1

WO2006130949A1 - Anti amyloid-related disease molecules and their uses

Info

Publication number: WO2006130949A1
Application number: PCT/CA2006/000702
Authority: WO
Inventors: Andrew Vaillant; Jean-Marc Juteau
Original assignee: Replicor Inc.
Priority date: 2005-06-08
Filing date: 2006-05-01
Publication date: 2006-12-14

Abstract

Oligonucleotides formulation comprising oligonucleotides having an anti amyloid-related disease activity that act by a sequence independent mechanism. The anti amyloid-related disease activity is greater for large oligonucleotides and for oligonucleotides with sulfur modification. The invention also relates to oligonucleotides and their use as therapeutic agents, and more particularly for their use and formulations for the treatment of amyloid-related diseases. The modified oligonucleotides are included in the present invention, for example, ONs containing modified backbones.

Description

ANTI AMYLOID-RELATED DISEASE MOLECULES AND THEIR USES

FIELD OF THE INVENTION

[0001] The present invention relates to oligonucleotides having an anti amyloid-related disease activity that act by a sequence independent mechanism. The anti amyloid-related disease activity is greater for large oligonucleotides and for oligonucleotides with sulfur modification.

BACKGROUND OF THE INVENTION

[0002] A review on amyloid-related disease or amyloidosis is found in US Pat. Application publication No 20050038117. For more precision, amyloidosis refers to a pathological condition characterized by the presence of amyloid fibrils. Amyloid is a generic term referring to a group of diverse protein deposits (intracellular or extracellular) which are seen in a number of different diseases. Amyloid-related diseases can either be restricted to one organ or spread to several organs. The first instance is referred to as "localized amyloidosis" while the second is referred to as "systemic amyloidosis." Some amyloid diseases can be idiopathic, but most of these diseases appear as a complication of a previously existing disorder. For example, primary amyloidosis (AL amyloid) can appear without any other pathology or can follow plasma cell dyscrasia or multiple myeloma. Secondary amyloidosis is usually seen associated with chronic infection (such as tuberculosis) or chronic inflammation (such as rheumatoid arthritis). A familial form of secondary amyloidosis is also seen in other types of familial amyloidosis, e.g., Familial Mediterranean Fever (FMF). This familial type of amyloidosis is genetically inherited and is found in specific population groups. In both primary and secondary amyloidosis, deposits are found in several organs and are thus considered systemic amyloid diseases. "Localized amyloidoses" are those that tend to involve a single organ system. Different amyloids are also characterized by the type of protein present in the deposit. For example, neurodegenerative diseases such as scrapie, bovine spongiform encephalitis, Creutzfeldt- Jakob disease, and the like are characterized by the appearance and accumulation of a protease-resistant form of a prion protein (referred to as AScr or PrP-27) in the central nervous system. Similarly, Alzheimer's disease, another neurodegenerative disorder, is characterized by neuritic plaques and neurofibrillary tangles. In this case, the amyloid plaques found in the parenchyma and the blood vessel is formed by the deposition of fibrillar A beta amyloid protein. Other diseases such as adult- onset diabetes (type II diabetes) are characterized by the localized accumulation of amyloid fibrils in the pancreas. Another type of amyloidosis is related to beta 2 microglobulin and is found in long-term hemodialysis patients.

[0003] Reactive or secondary amyloidosis (AA) is a manifestation of a number of diseases that provoke a sustained acute phase response. Such diseases include chronic inflammatory disorders, chronic local or systemic microbial infections, and malignant neoplasms. The most common form of reactive or secondary amyloidosis is seen as the result of long-standing inflammatory conditions. For example, patients with Rheumatoid Arthritis or Familial Mediterranean Fever (which is a genetic disease) can develop AA amyloidosis. The terms "AA amyloidosis" and "secondary AA amyloidosis" are used interchangeably. Deposition of AA fibrils can be widespread in the body, with a preference for parenchymal organs. The kidneys are usually a deposition site, and the liver and the spleen may also be affected. Deposition is also seen in the heart, gastrointestinal tract, and the skin.

[0004] Drugs that inhibit the degradation of acetylcholine within synapses are the mainstay of therapy in the treatment of Alzheimer's disease. Donepezil, rivastigmine, and galantamine are safe but have potentially troublesome cholinergic side effects, including nausea, anorexia, diarrhea, vomiting, and weight loss. Also, the cholinesterase inhibitor tacrine has been described. These drugs improve cognition, but evidence is less robust for benefits in delaying nursing home placement and improving functional ability and behaviors. Benefit for vitamin E, selegiline, estrogen in postmenopausal women and nonsteroidal anti-inflammatory drugs has been suggested.

[0005] The use of oligonucleotides (ONs), mainly antisense ONs, in the treatment of amyloid-related diseases has been described in the scientific literature.

[0006] Using antisense oligodeoxynucleotides (ODNs) that were prepared to the 5¹ terminus of amyloid precursor protein (APP) specifically to reduce the level of APP isoforms, it was shown that pretreatment of cells with these antisense ODNs diminished NGF-induced increases in cellular size and neurite length (Majocha et al., (1994) Cell MoI. Neurobiol. 14:425-437).

[0007] Kumar et al. tested antisense phosphorothioate-oligodeoxynucleotide (PS- ODN) in a mouse model of Alzheimer's and showed that antisense directed at the Abeta region of APP can reduce APP levels in the brain and reverse deficits in learning and memory (Kumar et al. (2000) Peptides 21 :1769-1775).

[0008] US patents No 5,837,449, 6,177,246, 5,670,634 and US patent application No 2003232435 describe the use of antisense ODNs to modulate the expression of genes involved in Alzeimer's disease and other amyloid plaque diseases.

[0009] Available therapies for treatment of amyloid diseases, mainly Alzheimer's disease, provide only temporary or partial clinical benefit. Although some pharmaceutical agents have been described that offer partial symptomatic relief, no comprehensive pharmacological therapy is currently available for the prevention or treatment of, for example, Alzheimer's disease. Thus, there is a need for compounds, methods of treatment and formulations to cure, prevent or control amyloid-related diseases.

SUMMARY OF THE INVENTION

[0010] One aim of the present invention is to provide a sequence independent ONs that could be used to treat amyloid-related diseases.

[0011] According to the present invention, there is provided an anti amyloid-related disease oligonucleotide formulation comprising at least one anti amyloid-related disease oligonucleotide and an excipient. The oligonucleotide has an activity against amyloid- related disease and the activity occurs principally by a sequence independent mode of action.

[0012] Another object of the present invention is to provide an oligonucleotide formulation, wherein said oligonucleotide is at least 15, 20, 25, 30, 35, 40, 45, 50, 60 or 80 nucleotides in length. [0013] In one embodiment of the present invention, the oligonucleotide formulation of contains an oligonucleotide which is 20-30, 30-40, 40-50, 50-60, 60-70, or 70-80 nucleotides in length.

[0014] Preferably, the oligonucleotide formulation has an oligonucleotide which is not complementary to any equal length portion of a genomic sequence. The genomic sequence is a genomic sequence of a human or non human animal.

[0015] In accordance with the present invention, the oligonucleotide used in the formulation comprises at least 10 contiguous nucleotides of randomer sequence, more preferably 20 nucleotides of randomer sequence, 30 nucleotides of randomer sequence, 40 nucleotides of randomer sequence or more preferably is a randomer oligonucleotide.

[0016] In a further embodiment, the oligonucleotide comprises a homopolymer sequence of at least 10 contiguous A nucleotides, or 10 contiguous T nucleotides, or 10 contiguous U nucleotides, or 10 contiguous G nucleotides, or 10 contiguous I nucleotide analogs or 10 contiguous C nucleotides.

[0017] Preferably, the oligonucleotide comprised in the oligonucleotide formulation is a homopolymer of C nucleotides.

[0018] In another embodiment of the present invention, the oligonucleotide formulation comprises an oligonucleotide having a poly AT sequence at least 10 nucleotides in length; a poly AC sequence at least 10 nucleotides in length; a polyAG sequence at least 10 nucleotides in length; a poly AU sequence at least 10 nucleotides in length; a polyAI sequence at least 10 nucleotides in length; a polyGC sequence at least 10 nucleotides in length; a polyGT sequence at least 10 nucleotides in length; a polyGU sequence at least 10 nucleotides in length; a polyGI sequence at least 10 nucleotides in length; a polyCT sequence at least 10 nucleotides in length; a polyCU sequence at least 10 nucleotides in length; a polyCI sequence at least 10 nucleotides in length; a polyTI sequence at least 10 nucleotides in length; a polyTU sequence at least 10 nucleotides in length; or a polyUI sequence at least 10 nucleotides in length. [0019] According to the present invention, the oligonucleotide formulation comprises an oligonucleotide having at least one phosphodiester linkage.

[0020] In a further embodiment, the oligonucleotide formulation comprises an oligonucleotide having at least one ribonucleotide.

[0021] Preferably, the oligonucleotide formulation comprises an oligonucleotide having at least one modification to its chemical structure, more preferably at least two different modifications to its chemical structure.

[0022] In accordance to the present invention, there is also provided an oligonucleotide formulation comprising an oligonucleotide having at least one sulfur modification

[0023] Preferably, the oligonucleotide formulation comprises an oligonucleotide having at least one phosphorothioated linkage; at least one phosphorodithioated linkage; or at least one boranophosphate linkage.

[0024] In a further embodiment, the oligonucleotide formulation comprises an oligonucleotide having at least one sulfur modified nucleobase moiety such as one sulfur modified ribose moiety, one 2' modification to the ribose moiety, one 2'-0 alkyl modified ribose moiety, one 2'-0 methyl modified ribose, one 2'-methoxyethyl modified ribose, or one 2'-FANA modified ribose.

[0025] In a further embodiment, the oligonucleotide formulation comprises an oligonucleotide having at least one methylphosphonate linkage.

[0026] In a further embodiment, the oligonucleotide formulation comprises an oligonucleotide having at least one portion consisting of glycol nucleic acid (GNA) with an acyclic propylene glycol phosphorothioate backbone.

[0027] In a further embodiment, the oligonucleotide formulation comprises an oligonucleotide having at least one locked nucleic acid moiety.

[0028] In another embodiment, the oligonucleotide formulation comprises an oligonucleotide having at least one phosphorodiamidate morpholino moiety. [0029] In another embodiment, the oligonucleotide formulation comprises an oligonucleotide having at least one abasic nucleic acid.

[0030] In a further embodiment, the oligonucleotide formulation comprises an oligonucleotide having a linker to form a concatemer of two or more oligonucleotide sequences.

[0031] According to the present invention, the oligonucleotide formulation of the present invention comprises an oligonucleotide linked or conjugated at one or more nucleotide residues, to a molecule modifying the characteristics of the oligonucleotide to obtain one or more characteristics selected from the group consisting of higher stability, lower serum interaction, higher cellular uptake, an improved ability to be formulated, a detectable signal, higher anti amyloid-related disease activity, better pharmacokinetic properties, specific tissue distribution and lower toxicity.

[0032] In a further embodiment, the oligonucleotide formulation comprises an oligonucleotide linked or conjugated to a PEG molecule; or linked or conjugated to a cholesterol molecule.

[0033] In a further embodiment, the oligonucleotide formulation comprises a double stranded oligonucleotide.

[0034] In another embodiment, the oligonucleotide formulation comprises an oligonucleotide having at least one base which is capable of hybridizing via non- Watson- Crick interactions.

[0035] According to an embodiment of the present invention, the oligonucleotide formulation comprises an oligonucleotide having a portion complementary to a genome. The oligonucleotide of the present invention has at most 90%, preferably 80%, more preferably 75% identity with the genomic sequence. In addition, the oligonucleotide has at most 60%, preferably 50%, more preferably 40% identity with a genomic sequence.

[0036] In a further embodiment, the oligonucleotide formulation comprises an oligonucleotide that binds to one or more amyloid protein. [0037] In a further embodiment, the oligonucleotide formulation comprises an oligonucleotide that interacts with one or more cellular components, wherein said interaction resulting in inhibition of a protein activity or expression.

[0038] In one embodiment of the present invention, the oligonucleotide formulation comprises an oligonucleotide wherein at least a portion of the sequence of said oligonucleotide is derived from a genome.

[0039] In a further embodiment, the oligonucleotide formulation comprises an oligonucleotide having an activity against amyloid-related disease, such as Alzeimer's disease.

[0040] Preferably, the targeted Alzheimer's disease is sporadic (non-hereditary) Alzheimer's disease, familial (hereditary) Alzheimer's disease, or early Alzheimer's disease.

[0041] Preferably, the targeted amyloid-related disease is Mild Cognitive Impairment or Mild-to-Moderate Cognitive Impairment.

[0042] In addition, the targeted amyloid-related disease is Beta 2-microglobulin-related (dialysis-related) amyloidosis.

[0043] Preferably, the targeted amyloid-related disease is Type II diabetes related amyloidosis. More preferably, the amyloid-related disease is cerebral amyloid angiopathy or hereditary cerebral hemorrhage. More preferably, the amyloid-related disease is senile dementia, Down's syndrome, inclusion body myositis, or age-related macular degeneration.

[0044] In accordance with the present invention, it is also provided an oligonucleotide mixture, comprising a mixture of at least two different anti amyloid-related disease oligonucleotide formulation according to the present invention.

[0045] In a further embodiment, the oligonucleotide mixture comprises a mixture oligonucleotide having of at least ten different anti amyloid-related disease oligonucleotide formulation according to the present invention. More preferably, the oligonucleotide mixture of the present invention comprises a mixture of at least 100 different anti amyloid-related disease oligonucleotide formulation; or at least 1000 different anti amyloid-related disease oligonucleotide formulation; or at least 10⁶ different anti amyloid-related disease oligonucleotide formulation according to the present invention.

[0046] In a further embodiment, the oligonucleotide mixture comprises oligonucleotides which are at least 10 nucleotides in length; at least 20 nucleotides in length; at least 30 nucleotides in length; at least 40 nucleotides in length; at least 50 nucleotides in length; at least 60 nucleotides in length; at least 70 nucleotides in length; are at least 80 nucleotides in length; or at least 120 nucleotides in length.

[0047] According to the present invention, it is also provided an anti amyloid-related disease pharmaceutical composition comprising a therapeutically effective amount of at least one pharmacologically acceptable, anti amyloid-related disease oligonucleotide formulation according to the present invention, and a pharmaceutically acceptable carrier.

[0048] In another embodiment, the anti amyloid-related disease pharmaceutical composition of the present invention is adapted for delivery by a mode selected from the group consisting of ocular administration, eye drop administration, oral ingestion, inhalation, subcutaneous injection, intramuscular injection, intrathecal, intracerebral, and intravenous injection.

[0049] In a further embodiment, the anti amyloid-related disease pharmaceutical composition further comprises a delivery system.

[0050] In a further embodiment, the anti amyloid-related disease pharmaceutical composition further comprises at least one other anti amyloid-related disease drug in combination.

[0051] In a further embodiment, the anti amyloid-related disease pharmaceutical composition further comprises a non-nucleotidic anti amyloid-related disease drug in combination. [0052] In another embodiment, the anti amyloid-related disease pharmaceutical composition further comprises an anti amyloid-related disease antisense oligonucleotide in combination.

[0053] In a further embodiment, the anti amyloid-related disease pharmaceutical composition further comprises an anti amyloid-related disease RNAi-inducing oligonucleotide.

[0054] According to the present invention, it is provided a use of a therapeutically effective amount of at least one pharmacologically acceptable anti amyloid-related disease oligonucleotide formulation according to the present invention, for treating or preventing amyloid related disease.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0055] The present invention involves the identification and the use of anti amyloid- related disease ONs that act by a sequence independent mechanism, and includes the discovery that the anti amyloid-related disease activity is greater for larger ONs and for ONs with sulfur modification.

[0056] The invention relates to ONs acting predominantly by a sequence independent mode of action for the treatment of amyloid-related diseases. The invention also relates to ONs and their use as therapeutic agents, and more particularly for their use and formulations for the treatment of amyloid-related diseases.

[0057] In the context of the present invention, unless specifically limited or specified the term "oligonucleotide (ON)" means oligodeoxynucleotide or oligodeoxyribonucleotide or oligoribonucleotide. Thus, "oligonucleotide" in the context of the present invention refers to an oligomer or polymer of ribonucleic acid (RNA) and/or deoxyribonucleic acid (DNA) and/or analogs thereof. This term includes oligonucleotides composed of naturally-occurring nucleobases, sugars and covalent internucleoside (backbone) linkages as well as oligonucleotides having non-naturally- occurring portions which function similarly. Examples of modifications that can be used are described herein. Oligonucleotides that include backbone and/or others modifications can also be referred to as oligonucleosides. Except otherwise specified, oligonucleotide definition includes homopolymers, heteropolymers, randomers, random sequence oligonucleotides, genomic-derived sequence oligonucleotides and oligonucleotides purified from natural sources.

[0058] As used herein in connection with the anti amyloid-related disease action of a material, the phrase "sequence independent activity" or "sequence independent mode of action" indicates that the mechanism by which the material exhibits an anti amyloid- related disease effect is not due to hybridization of complementary nucleic acid sequences, e.g., an antisense effect nor it is due to a sequence-specific aptameric activity. Conversely, a "sequence dependant mode of action or activity" means that the anti amyloid-related disease effect of a material involves hybridization of complementary nucleic acid sequences or involves a sequence-specific aptameric interaction.

[0059] As used herein the expression "anti amyloid-related disease" means inhibiting, reverting, curing, or preventing an amyloid-related disease. The expression anti amyloid- related disease also means inhibiting, reverting or preventing the formation of amyloid plaque or deposit. An anti amyloid-related disease compound can be used to treat a disease whose etiology is based on amyloid plaque formation.

[0060] As used herein the expression "amyloid-related disease", when referring to the term disease, means a disease involving unwanted formation of amyloid plaque or amyloid deposits or amyloid fibril formation. Amyloid-related disease also means amyloidosis; both terms can be use interchangeably.

[0061] The term amyloid includes beta-amyloid, amylin and any other form of amyloid. The term amyloid also refers to amyloidogenic proteins, peptides, or fragments thereof which can be soluble (e.g., monomeric or oligomeric) or insoluble (e.g., having fibrillary structure or in amyloid plaque).

[0062] As used herein in connection with ONs or other materials, the expression "anti amyloid-related disease" refers to an effect due to the presence of ONs or other material inhibiting, stopping, reverting, curing or preventing an amyloid-related disease compared to untreated cells, systems or organisms. In certain embodiments of the present invention, anti amyloid-related disease ONs will have anti amyloid-related disease activity against multiple diseases.

[0063] The expression "anti amyloid-related disease oligonucleotide formulation" refers to a preparation that includes at least one anti amyloid-related disease oligonucleotide that is adapted for use as an anti amyloid-related disease agent. The formulation includes the ON or ONs, and can contain other materials that do not interfere with their use as an anti amyloid-related disease agents in vivo. Such other materials can include without restriction diluents, excipients, carrier materials, delivery systems and/or other anti amyloid-related disease materials.

[0064] The amyloid-related disease targeted by the ONs of the present invention, formulation, pharmaceutical composition can be used to target for example Alzheimer's disease (e.g., sporadic, familial, or early AD), Down's syndrome, cerebral amyloid angiopathy, hereditary cerebral hemorrhage, mild cognitive impairment, sporadic inclusion body myositis, age-related macular degeneration (drusen associated, islet amyloid polypeptide (IAPP)-related amyloidosis), Type II diabetes related amyloidosis, primary (AL) amyloidosis , secondary (AA) amyloidosis, Beta 2-microglobulin-related (dialysis-related) amyloidosis. The amyloid-related disease targeting ONs, formulation, pharmaceutical composition or method of treatment can also target secondary amyloidosis caused, without limitation, by rheumatoid arthritis, juvenile chronic arthritis, ankylosing spondylitis, psoriasis, psoriatic arthropathy, Reiter's syndrome, Adult Still's disease, Behcet's syndrome, Crohn's disease, chronic microbial infections (such as leprosy, tuberculosis), bronchiectasis, decubitus ulcers, chronic pyelonephritis, osteomyelitis, Whipple's disease, Castleman's disease, Schnitzler's syndrome, malignant neoplasms (including Hodgkin's lymphoma, renal carcinoma, carcinomas of gut, lung and urogenital tract, basal cell carcinoma, and hairy cell leukemia) and hereditary systemic amyloidoses. Other diseases or conditions related to amyloid targeted by ON of this invention are nodular AL amyloid, cutaneous amyloid, endocrine amyloid, tumor-related amyloid, familial amyloid polyneuropathy (FAP), senile systemic amyloidosis, tenosynovium, familial amyloidosis, Ostertag-type, non-neuropathic amyloidosis, cranial neuropathy, hereditary cerebral hemorrhage, familial dementia, chronic dialysis; Gerstmann-Strussler-Scheinker syndrome, familial Mediterranean fever, Muckle-Well's syndrome, nephropathy, deafness, urticaria, limb pain, cardiomyopathy, cutaneous deposits, multiple myeloma, benign monoclonal gammopathy, maccoglobulinaemia, myeloma associated amyloidosis, medullary carcinomas of the thyroid, isolated atrial amyloid, and diabetes.

[0065] The present invention involves the discovery that ONs, e.g., oligodeoxynucleotides (ODNs), including modified oligonucleotides, can have a therapeutic application through a sequence independent mode of action. It is not necessary for the oligonucleotide to be complementary to any sequence or to have a particular distribution of nucleotides in order to have activity. Such an oligonucleotide can even be prepared as a randomer, such that there will be at most a few copies of any particular sequence in a preparation, e.g., in a 15 micromol randomer preparation of an ON having 32 or more nucleotides in length.

[0066] It is also disclosed in the present invention that different length oligonucleotides have different activity. For example, present results indicate that the length of oligonucleotide that produces maximal effect when modified with sulfur linkages is typically in the range of 30-120 nucleotides but not restricted to these length. In view of the present invention, another embodiment concerns the properties of oligonucleotides. Further, the present invention provides oligonucleotide agents that can have activity against diseases and conditions described herein. Such agents are particularly advantageous in view of the limited therapeutic options currently available.

[0067] Therefore, the ONs, e.g., ODNs, of the present invention are useful in therapy for treating or preventing diseases and conditions described herein. Such treatments are applicable to many types of patients and treatments, including, for example, the prophylaxis or treatment of diseases and conditions described herein

[0068] In one embodiment of the invention, the activity of the oligonucleotides, e.g., purified oligonucleotides, occurs principally by a sequence independent (e.g., non- sequence complementary or non-sequence dependant aptameric activity) mode of action, and formulations containing such oligonucleotides.

[0069] Oligonucleotides useful in the present invention can be of various lengths, e.g., at least 6, 10, 14, 15, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 50, 60, 70, 80, 90, 100, 110, 120, 140, 160, or more nucleotides in length. Likewise, the length of the oligonucleotide can be within a range, e.g., a range defined by taking any two of the preceding listed values as inclusive end points of the range, for example 10-20, 20-30, 20-40, 30-40, 30-50, 40-50, 40-60, 40-80, 50-60, 50-70, 60-70, 70-80, 60-120, and 80-120 nucleotides.

Oligonucleotides compositions and modifications

[0070] According to one embodiment of the present invention, the nucleotide can include various modifications, e.g., stabilizing modifications, and thus can include at least one modification in the phosphodiester linkage and/or on the sugar, and/or on the base. For example, the oligonucleotide can include one or more phosphorothioate linkages, phosphorodithioate linkages, and/or methylphosphonate linkages. Different chemically compatible modified linkages can be combined, e.g., modifications where the synthesis conditions are chemically compatible. While modified linkages are useful, the oligonucleotides can include phosphodiester linkages, e.g., include at least one phosphodiester linkage, or at least 5, 10, 20, 30% or more phosphodiester linkages. Additional useful modifications include, without restriction, modifications at the T- position of the sugar, such as 2'-O-alkyl modifications such as 2'-O-methyl modifications, 2'-amino modifications, 2'-halo modifications such as 2'-fluoro; acyclic nucleotide analogs. In another embodiment, the oligonucleotide has modified linkages throughout, e.g., phosphorothioate; has a 3'- and/or 5'-cap; includes a terminal 3 '-5' linkage; the oligonucleotide is or includes a concatemer consisting of two or more oligonucleotide sequences joined by a linker(s).

[0071] Such modified ONs include, for example, ONs containing modified backbones or non-natural internucleoside linkages. ONs having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone.

[0072] Such modified ON backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters aminoalkylphosphotri-esters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates, 5'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'- amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates, carboranyl phosphate and borano-phosphates having normal 3 '-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3' to 3', 5 ' to 5' or 2' to 2' linkage. Oligonucleotides having inverted polarity typically include a single 3' to 3' linkage at the 3 '-most internucleotide linkage i.e. a single inverted nucleoside residue which may be abasic (the nucleobase is missing or has a hydroxyl group in place thereof). Various salts, mixed salts and free acid forms are also included.

[0073] Preparation of oligonucleotides with phosphorus-containing linkages as indicated above are described, for example, in U.S. Pat Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 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; 5,194,599; 5,565,555; 5,527,899; 5,721,218; 5,672,697 and 5,625,050.

[0074] Some exemplary modified ON backbones that do not include a phosphodiester linkage have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; riboacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, 0, S and CH₂ component parts. Particularly advantageous are backbone linkages that include one or more charged moieties. Examples of U.S. patents describing the preparation of the preceding oligonucleotides include U.S. 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; 5,434,257; 5,466,677; 5,470,967; 5,489, 677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439.

[0075] Modified ONs may also contain one or more substituted sugar moieties. For example, such oligonucleotides can include one of the following 2 '-modifications: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S— or N-alkynyl; or O-alkyl-O- alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted Ci to Cio alkyl or C₂ Io Cio alkenyl and alkynyl, or 2'-O-(O-carboran-l-yl)methyl. Particular examples are O[(CH₂)_nO]_mCH₃, O(CH₂)~OCH₃, O(CH₂)_nNH₂, O(CH₂)_nCH₃, O(CH₂)_nONH₂, and O(CH₂)_nON [(CH₂)_nCH₃)]₂, where n and m are from 1 to 10. Other exemplary ONs include one of the following 2 '-modifications: Ci to Cio lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃. OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an ON, or a group for improving the pharmacodynamic properties of an ON. Examples include 2'-methoxyethoxy (2'-0 — CH₂CH₂OCH₃, also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al, HeIv. Chim. Acta (1995) 78: 486-504) i.e., an alkoxyalkoxy group; 2'-dimethy-laminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as T- DMAOE; and 2'-dimethylaminoethoxyethoxy (also known as 2'-O- dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O— CH₂- O— CH₂- N(CH₂)₂.

[0076] Other modifications include Locked Nucleic Acids (LNAs) in which the T- hydroxyl group is linked to the 3' or 4' carbon atom of the sugar ring thereby forming a bicyclic sugar moiety. The linkage can be a methelyne ( — CH₂ — )~ group bridging the T oxygen atom and the 4' carbon atom wherein n is 1 or 2. LNAs and preparation thereof are described in international patent application publication Nos WO 98/39352 and WO 99/14226.

[0077] Other modifications include sulfur-nitrogen bridge modifications, such as locked nucleic acid as described in Orum et al. ((2001) Curr. Opin. MoI. Ther. 3:239-

243).

[0078] Other modifications include 2'-methoxy (2'-0-CH₃), 2'-methoxyethyl (2'O- CH₂-CH₃ ), 2'-ethyl, 2'-ethoxy, 2'-aminopropoxy (2'-OCH₂CH₂CH₂NH₂), 2'-allyl (T- CH₂-CH=CH₂), 2'-O-allyl (2'-0-CH₂-CH=CH₂) and 2'-fluoro (2'-F).

[0079] The 2 '-modification may be in the arabino (up) position or ribo (down) position. Similar modifications may also be made at other positions on the ON, particularly the 3' position of the sugar on the 3' terminal nucleotide or in 2 '-5' linked oligonucleotides and the 5' position of the 5' terminal nucleotide. ONs may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Exemplary U.S. patents describing the preparation of such modified sugar structures include, for example, U.S. patents Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393, 878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567, 811 ; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627, 053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,792, 747; and 5,700,920.

[0080] Still other modifications include an ON concatemer consisting of multiple ON sequences joined by a linker(s). The linker may, for example, consist of modified nucleotides or non-nucleotide units. In some embodiments, the linker provides flexibility to the ON concatemer. Use of such ON concatemers can provide a facile method to synthesize a final molecule, by joining smaller ON building blocks to obtain the desired length. For example, a 12 carbon linker (C12 phosphoramidite) can be used to join two or more ON concatemers and provide length, stability, and flexibility.

[0081] As used herein, "unmodified" or "natural" bases (nucleobases) include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). ONs may also include base modifications or substitutions. Modified bases include other synthetic and naturally-occurring bases such as 5- methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2- aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2- thiocytosine, 5-halouracil and cytosine, 5-propynyl( — C≡C — CH₃) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5- uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F- adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7- deazaadenine and 3-deazaguanine and 3-deazaadenine. Additional modified bases include tricyclic pyrimidines such as phenoxazine cytidine(lH-pyrimido[5,4- b][l,4]benzoxazin-2(3H)-one), phenothiazine cytidine (lH-pyrimido[5,4- b][l,4]benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g. 9-(2-aminoethoxy)-H-pyrimido [5,4-b][l,4]benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine (H- pyrido[3',2':4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified bases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobases include those described in U.S. patent No. 3,687,808; those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons, 1990, those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613; and those disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., ed., CRC Press, 1993.

[0082] Another modification includes phosphorodithioate linkages. Knowing that phosphorodithioate ONs (PS2-ONs) and PS-ONs have a similar binding affinity to proteins (Tonkinson et al. (1994) Antisense Res. Dev. 4:269-278) (Cheng et al. (1997) J. MoI. Recogn. 10:101-107) and knowing that a possible mechanism of action of ONs is binding to protein involved in amyloid-related disease, it could be desirable to include phosphorodithioate linkages on the anti amyloid-related disease ONs described in this invention.

[0083] Another approach to modify ONs is to produce stereodefmed or stereo-enriched ONs as described in Yu at al ((2000) Bioorg. Med. Chem. 8:275-284) and in Inagawa et al. ((2002) FEBS Lett. 25:48-52). ONs prepared by conventional methods consist of a mixture of diastereomers by virtue of the asymmetry around the phosphorus atom involved in the internucleotide linkage. This may affect the stability of the binding between ONs and targets such as proteins involved in amyloid-related disease. Previous data showed that protein binding is significantly stereo-dependent (Yu et al.). Thus, using stereodefmed or stereo-enriched ONs could improve their protein binding properties and improve their anti amyloid-related disease efficacy.

[0084] The incorporation of modifications such as those described above can be utilized in many different incorporation patterns and levels. That is, a particular modification need not be included at each nucleotide or linkage in an ON, and different modifications can be utilized in combination in a single ON, or even on a single nucleotide.

[0085] As examples and in accordance with the description above, modified oligonucleotides containing phosphorothioate or dithioate linkages may also contain one or more substituted sugar moieties particularly modifications at the sugar moieties including, without restriction, 2'-ethyl, 2'-ethoxy, 2'-methoxy, 2'-aminopropoxy, 2'-allyl, 2'-fluoro, 2'-pentyl, 2'-propyl, 2'-dimethylaminooxyethoxy, and T- dimethylaminoethoxyethoxy. The 2'-modification may be in the arabino (up) position or ribo (down) position. A preferred 2'-arabino modification is 2'-fluoro. Similar modifications may also be made at other positions on the ON, particularly the 3' position of the sugar on the 3' terminal nucleotide or in 2 '-5' linked oligonucleotides and the 5' position of 5' terminal nucleotide. ONs may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Moreover ONs may have a structure of or comprise a portion consisting of glycol nucleic acid (GNA) with an acyclic propylene glycol phosphodiester backbone (Zhang L, et al. (2005) J. Am. Chem. Soc.

127(12):4174-5). Such GNA may comprise phosphorothioate linkages and may comprise only pyrimidine bases.

[0086] The incorporation of phosphorothioate linkages and ribonucleotide modifications, including 2'-O-methyl and other 2' sugar modifications, into oligonucleotides of this invention, may be useful for improving characteristics of sequence-independant anti amyloid-related disease oligonucleotides. Results demonstrate that modification at the 2'-position of each ribose reduces the general interaction of the PS-ONs with serum proteins and renders them significantly more resistant to low pH. These properties promise to increase the pharmacokinetic performance and reduce the toxic side effects normally seen with PS-ONs. For example, their pH resistance makes them more suitable for oral delivery. Also their lowered interaction with serum proteins promises to improve their pharmacokinetic behaviour without affecting their anti amyloid-related disease activity. Thus, oligonucleotides having each linkage phosphorothioated and each ribonucleotide modified at the 2'-position of the ribose, e.g., 2'-O-methyl modifications, may have anti amyloid-related disease activity but do not trigger RNase H activity , a property desirable for traditional antisense ONs but completely dispensable for the activity described in this present invention. Results also demonstrate that modifications at the 2'-position of each ribose of PS-ONs renders the ON more resistant to nucleases in comparison with a PS-ON comprising the same modifications but only at both ends (gapmer). Gapmers are preferentially used in the antisense technology. Nuclease resistance of PS-ONs including modifications at the T- position of each ribose could display beneficial properties, such as improved pharmakokinetics and/or oral availability.

[0087] In addition, while PS-ONs that include modifications at the 2'-position of each ribose show the desirable properties, PS-ONs with substantial numbers of modifications at the 2'-position of ribose could also display the desirable properties, e.g., modification at least 50 % of the riboses and more preferably 80% or even more. [0088] The present invention further provides an oligonucleotide, wherein said oligonucleotide is linked or conjugated at one or more nucleotide residues, to a molecule modifying the characteristics of the oligonucleotide to obtain one or more characteristics selected from the group consisting of higher stability, lower serum interaction, higher cellular uptake, higher protein interaction, an improved ability to be formulated for delivery, a detectable signal, higher activity, better pharmacokinetic properties, specific tissue distribution, lower toxicity.

[0089] In another embodiment, the oligonucleotide includes at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or 100% modified linkages, e.g., phosphorothioate, phosphorodithioate, and/or methylphosphonate.

[0090] In a further embodiment, at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 95%, or all of the nucleotides are modified at the 2'-position of the ribose, e.g., 2'-OMe, T- fluoro, 2'-amino.

[0091] In another embodiment, modified linkages are combined with 2 '-modifications in oligonucleotides, for example, at least 30% modified linkages and at least 30% T- modifications; or respectively at least 40% and 40%, at least 50% and 50%, at least 60% and 60%, at least 70% and 70%, at least 80% and 80%, at least 90% and 90%, or further 100% and 100%. In a further aspect of the invention, the oligonucleotide includes at least 30, 40, 50, 60, 70, 80, 90, or 100% modified linkages and at least 30, 40, 50, 60, 70, 80, 90, or 100% 2 '-modifications. In a further embodiment, it is included each combination of listed modified linkage percentage and 2 '-modification percentage (e.g., at least 50% modified linkage and at least 80% 2 '-modifications, and at least 80% modified linkages and 100% 2 '-modifications). In a particular embodiment, the modified linkages are phosphorothioate linkages; the modified linkages are phosphorodithioate linkages; the T- modifications are 2'-OMe; the 2 '-modifications are 2'-fluoro; the 2 '-modifications are a combination of 2'-OMe and 2'-fluoro; the modified linkages are phosphorothioate linkages and the 2 '-modifications are 2'-OMe; the modified linkages are phosphorothioate linkages and the 2 '-modifications are 2'-fluoro; the modified linkages are phosphorodithioate linkages and the 2 '-modifications are 2'-OMe; the modified linkages are phosphorodithioate linkages and the 2 '-modifications are 2'-fluoro; the modified linkages are phosphorodithioate linkages and the 2 '-modifications are a combination of 2'-0Me and 2'-fluoro. In a certain embodiment of the present invention, the oligonucleotides described herein combine a particular percentage of modified linkages and a particular percentage of 2'-modifications, the oligonucleotide is at least 6, 10, 14, 15, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 50, 60, 70, 80, 90, 100, 110, 120, 140, 160, or more nucleotides in length, or is in a length range defined by taking any two of the specified lengths as inclusive endpoints of the range.

[0092] In a further embodiment, all but 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the internucleotidic linkages and/or 2'-positions of the ribose moiety are modified, e.g., with linkages modified with phosphorothioate, phosphorodithioate, or methylphosphonate linkages and/or 2'-0Me, 2'-fluoro, and/or 2'-amino modifications of the ribose moiety.

[0093] In another embodiment, the oligonucleotide includes at least 1 , 2, 3, or 4 ribonucleotides, or at least 10, 20, 30, 40, 50, 60, 70, 80, 90%, or even 100% ribonucleotides.

[0094] In another embodiment, the oligonucleotide includes non-nucleotide groups in the chain (i.e., form part of the chain backbone) and/or as side chain moieties, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or even more, or up to 5, 10, 20% or more of the chain moieties and/or side chain moieties.

[0095] In a certain embodiment, the oligonucleotide is free of self-complementary sequences longer than 5, 8, 10, 15, 20, 25, 30 nucleotides; the oligonucleotide is free of catalytic activity, e.g., cleavage activity against RNA; the oligonucleotide does not induce an RNA interference mechanism.

[0096] In particular embodiments, the oligonucleotide binds protein involved in a disease or condition described in the present invention ; the sequence of the oligonucleotide (or a portion thereof, e.g., at least 20, 30, 40, 50, 60, 70% or more) is derived from a genome; the activity of an oligonucleotide with a sequence derived from a genome is not superior to a randomer oligonucleotide or a random oligonucleotide of the same length; the oligonucleotide includes a portion complementary to a genome sequence and a portion not complementary to a genome sequence; unless otherwise indicated, the sequence of the oligonucleotide includes A(x), C(x), G(x), T(x), U(x), I(x), AC(x), AG(x), AT(x), AU(x), CG(x), CT(x), CU(x), GT(x), GU(x), TU(x), AI(x), IC(x), IG(x), IT(x) IU (x) where x is 2, 3, 4, 5, 6, ... 60, ... 120 (in a particular embodiment the oligonucleotide is at least 6, 10, 14, 15, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 50, 60, 70, 80, 90, 100, 110, 120, 140, 160, or more nucleotides in length or is in a range defined by taking any two of the listed values as inclusive endpoints, or the length of the specified repeat sequence is at least a length or in a length range just specified); the oligonucleotide includes a combination of repeat sequences (e.g., repeat sequences as specified above), including, for example, each combination of the above monomer and/or dimer repeats taken 2, 3, or 4 at a time; the oligonucleotide is single stranded (RNA or DNA); the oligonucleotide is double stranded (RNA or DNA); the oligonucleotide includes at least one Gquartet or CpG portion; the oligonucleotide includes a portion complementary to a mRNA and is at least 29, 37, or 38 nucleotides in length (or other length as specified above); the oligonucleotide includes at least one non- Watson-Crick oligonucleotide and/or at least one nucleotide that participates in non- Watson-Crick binding with another nucleotide and/or at least one nucleotide that cannot form base pairs with other nucleotides; the oligonucleotide is a random oligonucleotide, the oligonucleotide is a randomer or includes a randomer portion, e.g., a randomer portion that has a length of at least 5, 10, 15, 20, 25, 30, 35, 40 or more contiguous oligonucleotides or a length as specified above for oligonucleotide length or at least 10, 20, 30, 40, 50, 60, 70, 80, 90% or all the nucleotides are randomer; the oligonucleotide is linked or conjugated at one or more nucleotide residues to a molecule that modifies the characteristics of the oligonucleotide, e.g. to provide higher stability (such as stability in serum or stability in a particular solution), lower serum interaction, higher cellular uptake, higher protein interaction, improved ability to be formulated for delivery, a detectable signal, improved pharmacokinetic properties, specific tissue distribution, and/or lower toxicity. [0097] It was also discovered that phosphorothioated ONs containing only (or at least primarily) pyrimidine nucleotides, including cytosine and/or thymidine and/or other pyrimidines are resistant to low pH and polycytosine oligonucleotides showed increased resistance to a number of nucleases, thereby providing two important characteristics for oral administration of an ON. Thus, in a certain embodiment, the oligonucleotide has at least 80, 90, or 95, or 100% modified internucleotidic linkages (e.g., phosphorothioate or phosphorodithoiate) and the pyrimidine content is more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, or 100%, i.e.; is a pyrimidine oligonucleotide or the cytosine content is more than 50%, more than 60%, more than 70%, more than 80%, more than 90% or 100% i.e. is a polycytosine oligonucleotide. In certain embodiments, the length is at least 29, 30, 32, 34, 36, 38, 40, 45, 50, 60, 70, or 80 nucleotides, or is in a range of 20-28, 25-35, 29-40, 30-40, 35-45, 40-50, 45-55, 50-60, 55-65, 60-70, 65-75, or 70-80, or is in a range defined by taking any two of the listed values as inclusive endpoints of the range. In particular embodiment, the oligonucleotide is at least 50, 60, 70, 80, or 90% cytosine; at least 50, 60, 70, 80, or 90% thymidine (and may have a total pyrimidine content as listed above). In particular embodiments, the oligonucleotide contains a listed percentage of either cytosine (C) or thymidine (T). Also in certain embodiments, the oligonucleotide includes at least 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, or more contiguous pyrimidine nucleotides, e.g., as C nucleotides, T nucleotides, or CT dinucleotide pairs. In a certain embodiment, the pyrimidine oligonucleotide consists only of pyrimidine nucleotides; includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 non-pyrimidine moieities; includes 1-5, 6-10, 11-15, or at least 16 non- pyrimidine backbone moieties; includes further at least one, 1-20, 1-5, 6-10, 11-15, or 16- 20 non-nucleotide moieties; includes at least one, 1-20, 1-5, 6-10, 11-15, or 16-20 purine nucleotides. In one embodiment in which non-nucleotide moieities are present, the linkages between such moieties or between such moieties and nucleotides are at least 25, 35, 50, 70, 90, or 100 % as resistant to acidic conditions as PS linkages in a 40-mer polyC oligonucleotide as evaluated by gel electrophoresis under conditions appropriate for the size and chemistry of the oligonucleotide.

[0098] In another embodiment of the invention, oligonucleotides can also be used in combinations, e.g., as a mixture. Such combinations or mixtures can include, for example, at least 2, 3, 4, 5, 10, 20, 50, 100, 1000, 10000, 100,000, 1,000,000, or more different oligonucleotides. Such combinations or mixtures can, for example, be different sequences and/or different lengths and/or different modifications and/or different linked or conjugated molecules. In another embodiment of such combinations or mixtures of the invention, pluralities of oligonucleotides have a minimum length or are in a length range as specified above for oligonucleotides. In a particular embodiment of such combinations or mixtures, at least one, or a plurality, or each of the oligonucleotides can have any of the other properties specified herein for individual oligonucleoties (which can also be in any consistent combination).

[0099] In a further embodiment, the sequence of the oligonucleotide is not perfectly complementary to any equal length portion of the a genome sequence, or has less than 95, 90, 80, 70, 60, or 50% complementarity to any equal length portion of the genomic sequence, the oligonucleotide sequence does not consist essentially of polyA, polyC, polyG, polyT, Gquartet, or a TG-rich sequence.

[0100] As used in connection with the present oligos, the term "TG-rich" indicates that the sequence of the oligonucleotide consists of at least 50 percent T and G nucleotides, or if so specified, at least 60, 70, 80, 90, or 95% T and G, or even 100%.

[0101] In a related aspect, the invention provides a mixture of oligonucleotides that includes at least two different oligonucleotides as described herein, e.g., at least 2, 3, 4, 5, 7, 10, 50, 100, 1000, 10,000, 100,000, 1,000,000, or even more.

[0102] Specification of particular lengths for oligonucleotides, e.g., at least 20 nucleotides in length, means that the oligonucleotide includes at least 20 linked nucleotides. Unless clearly indicated to the contrary, the oligonucleotide may also include additional, non-nucleotide moieties, which may form part of the backbone of the oligonucleotide chain. Unless otherwise indicated, when non-nucleotide moeities are present in the backbone, at least 10 of the linked nucleotides are contiguous.

[0103] As used herein in connection with the action of an oligonucleotide, "sequence independent mode of action" indicates that the particular biological activity is not dependent on a particular oligonucleotide sequence in the oligonucleotide. For example, the activity does not depend on sequence dependent hybridization such as with antisense activity, or a particular sequence resulting in a sequence dependent aptameric interaction. Similarly, the phrase "non-sequence complementary mode of action" indicates that the mechanism by which the material exhibits an effect is not due to hybridization of complementary nucleic acid sequences, e.g., an antisense effect. Conversely, a "sequence complementary mode of action" means that the effect of a material involves hybridization of complementary nucleic acid sequences or sequence specific aptameric interaction. Thus, indicating that the activity of a material is due to a sequence independent mode of action" or that the activity is "not primarily due to a sequence complementary mode of action" means that the activity of the oligonucleotide satisfies at least one of the 4 tests provided herein.

[0104] In another embodiment of the present invention, the oligonucleotide satisfies test 1 or test 2, defined hereinafter; the oligonucleotide satisfies a combination two of the tests, i.e., tests 1 & 2.

[0105] A related aspect concerns an oligonucleotide randomer or randomer formulation that contains at least one randomer, where the activity of the randomer occurs principally by a sequence independent, e.g., non-sequence complementary mode of action. Such a randomer formulation can, for example, include a mixture of randomers of different lengths, e.g., at least 2, 3, 5, 10, or more different lengths, or other mixtures as described herein.

[0106] The phrase "derived from a genome" indicates that a particular sequence has a nucleotide base sequence that has at least 70% identity to a genomic nucleotide sequence or its complement (e.g., is the same as or complementary to a genomic sequence), or is a corresponding RNA sequence. In another embodiment of the present invention, the expression "derived from a genome" indicates that the sequence is at least 70% identical to a genomic sequence of a particular gene involved in the disease or condition against which the oligonucleotide is directed, or to its complementary sequence. In particular embodiments, the identity is at least 80, 90, 95, 98, 99, or 100%. Genome can be from an animal, e.g. a human, from a microorganism, e.g. a virus, a bacteria, a parasite, or from plant.

[0107] In yet another aspect, the invention provides a use for the prophylaxis or treatment in an acidic environment in a subject, comprising administering to a subject in need of such a treatment a therapeutically effective amount of at least one pharmacologically acceptable pharmaceutical composition of the invention, said composition being adapted for administration to an acidic in vivo site.

[0108] In a further embodiment of the present invention, the oligonucleotide is a polyp yrimidine oligonucleotide (or a formulation or pharmaceutical composition containing such polypyrimidine oligonucleotide), which may be adapted for oral or vaginal administration, e.g., as described herein.

[0109] In a further embodiment involving oligonucleotide formulations, pharmaceutical compositions, treatment and prophylactic methods and/or treatment and prophylactic uses described herein, the oligonucleotide(s) having a sequence independent mode of action is not associated with a transfection agent; the oligonucleotide(s) having a sequence independent mode of action does not need to be, and thus is not encapsulated in liposomes and/or non-liposomal lipid particles. In addition, the oligonucleotide(s) having a sequence independent mode of action is in a pharmaceutical composition or is administered in conjunction with (concurrently or sequentially) an oligonucleotide that acts principally by a sequence dependent mode of action, e.g., antisense oligonucleotide or siRNA, where the oligonucleotide(s) having a sequence dependent mode of action can be associated with a transfection agent and/or encapsulated in liposomes and/or non- liposomal lipid particles.

[0110] In yet another aspect, the invention provides a polymer mix that includes at least one oligonucleotide and at least one non-nucleotide polymer.

[0111] According to one embodiment of the present invention, the invention provides an oligonucleotide randomer, where the randomer is at least 6 nucleotides in length. In a further embodiment, the randomer has a length as specified above for oligonucleotides; the randomer includes at least one phosphorothioate linkage, the randomer includes at least one phosphorodithioate linkage or other modification as listed herein; the randomer oligonucleotides include at least one non-randomer segment (such as a segment complementary to a selected nucleic acid sequence), which can have a length as specified above for oligonucleotides; the randomer is in a preparation or pool of preparations containing at least 5, 10, 15, 20, 50, 100, 200, 500, or 700 micromol, 1, 5, 7, 10, 20, 50, 100, 200, 500, or 700 mmol, or 1 mole of randomer, or a range defined by taking any two different values from the preceding as inclusive end points, or is synthesized at one of the listed scales or scale ranges.

[0112] In connection with modifying characteristics of an oligonucleotide by linking or conjugating with another molecule or moiety, the modifications in the characteristics are evaluated relative to the same oligonucleotide without the linked or conjugated molecule or moiety.

[0113] In another embodiment of the present invention, considering the volumes and concentrations of ONs used in tests disclosed in the present invention, it is theoretically impossible that a particular sequence is present at more than 1 copy in the mixture. This means than there can be no antisense or sequence-specific aptameric effect in these ONs randomers. In all examples disclosed in the present invention, should the amyloid-related disease inhibition effect be caused by the sequence-specificity of the ONs, such effect would thus have to be caused by only one molecule, a result that does not appear possible. For example, for an ON randomer 40 bases in length, any particular sequence in the population would theoretically represent only 1/4⁴⁰ or 1/8.27X10^"25 of the total fraction. Given that 1 mole = 6.022X10²³ molecules, and the fact that our largest synthesis is currently done at the 15 micromole scale, all possible sequences will not be present and also, each sequence is present most probably as only one copy.

[0114] Therefore, ONs have been tested for potential broad-spectrum activity against many types of diseases involving amyloidosis. .Several ON randomers of different sizes and chemical type were selected to be tested in vitro for binding to the amyloid proteins, amyloid-beta and amylin. It is disclosed in the present invention that ONs interact with beta-amyloid and amylin and that this interaction is sequence independent, dependent on size, and chemical (i.e. sulfur) modification. The results suggest that ONs have an anti amyloid-related disease activity by binding to the amyloid and amylin and therefore preventing, inhibiting or reversing amyloid plaque formation, deposition, polymerization or aggregation.

[0115] The data reported herein demonstrates that ONs have a therapeutic activity in slowing the progression of plaque formation in a mouse model of Alzheimer's disease. This data shows that ONs of this invention can be used in a treatment for an amyloidal- related disease such as, but without restriction, Alzheimer's disease.

[0116] One skilled in the art applying the teaching of the present invention could also use ONs with different chemical modifications. A modification of the ON, such as, but not limited to, a phosphorothioate modification or other sulfur modifications, appears to be beneficial for anti amyloid-related disease activity. Such sulfur modifications may include without restriction mono and diphosphorothioation of the phosphodiester linkage, 4' or 5' thiolation of the uracil moiety, 5' thiolation of the cytidine moiety, 2' or 4' thiolation of the thymine moiety, 6' thiolation of the guanine moiety, sulfur modifications to any other nucleobase moiety and sulfur modifications to the ribose moiety of any nucleotide or combinations of any of the above mentioned modifications. Moreover, ONs may have more than one sulfur substitution on each nucleotide, which can potentially increase the activity. Finally, any single or multiple sulfur substitution may be combined with other modifications known to improve properties of ONs. ONs of this invention may also have chemical modifications including without restriction: any 2' ribose modification including 2'-0 methyl, 2'-fluorine, 2'-FANA, 2'-methoxyethyl, locked nucleic acids, methylphosphonates, boraophosphates and phosphorodiamidate morpholino oligomers. Moreover ONs may have a structure of or comprise a portion consisting of glycol nucleic acid (GNA) with an acyclic propylene glycol phosphodiester backbone capable of forming stable antiparallel duplexes following the Watson-Crick base pairing rules (Zhang L, et al (2005) J. Am. Chem. Soc. 127(12):4174-5). Such GNA may comprise phosphorothioate linkages or other appropriate modifications as described above. Oligonucleotides formulations and pharmaceutical compositions

[0117] The invention also provides an pharmaceutical composition that includes a therapeutically effective amount of a pharmacologically acceptable, oligonucleotide or mixture of oligonucleotides as described herein, e.g., at least 15 nucleotides in length or other length as listed herein, where the activity of the oligonucleotide occurs principally by a sequence independent, e.g., non-sequence complementary or non-sequence dependent aptamer, mode of action, and a pharmaceutically acceptable carrier. In another embodiment, the oligonucleotide or a combination or mixture of oligonucleotides is as specified above for individual oligonucleotides or combinations or mixtures of oligonucleotides. In a further embodiment, the pharmaceutical compositions are approved for administration to a human, or a non-human animal such as a non-human primate.

[0118] As used herein, the term "pharmaceutical composition" refers to an anti amyloid-related disease ON formulation that includes a physiologically or pharmaceutically acceptable carrier or excipient. Such compositions can also include other components that do not make the composition unsuitable for administration to a desired subject, e.g., a human.

[0119] As used in connection with an anti amyloid-related disease formulation, pharmaceutical composition, or other material, the phrase "adapted for use as an anti amyloid-related disease agent" indicates that the material exhibits an anti amyloid-related disease effect and does not include any component or material that makes it unsuitable for use in inhibiting such disease in an in vivo system, e.g., for administering to a subject such as a human subject.

[0120] As used herein in connection with administration of an anti amyloid-related disease material, the term "subject" refers to a living higher organism, including, for example, animals such as mammals, e.g., humans, non-human primates, non-human animals and plants, e.g., fruit trees.

[0121] According to the invention, the pharmaceutical composition can be formulated for delivery by a mode selected from the group consisting of but not restricted to oral ingestion, oral mucosal delivery, intranasal drops or spray, intraocular injection, subconjunctival injection, eye drops, ear drops, by inhalation, intratracheal injection or spray, intrabronchial injection or spray, intrapleural injection, intraperitoneal injection perfusion or irrigation, intrathecal injection or perfusion, intracranial injection or perfusion, intramuscular injection, intravenous injection or perfusion, intraarterial injection or perfusion, intralymphatic injection or perfusion, subcutaneous injection or perfusion, intradermal injection, topical skin application, by organ perfusion, by topical application during surgery, intratumoral injection, topical application, gastric injection perfusion or irrigation, enteral injection or perfusion, colonic injection perfusion or irrigation, rectal injection perfusion or irrigation, by rectal suppository or enema, by urethral suppository or injection, intravesical injection perfusion or irrigation, or intraarticular injection.

[0122] Pharmaceutical compositions and formulations for administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Other formulations include those in which the ONs of the invention are in mixed with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants. Preferred lipids and liposomes include neutral (e.g. dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline) negative (e.g. dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g. dioleoyltetramethylaminopropyl DOTAP, dioleoylphosphatidyl ethanolamine DOTMA) and other delivering agents or molecules. ONs may be encapsulated within liposomes or may form complexes thereto, in particular to cationic liposomes. Alternatively, ONs may be complexed to lipids, in particular to cationic lipids. Preferred fatty acids and esters include but are not limited to arachidonic acid, oleic acid, eicosanoic acid, laurie acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1 -monocaprate, 1- dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a Ci.io alkyl ester (e.g. isopropylmyristate IPM), monoglyceride, diglyceride or pharmaceutically acceptable salt thereof. [0123] Compositions and formulations for oral administration include powders or granules, microparticulates, nanoparticulates, suspensions or solutions in water or nonaqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavoring agents, diluents, emulsifϊers, dispersing aids or binders may be desirable. Preferred oral formulations are those in which oligonucleotides of the invention are administered in conjunction with one or more penetration enhancers surfactants and chelators. Exemplary surfactants include fatty acids and/or esters or salts thereof, bile acids and/or salts thereof. Exemplary bile acids/salts include chenodeoxycholic acid (CDCA) and ursodeoxychenedeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate, sodium glycodihydrofusidate. Exemplary fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1 - monocaprate, 1 -dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a monoglyceride, a diglyceride or a pharmaceutically acceptable salt thereof (e.g. sodium). Also preferred are combinations of penetration enhancers, for example, fatty acids/salts in combination with bile acids/salts. A particularly preferred combination is the sodium salt of lauric acid, capric acid and UDCA. Further exemplary penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. Oligonucleotides of the invention may be delivered orally in granular form including sprayed dried particles, or complexed to form micro or nanoparticles. ON complexing agents include poly-amino acids; polyimines; polyacrylates; polyalkylacrylates, polyoxethanes, polyalkylcyanoacrylates; cationized gelatins, albumins, starches, acrylates, polyethyleneglycols (PEG) and starches; polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans, celluloses, and starches. Particularly advantageous complexing agents include chitosan, N-trimethytchitosan, poly-L-lysine, polyhistidine, polyorithine, polyspermines, protamine, polyvinylpyridine, polythiodiethylamino-methylethylene P(TDAE), polyaminostyrene (e.g. p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate), poly(butylcyanoacrylatc), poly(isobutylcyanoacrylate), poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate, DEAE- acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate, polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolic acid (PLGA), alginate, and polyethylene glycol (PEG).

[0124] Compositions and formulations for parenteral administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.

[0125] In particular embodiments, the composition includes a delivery system, e.g., targeted to specific cells or tissues; a liposomal formulation, another drug, e.g., a non- nucleotide polymer, an antisense molecule, a siRNA, or a small molecule drug.

[0126] As used herein, the term "delivery system" refers to a component or components that, when combined with an ON as described herein, facilitates the transfer of ONs inside cells, increases the amount of ONs that contact the intended location in vivo, and/or extends the duration of its presence at the target or increases its circulating lifetime in vivo, e.g., by at least 10, 20, 50, or 100%, or even more as compared to the amount and/or duration in the absence of the delivery system. The term delivery system also means encapsulation system or encapsulation reagent. To encapsulate ONs means to put in contact an ON with a delivery system or an encapsulation reagent. An ON in contact with a delivery system can be referred to as an "encapsulated ON".

[0127] In particular embodiments, the oligonucleotide, oligonucleotide preparation, oligonucleotide formulation, or pharmaceutical composition has an in vitro IC₅₀ or EC₅₀ of 10, 5, 2, 1, 0.50, 0.20, 0.10, 0.09. 0.08, 0.07, 0.75, 0.06, 0.05, 0.045, 0.04, 0.035, 0.03, 0.025, 0.02, 0.015, or 0.01 μM or less.

[0128] In a further embodiment, the pharmaceutical composition contains at least one polypyrimidine oligonucleotide as described herein. In view of the resistance to low pH discovered for polypyrimidine oligonucleotides; in a further embodiment, such a composition is adapted for delivery to an acidic in vivo site, e.g., oral delivery or vaginal delivery. [0129] As used in relation to in vivo administration of the present oligonucleotides and compositions, the term "acidic site" means a site that has a pH of less than 7. Examples include the stomach (pH generally 1-2), the vagina (pH generally 4-5 but may be lower), and to a lesser degree, the skin (pH generally 4-6).

[0130] As used herein, the phrase "adapted for oral delivery" and like terms indicate that the composition is sufficiently resistant to acidic pH to allow oral administration without a clinically excessive loss of activity, e.g., an excessive first pass loss due to stomach acidity of less than 50% (or is indicated, less than 40%, 30%, 20%, 10%, or 5%).

[0131] As used herein in connection with agents and drugs or test compounds, the term "small molecule" means that the molecular weight of the molecule is 1500 daltons or less. In some cases, the molecular weight is 1000, 800, 600, 500, or 400 daltons or less.

[0132] In another aspect, the invention provides a kit that includes at least one oligonucleotide, oligonucleotide mixture, oligonucleotide formulation, or pharmaceutical composition that includes such oligonucleotide, oligonucleotide mixture, or oligonucleotide formulation in a labeled package, where the activity of the oligonucleotide occurs principally by a sequence independent e.g., non-sequence complementary or non-sequence dependent aptameric, mode of action and the label on the package indicates that the oligonucleotide can be used against at least one amyloid- related disease or condition.

[0133] In a particular embodiment, the kit includes a pharmaceutical composition that includes at least one oligonucleotide as described herein. In one embodiment, the kit contains a mixture of at least two different oligonucleotides. In another embodiment, the oligonucleotide is adapted for in vivo use in an animal and/or the label indicates that the oligonucleotide or composition is acceptable and/or approved for use in an animal; wherein the animal is a mammal, such as human, or a non-human mammal such as bovine, porcine, a ruminant, ovine, or equine; the animal is a non-human animal; wherein the animal is a bird; the kit is approved by a regulatory agency such as the U.S. Food and Drug Administration or equivalent agency for use in an animal, e.g., a human. [0134] In a particular embodiment, the different random oligonucleotides comprises randomers of different lengths; the random oligonucleotides can have different sequences or can have sequence in common, such as the sequence of the shortest oligos of the plurality; and/or the different random oligonucleotides comprise a plurality of oligonucleotides comprising a randomer segment at least 5 nucleotides in length or the different random oligonucleotides include a plurality of randomers of different lengths. Other oligonucleotides, e.g., as described herein oligonucleotides, can be tested in a particular system.

[0135] In the present invention, the term "randomer" is intended to mean a single stranded nucleic acid polymer, modified or not, having degenerate sequences at every position, such as NNNNNNNNNN. Each degenerate nucleotide position actually exists as a random population of the five naturally occurring bases on the nucleotide (adenine, guanine, cytosine, thymine, and uracil) at this particular position, resulting in a completely degenerate pool of ONs of the same size but having no sequence identity as a population. Randomers can also include nucleobases which do not occur naturally including without restriction hypoxanthine, xanthosine, imidazole, 2-aminopurines or 5- nitroindole. The term randomer can apply to a sequence or a portion of a sequence.

[0136] In the present invention, the term degenerate means that a sequence is made of a mix of nucleotides. A completely degenerate sequence means that A, C, G, and T (or other nucleobases) are randomly used at each position of the sequence and nucleotide position are identified by N. A degenerate sequence means also that at least two nucleobases are randomly used at each position of the sequence. Degenerate can apply to a sequence, a portion of a sequence or one nucleotide position in a sequence.

[0137] In yet another aspect, the invention provides a method for the prophylaxis or treatment in a subject by administering to a subject in need of such treatment a therapeutically effective amount of at least one pharmacologically acceptable oligonucleotide as described herein, e.g., a sequence independent oligonucleotide of at least 15 nucleotides in length, or an pharmaceutical composition or formulation or mixture containing such oligonucleotide(s). In a further embodiment, the invention provides a use for the prophylaxis or treatment in a subject by administering to a subject in need of such treatment a therapeutically effective amount of at least one pharmacologically acceptable oligonucleotide as described herein, e.g., a sequence independent oligonucleotide of at least 15 nucleotides in length, or an pharmaceutical composition or formulation or mixture containing such oligonucleotide(s). In a further embodiment, the disease or condition targeted be any of those listed herein as suitable for inhibition using the present invention; the subject is a type of subject as indicated herein, e.g., human, non-human animal, non-human mammal, bird, plant, and the like; the treatment is for a condition or a disease as intended in the present invention.

[0138] The term "therapeutically effective amount" refers to an amount that is sufficient to affect a therapeutically or prophylactically significant reduction amyloid- related disease when administered to a typical subject of the intended type. In aspects involving administration of an anti amyloid-related disease ON to a subject, typically the ON, formulation, or composition should be administered in a therapeutically effective amount.

[0139] According to the present invention, the anti-amyloid-related disease ON, ON formulation, ON pharmaceutical composition or use of ON for treatment described herein prevent, reverse or inhibit amyloid protein assembly into insoluble fibrils which, in vivo, are deposited in various organs, or it favors clearance of pre-formed deposits or slows deposition in patients already having deposits. In another embodiment, the ON may also prevent the amyloid protein, in its soluble, oligomeric form or in its fibrillar form, from binding or adhering to a cell surface and causing cell damage or toxicity. In yet another embodiment, the ON may block amyloid-induced cellular toxicity or macrophage activation. In another embodiment, the ON may block amyloid-induced neurotoxicity or microglial activation. In another embodiment, the ON protects cells from amyloid induced cytotoxicity of B-islet cells to protect against amyloidal induced renal diseases. In another embodiment, the ON may enhance clearance from a specific organ, e.g., the brain or it decreases concentration of the amyloid protein in such a way that amyloid fibril formation is prevented in the targeted organ. [0140] In a yet another embodiment, the anti-amyloid-related disease ON, ON formulation, ON pharmaceutical composition or use of the ON for treatment described herein may be administered therapeutically or prophylactically to treat diseases associated with amyloid fibril formation, aggregation or deposition. The ONs of the invention may act to ameliorate the course of an amyloid related disease using, without limitation, any of the following mechanisms: slowing the rate of amyloid fibril formation or deposition; lessening the degree of amyloid deposition; inhibiting, reducing, or preventing amyloid fibril formation; inhibiting neurodegeneration or cellular toxicity induced by amyloid; inhibiting amyloid induced inflammation; enhancing the clearance of amyloid; or favoring the degradation of amyloid protein prior to its organization in fibrils, and preventing the association of amyloid proteins and or fibrils with cells.

[0141] In a yet another embodiment, the anti-amyloid-related disease ON, ON formulation, ON pharmaceutical composition or use of the ON for treatment described herein may be administered therapeutically or prophylactically to treat diseases associated with amyloid-beta fibril formation, aggregation or deposition. The ONs of the invention may act to ameliorate the course of an amyloid-beta related disease using, without limitation, any of the following mechanisms: slowing the rate of amyloid-beta fibril formation or deposition; lessening the degree of amyloid-beta deposition; inhibiting, reducing, or preventing amyloid-beta fibril formation; inhibiting neurodegeneration or cellular toxicity induced by amyloid-beta; inhibiting amyloid-beta induced inflammation; enhancing the clearance of amyloid-beta from the brain; or favoring the degradation of amyloid-beta protein prior to its organization in fibrils.

[0142] The present oligonucleotides can be prepared in an ON formulation or pharmaceutical composition. Thus, the present ONs may also be mixed, encapsulated, conjugated or otherwise associated with other molecules, molecule structures or mixtures of compounds, as for example, liposomes, receptor targeted molecules, oral, rectal, topical or other formulations, for assisting in uptake, distribution and/or absorption. Exemplary United States patents that describe the preparation of such uptake, distribution and/or absorption assisting formulations include, for example, U.S. patents Nos. 5,108,921; 5,354,844; 5,416,016; 5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721; 4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170; 5,264,221 ; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854; 5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948; 5,580,575; and 5,595,756.

[0143] The ONs, formulations, and compositions of the invention include any pharmaceutically acceptable salts, esters, or salts of such esters, or any other compound which, upon administration to an animal including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to prodrugs and pharmaceutically acceptable salts of the compounds of the invention, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents

[0144] The term "prodrug" indicates a therapeutic agent that is prepared in an inactive form that is converted to an active form (i.e., drug) within the body or cells thereof by the action of endogenous enzymes or other chemicals and/or conditions. In particular embodiments, prodrug versions of the present oligonucleotides are prepared as SATE [(S-acetyl-2-thioethyl) phosphate] derivatives according to the methods disclosed in Gosselin et al. (international patent application publication No WO 93/24510) and in Imbach et al. (international patent application publication No WO 94/26764) and U.S. patent No 5,770,713.

[0145] The term "pharmaceutically acceptable salts" refers to physiologically and pharmaceutically acceptable salts of the present compounds: i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto. Many such pharmaceutically acceptable salts are known and can be used in the present invention.

[0146] For ONs, useful examples of pharmaceutically acceptable salts include but are not limited to salts formed with cations such as sodium, potassium, ammonium, magnesium, calcium, polyamines such as spermine and spermidine, etc.; acid addition salts formed with inorganic acids, for example hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like; salts formed with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid, and the like; and salts formed from elemental anions such as chlorine, bromine, and iodine.

[0147] Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids.

[0148] The pharmaceutical formulations of the present invention, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaking the product.

[0149] The compositions of the present invention may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.

[0150] In one embodiment of the present invention, the pharmaceutical compositions may be formulated and used as foams. Pharmaceutical foams include formulations such as, but not limited to, emulsions, microemulsions, creams, jellies and liposomes. While basically similar in nature these formulations vary in the components and the consistency of the final product. The preparation of such compositions and formulations is generally known to those skilled in the pharmaceutical and formulation arts and may be applied to the formulation of the compositions of the present invention.

Emulsions

[0151] The formulations and compositions of the present invention may be prepared and formulated as emulsions. Emulsions are typically heterogenous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 μm in diameter. (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (lids.), 1988, Marcel Dekker, Inc., New York, N. Y., volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N. Y., Volume 1, p. 245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N. Y., volume 2, p. 335; Higuchi et at., in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 301). Emulsions are often biphasic systems comprising of two immiscible liquid phases intimately mixed and dispersed with each other. In general, emulsions may be either water-in-oil (w/o) or of the oil-in- water (o/w) variety. When an aqueous phase is finely divided into and dispersed as minute droplets into a bulk oily phase the resulting composition is called a water-in-oil (w/o) emulsion. Alternatively, when an oily phase is finely divided into and dispersed as minute droplets into a bulk aqueous phase the resulting composition is called an oil-in-water (o/w) emulsion. Emulsions may contain additional components in addition to the dispersed phases and the active drug which may be present as a solution in either the aqueous phase, oily phase or itself as a separate phase. Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, and anti-oxidants may also be present in emulsions as needed. Pharmaceutical emulsions may also be multiple emulsions that are comprised of more than two phases such as, for example, in the case of oil-in- water-in-oil (o/w/o) and water-in-oil-in- water (w/o/w) emulsions. Such complex formulations often provide certain advantages that simple binary emulsions do not. Multiple emulsions in which individual oil droplets of an o/w emulsion enclose small water droplets constitute a w/o/w emulsion. Likewise a system of oil droplets enclosed in globules of water stabilized in an oily continuous provides an o/w/o emulsion. [0152] Emulsions are characterized by little or no thermodynamic stability. Often, the dispersed or discontinuous phase of the emulsion is well dispersed into the external or continuous phase and maintained in this form through the means of emulsifiers or the viscosity of the formulation. Either of the phases of the emulsion may be a semisolid or a solid, as is the case of emulsion-style ointment bases and creams. Other means of stabilizing emulsions entail the use of emulsifiers that may be incorporated into either phase of the emulsion. Emulsifiers may broadly be classified into four categories: synthetic surfactants, naturally occurring emulsifiers, absorption bases, and finely dispersed solids (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

[0153] Synthetic surfactants, also known as surface active agents, have found wide applicability in the formulation of emulsions and have been reviewed in the literature (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N. Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York, N. Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic and comprise a hydrophilic and a hydrophobic portion. The ratio of the hydrophilic to the hydrophobic nature of the surfactant has been termed the hydrophile/lipophile balance (HLB) and is a valuable tool in categorizing and selecting surfactants in the preparation of formulations. Surfactants may be classified into different classes based on the nature of the hydrophilic group: non-ionic, anionic, cationic and amphoteric (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285).

[0154] Naturally occurring emulsifiers used in emulsion formulations include lanolin, beeswax, phosphatides, lecithin and acacia. Absorption bases possess hydrophilic properties such that they can soak up water to form w/o emulsions yet retain their semisolid consistencies, such as anhydrous lanolin and hydrophilic petrolatum. Finely divided solids have also been used as good emulsifiers especially in combination with surfactants and in viscous preparations. These include polar inorganic solids, such as heavy metal hydroxides, nonswelling clays such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate and colloidal magnesium aluminum silicate, pigments and nonpolar solids such as carbon or glyceryl tristearate.

[0155] A large variety of non-emulsifying materials is also included in emulsion formulations and contributes to the properties of emulsions. These include fats, oils, waxes, fatty acids, fatty alcohols, fatty esters, humectants, hydrophilic colloids, preservatives and antioxidants (Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N. Y., volume 1, p. 335; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N. Y., volume 1, p. 199).

[0156] Hydrophilic colloids or hydrocolloids include naturally occurring gums and synthetic polymers such as polysaccharides (for example, acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (for example, carboxymethylcellulose and carboxypropylcellulose), and synthetic polymers (for example, carbomers, cellulose ethers, and carboxyvinyl polymers). These disperse or swell in water to form colloidal solutions that stabilize emulsions by forming strong inter- facial films around the dispersed-phase droplets and by increasing the viscosity of the external phase.

[0157] Since emulsions often contain a number of ingredients such as carbohydrates, proteins, sterols and phosphatides that may readily support the growth of microbes, these formulations often incorporate preservatives. Commonly used preservatives included in emulsion formulations include methyl paraben, propyl paraben, quaternary ammonium salts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boric acid, Antioxidants are also commonly added to emulsion formulations to prevent deterioration of the formulation. Antioxidants used may be free radical scavengers such as tocopherols, alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulfϊte, and antioxidant synergists such as citric acid, tartaric acid, and lecithin.

[0158] The application of emulsion formulations via dermatological, oral and parenteral routes and methods for their manufacture has been reviewed in the literature (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N. Y., volume 1, p. 199). Emulsion formulations for oral delivery have been very widely used because of reasons of ease of formulation, efficacy from an absorption and bioavailability standpoint. (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N. Y., volume 1, p. 245; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N. Y., volume 1, p. 199). Mineral- oil base laxatives, oil-soluble vitamins and high fat nutritive preparations are among the materials that have commonly been administered orally as o/w emulsions.

[0159] In one embodiment of the present invention, the compositions of ONs are formulated as microemulsions. A microemulsion may be defined as a system of water, oil and amphiphile which is a single optically isotropic and thermodynamically stable liquid solution (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N. Y., volume 1, p. 245). Typically micro- emulsions are systems that are prepared by first dispersing oil in an aqueous surfactant solution and then adding a sufficient amount of a fourth component, generally an intermediate chain-length alcohol to form a transparent system. Therefore, microemulsions have also been described as thermodynamically stable, isotropically clear dispersions of two immiscible liquids that are stabilized by interfacial films of surface- active molecules (Leung and Shah, in: Controlled Release of Drugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185-215). Microemulsions commonly are prepared via a combination of three to five components that include oil, water, surfactant, cosurfactant and electrolyte. Whether the microemulsion is of the water-in-oil (w/o) or an oil-in- water (o/w) type is dependent on the properties of the oil and surfactant used and on the structure and geometric packing of the polar heads and hydrocarbon tails of the surfactant molecules (Schott, in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).

[0160] The phenomenological approach utilizing phase diagrams has been extensively studied and has yielded a comprehensive knowledge, to one skilled in the art, of how to formulate microemulsions (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N. Y., volume 1, p. 245; Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N. Y., volume 1, p. 335). Compared to conventional emulsions, microemulsions offer the advantage of solubilizing water-insoluble drugs in a formulation of thermodynamically stable droplets that are formed spontaneously.

[0161] Surfactants used in the preparation of microemulsions include, but are not limited to, ionic surfactants, non-ionic surfactants, Brij 96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sequioleate (SO750), decaglycerol decaoleate (DA0750), alone or in combination with cosurfactants. The cosurfactant, usually a short-chain alcohol such as ethanol, 1-propanol, and 1-butanol, serves to increase the interfacial fluidity by penetrating into the surfactant film and consequently creating a disordered film because of the void space generated among surfactant molecules. Microemulsions may, however, be prepared without the use of cosurfactants and alcohol-free self-emulsifying microemulsion systems are known in the art. The aqueous phase may typically be, but is not limited to, water, an aqueous solution of the drug, glycerol, PEG300, PEG400, polyglycerols, propylene glycols, and derivatives of ethylene glycol. The oil phase may include, but is not limited to, materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain (C8-C12) mono, di, and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils and silicone oil.

[0162] Microemulsions are particularly of interest from the standpoint of drug solubilization and the enhanced absorption of drugs. Lipid based microemulsions (both o/w and w/o) have been proposed to enhance the oral bioavailability of drugs, including peptides (Constantinides et al, Pharmaceutical Research, 1994, 11, 1385-1390; Ritschet, Met/i. Find. Exp. Clin. PharmacoL, 1993, 13, 205). Micro-emulsions afford advantages of improved drug solubilization, protection of drug from enzymatic hydrolysis, possible enhancement of drug absorption due to surfactant- induced alterations in membrane fluidity and permeability, ease of preparation, ease of oral administration over solid dosage forms, improved clinical potency, and decreased toxicity (Constantinides et al. , Pharmaceutical Research, 1994, 11, 1385; Ho et al, J. Pharm. Set, 1996, 85, 138-143). Often microemulsions may form spontaneously when their components are brought together at ambient temperature. This may be particularly advantageous when formulating thermolabile drugs, peptides or oligonucleotides. Microemulsions have also been effective in the transdermal delivery of active components in both cosmetic and pharmaceutical applications. It is expected that the microemulsion compositions and formulations of the present invention will facilitate the increased systemic absorption of ONs and nucleic acids from the gastrointestinal tract.

[0163] Microemulsions of the present invention may also contain additional components and additives such as sorbitan monostearate (Grill 3), Labrasol, and penetration enhancers to improve the properties of the formulation and to enhance the absorption of the oligonucleotides and nucleic acids of the present invention. Penetration enhancers used in the microemulsions of the present invention may be classified as belonging to one of five broad categories: surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al. , Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92).

Liposomes

[0164] There are many organized surfactant structures besides microemulsions that have been studied and used for the formulation of drugs. These include monolayers, micelles, bilayers and vesicles. Vesicles offer specificity and extended duration of action for drug delivery. Thus, as used herein, the term "liposome" refers to a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayers, i.e., liposomes are unilamellar or multilamellar vesicles which have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion typically contains the composition to be delivered. In order to cross intact mammalian skin, lipid vesicles must pass through a series of fine pores, each with a diameter less than 50 nm, under the influence of a suitable transdermal gradient. Therefore, it is desirable to use a liposome which is highly deformable and able to pass through such fine pores. Additional factors for liposomes include the lipid surface charge, and the aqueous volume of the liposomes.

[0165] Further advantages of liposomes include; liposomes obtained from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water and lipid soluble drugs; liposomes can protect encapsulated drugs in their internal compartments from metabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N. Y., volume 1, p. 245).

[0166] For topical administration, there is evidence that liposomes present several advantages over other formulations. Such advantages include reduced side-effects related to high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target.

[0167] One major type of liposomal composition includes phospholipids other than naturally-derived phosphatidylcholine. Neutral liposome compositions, for example, can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions generally are formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are formed primarily from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposomal composition is formed from phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC. Another type is formed from mixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

[0168] Liposomes also include "sterically stabilized" liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome include one or more glycolipids, such as monosialoganglioside G_MI, or is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. Without being bound by any particular theory, it is believed that for sterically stabilized liposomes containing gangliosides, sphingomyelin, or PEG-derivatized lipids, the increase in circulation half-life of these sterically stabilized liposomes is due to a reduced uptake into cells of the reticuloendothelial system (RES) (Allen et al, FEBS Lett, 1987, 223, 42; Wu et al, Cancer Research, 1993, 53, 3765).

[0169] Various liposomes that include one or more glycolipids have been reported in Papahadjopoulos et al, Ann. N. Y. Acad. Sci., 1987, 507, 64 (monosiatoganglioside G_{M I} , galactocerebroside sulfate and phosphatidylinositol); Gabizon et al., Proc. Natl. Acad. Sci. USA., 1988, 85, 6949,;Allen et al, US. Patent No 4,837,028 and International Application Publication No WO 88/04924 (sphingomyelin and the ganglioside G_MI or a galactocerebroside sulfate ester); Webb et al, U.S. patent No 5,543,152 (sphingomyelin); Lim et al, International Application Publication No WO 97/13499 (1,2-sn- dimyristoylphosphatidylcholine).

[0170] Liposomes that include lipids derivatized with one or more hydrophilic polymers, and methods of preparation are described, for example, in Sunamoto et al, Bull. Chem. Soc. Jpn., 1980, 53, 2778 (a nonionic detergent, 2C₁₂15G, that contains a PEG moiety); Ilium et al, FEBS Lett., 1984, 167, 79 (hydrophilic coating of polystyrene particles with polymeric glycols); Sears, U.S. patents Nos. 4,426,330 and 4,534, 899 (synthetic phospholipids modified by the attachment of carboxylic groups of polyalkylene glycols (e.g., PEG)); Klibanov et al, FEBS Lett., 1990, 268, 235 (phosphatidylethanolamine (PE) derivatized with PEG or PEG stearate); Blume et al, Biochimica et Biophysica Acta, 1990, 1029, 91 (PEG-derivatized phospholipids, e.g., DSPE-PEG, formed from the combination of distearoylphosphatidylethanolamine (DSPE) and PEG); Fisher, European Patent No. EP 0 445 131 Bl and Internaltional application publication No WO 90/04384 (covalently bound PEG moieties on liposome external surface); Woodle et al, U.S. patents . Nos. 5,013,556 and 5,356,633, and Martin et al, U.S. patents. No. 5,213,804 and European Patent No. EP 0 496 813 Bl (liposome compositions containing 1-20 mole percent of PE derivatized with PEG); Martin et al, International application publication No WO 91/05545 and U.S. patent No. 5,225,212 and in Zalipsky et al. , International application publication No WO 94/20073 (liposomes containing a number of other lipid-polymer conjugates); Choi et al, International application publication No WO 96/10391 (liposomes that include PEG-modified ceramide lipids); Miyazaki et al., U.S. patent No. 5,540,935, and Tagawa et al, U.S. Patent No. 5,556,948 (PEG-containing liposomes that can be further derivatized with functional moieties on their surfaces).

[0171] Liposomes that include nucleic acids have been described, for example, in Thierry et al., International application publication No WO 96/40062 (methods for encapsulating high molecular weight nucleic acids in liposomes); Tagawa et al, U.S. patent No 5,264,221 (protein-bonded liposomes containing RNA); Rahman et al, U.S. patent No 5,665,710 (methods of encapsulating oligodeoxynucleotides in liposomes); Love et al. , International application publication No WO 97/04787 (liposomes that include antisense oligonucleotides).

[0172] Another type of liposome, transfersomes, is highly deformable lipid aggregates which are attractive for drug delivery vehicles. (Cevc et al, 1998, Biochim Biophys Acta. 1368(2):201-15.) Transfersomes may be described as lipid droplets which are so highly deformable that they can penetrate through pores which are smaller than the droplet. Transfersomes are adaptable to the environment in which they are used, for example, they are shape adaptive, self-repairing, frequently reach their targets without fragmenting, and often self-loading. Transfersomes can be made, for example, by adding surface edge- activators, usually surfactants, to a standard liposomal composition.

Surfactants

[0173] Surfactants are widely used in formulations such as emulsions (including microemulsions) and liposomes. The most common way of classifying and ranking the properties of the many different types of surfactants, both natural and synthetic, is by the use of the hydrophile/lipophile balance (HLB). The nature of the hydrophilic group (also known as the "head") provides the most useful means for categorizing the different surfactants used in formulations (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285). [0174] If the surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants are widely used in pharmaceutical and cosmetic products and are usable over a wide range of pH values, and with typical HLB values from 2 to about 18 depending on structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters; and nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylated block polymers are also included in this class. The polyoxyethylene surfactants are the most commonly used members of the nonionic surfactant class.

[0175] Surfactant molecules that carry a negative charge when dissolved or dispersed in water are classified as anionic. Anionic surfactants include carboxylates such as soaps, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isothionates, acyl laurates and sulfosuccinates, and phosphates. The alkyl sulfates and soaps are the most commonly used anionic surfactants.

[0176] Surfactant molecules that carry a positive charge when dissolved or dispersed in water are classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines, with the quaternary ammonium salts used most often.

[0177] Surfactant molecules that can carry either a positive or negative charge are classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines and phosphatides.

[0178] The use of surfactants in drug products, formulations and in emulsions has been reviewed in Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

Penetration Enhancers

[0179] According to the present invention, penetration enhancers are used in or with a composition to increase the delivery of nucleic acids, particularly ONs across membranes of animals. Most drugs are present in solution in both ionized and nonionized forms. However, usually only lipid soluble or lipophilic drugs readily cross cell membranes. It has been discovered that even non-lipophilic drugs may cross cell membranes if the membrane to be crossed is treated with a penetration enhancer. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also enhance the permeability of lipophilic drugs.

[0180] Penetration enhancers may be classified as belonging to one of five broad categories, i.e., surfactants, fatty acids, bile salts, chelating agents, and non-chelating nonsurfactants (Lee et al, Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92). Each of these classes of penetration enhancers is described below in greater detail.

[0181] In connection with the present invention, surfactants (or "surface-active agents") are chemical entities which, when dissolved in an aqueous solution, reduce the surface tension of the solution or the interfacial tension between the aqueous solution and another liquid, with the result that absorption of ONs through the mucosa is enhanced. These penetration enhancers include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (Lee et al, Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92); and perfluorochemical emulsions, such as FC-43. Takahashi et al. (J. Pharm. Pharmacol., 1988, 40, 252), each of which is incorporated herein by reference in its entirety.

[0182] Fatty acids: Various fatty acids and their derivatives which act as penetration enhancers include, for example, oleic acid, lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein (1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glycerol 1 -monocaprate, 1 -dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, Ci-io alkyl esters thereof (e.g., methyl, isopropyl and t-butyl), and mono- and diglycerides thereof (L e. , oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc.) (Lee et al, Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; El Hariri et al, J. Pharm. Pharmacol., 1992, 44, 651-654), each of which is incorporated herein by reference in its entirety. [0183] The physiological role of bile includes the facilitation of dispersion and absorption of lipids and fat-soluble vitamins (Brunton, Chapter 38 in: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, New York, 1996, pp. 934-935). Various natural bile salts, and their synthetic derivatives, act as penetration enhancers. Thus the term "bile salts" in the present invention includes any of the naturally occurring components of bile as well as any of their synthetic derivatives. The bile salts of the invention include, for example, cholic acid (or its pharmaceutically acceptable sodium salt, sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid (sodium glucholate), glycholic acid (sodium glycocholate), glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid (sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodium tauro-24,25- dihydro-fusidate (STDHF), sodium glycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Yamamoto et al., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al, J. Pharm. ScL, 1990, 79, 579-583).

[0184] In the present context, chelating agents can be regarded as compounds that remove metallic ions from solution by forming complexes therewith, with the result that absorption of ONs through the mucosa is enhanced. With regards to their use as penetration enhancers in the present invention, chelating agents have the added advantage of also serving as DNase inhibitors, as most characterized DNA nucleases require a divalent metal ion for catalysis and are thus inhibited by chelating agents (Jarrett, J. Cliromatogr., 1993, 618, 315-339). Without limitation, chelating agents include disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5- methoxysalicylate and homovanilate), N-acyl derivatives of collagen, laureth-9 and N- amino acyl derivatives of beta-diketones (enamines)(Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Buur et al, J. Control ReI., 1990, 14,

43-51).

[0185] As used herein, non-chelating non- surfactant penetration enhancing compounds are compounds that do not demonstrate significant chelating agent or surfactant activity, but still enhance absorption of oligonucleotides through the alimentary mucosa (Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). Examples of such penetration enhancers include unsaturated cyclic ureas, 1 -alkyl- and 1- alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92); and nonsteroidal anti-inflammatory agents such as diclofenac sodium, indomethacin and phenylbutazone (Yamashita et ah, J. Pharm. Pharmacol., 1987, 39, 621-626).

[0186] Other agents may be utilized to enhance the penetration of the administered nucleic acids, including glycols such as ethylene glycol and propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenes such as limonene and menthone.

Carriers

[0187] Certain compositions of the present invention also incorporate carrier compounds in the formulation. As used herein, "carrier compound" or "carrier" can refer to a nucleic acid, or analog thereof, which is inert (i.e., does not possess biological activity per se) but is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of a nucleic acid having biological activity by, for example, degrading the biologically active nucleic acid or promoting its removal from circulation. The co administration of a nucleic acid and a carrier compound, often with an excess of the latter substance, can result in a substantial reduction of the amount of nucleic acid recovered in the liver, kidney or other extra circulatory reservoirs. For example, the recovery of a partially phosphorothioated ON in hepatic tissue can be reduced when it is co administered with polyinosinic acid, dextran sulfate, polycytidic acid or 4-acetamido- 4'isothiocyano-stilbene-2,2-disulfonic acid (Miyao e? α/.,AntisenseRes. Dev., 1995, 5, 115-121; Takakura et al, Antisense & NucL Acid Drug Dev., 1996, 6, 177-183), each of which is incorporated herein by reference in its entirety. Excipients

[0188] In contrast to a carrier compound, a "pharmaceutical carrier" or "excipient" is a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal, and is typically liquid or solid. A pharmaceutical carrier is generally selected to provide for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given pharmaceutical composition, in view of the intended administration mode. Typical pharmaceutical carriers include, but are not limited to, binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycotate, etc.); and wetting agents (e.g., sodium lauryl sulphate, etc.).

[0189] Pharmaceutically acceptable organic or inorganic excipients suitable for non- parenteral administration which do not deleteriously react with nucleic acids can also be used to formulate the compositions of the present invention. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.

[0190] Formulations for topical administration of nucleic acids may include sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions of the nucleic acids in liquid or solid oil bases. The solutions may also contain buffers, diluents and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can be used. Other Pharmaceutical Composition Components

[0191] The present compositions may additionally contain other components conventionally found in pharmaceutical compositions, at their art-established usage levels. Thus, for example, the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present invention. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.

[0192] Aqueous suspensions may contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran, and/or stabilizers.

[0193] In another embodiment of the present invention, there is provided a pharmaceutical composition containing (a) one or more anti amyloid-related disease ONs and (b) one or more other chemotherapeutic agents used which function by similar or different mechanisms. Examples of such chemotherapeutic agents include but are not limited to maltose tetrapalmitate, maltose tripalmitate, daunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide, cytosine arabinoside, bis-chloroethylnitrosurea, busulfan, mitomycin C, actinomycin D, mithramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine, pentamethytmetamine, mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosurea, nitrogen mustards, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-azacytidine, hydroxyurea, deoxycoformycin, 4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5- FU), 5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol, vincristine, vinblastine, etoposide (VP- 16), trimetrexate, irinotecan, topotecan, gemcitabine, teniposide, cisplatin, and diethylstilbestrol (DES)(The Merck Manual of Diagnosis and Therapy, 15th Ed. 1987, pp. 1206-1228, Berkow et al, eds., Rahway, NJ). When used with the compounds of the invention, such chemotherapeutic agents may be used individually (e.g., 5-FU and an ON), sequentially (e.g., 5-FU and an ON for a period of time followed by MTX and ON), or in combination with one or more other such chemotherapeutic agents (e.g., 5-EU, MTX and an ON, or 5-FU, radiotherapy and an ON). Chemotherapeutic agents can be anti-inflammatory drugs, including but not limited to nonsteroidal anti-inflammatory drugs and corticosteroids. Other specific agents used to treat amyloidogenic disorders include donepezil, rivastigmine, and galantamine, the cholinesterase inhibitor tacrine vitamin E, selegiline and estrogen in postmenopausal women.

[0194] The pharmaceutical composition or ON formulation of the invention may further contain other drugs for the treatment of amyloid-related diseases. Such additional factors and/or agents may be included in the pharmaceutical composition, for example, to produce a synergistic effect with the ONs of the invention.

[0195] In another embodiment of the invention, anti amyloid-related disease ONs demonstrating low, preferably the lowest possible, homology with the human (or other subject organism's) genome is designed. One goal is to obtain an ON that will show the lowest toxicity due to interactions with human or animal genome sequence(s) and/or mRNAs. The first step is to produce the desired length sequence of the ON, e.g., by aligning nucleotides A, C, G, T/U in a random fashion, manually or, more commonly, using a computer program. The second step is to compare the ON sequence with a library of human sequences such as GenBank and/or the Ensemble Human Genome Database. The sequence generation and comparison can be performed repetitively, if desired, to identify a sequence or sequences having a desired low homology level with the subject genome. It is desirable for the ON sequence to have the lowest homology possible with the entire genome, while also minimizing self interaction. The last step is to test the ON in an assay to measure anti amyloid-related disease activity. [0196] In another embodiment, sequence independent ON sequence portion(s) is/are coupled with antisense sequence portion(s) to increase the activity of the final ON. The non-specific portion of the ON is described in the present invention. The antisense portion can be complementary to a amyloid pathogeneses related gene mRNA or to other genes important for the progression of amyloid-related diseases.

[0197] In further embodiment, sequence independent sequence portion(s) is/are coupled with a G-rich motif ON portion(s) to improve the activity of the final ON. The non-specific portion of the ON is described in the present invention. The G-rich motif portion can, as non-limiting examples, include, CpG, Gquartet, and/or CG that are described in the literature as stimulators of the immune system.

[0198] Another approach is to use an ON composed of one or more types of non- Watson-Crick nucleotides/nucleosides. Such ONs can mimic PS-ONs and other modifications with some of the following characteristics similar to PS-ONs: a) the total charge; b) the space between the units; c) the length of the chain; d) a net dipole with accumulation of negative charge on one side; e) the ability to bind to proteins f) the ability to be used with delivery systems, h) an acceptable therapeutic index, i) an anti amyloid-related disease activity. The ON can have a phosphorothioate backbone but is not limited to it. Examples of non- Watson-Crick nucleotides/nucleosides are described in Kool, 2002, Ace. Chem. Res. 35:936-943; and Takeshita et al. , (1987) J Biol. Chem. 262:10171-10179 where ONs containing synthetic abasic sites are described. Another embodiment of the present invention is to use a polymer mimicking the activity of ONs described in the present invention to obtain inhibition of amyloid-related disease activity. As described in the literature, several anionic polymers were shown to bind to proteins. These polymers belong to several classes: (1) sulfate esters of polysaccharides (dextrin and dextran sulfates; cellulose sulfate); (2) polymers containing sulfonated benzene or naphthalene rings and naphthalene sulfonate polymers; (3) polycarboxylates (acrylic acid polymers); and acetyl phthaloyl cellulose (Neurath et al. (2002) BMC Infect Dis 2:27); and (4) abasic ONs (Takeshita et al. , 1987, J. Biol. Chem. 262:10171-10179). Other examples of non-nucleotide protein binding polymers are described in the literature. The polymers described herein can mimic ONs described in this invention and may have some or all of the following characteristics similar to ONs: a) the length of the chain; b) a net dipole with accumulation of negative charge on one side; c) the ability to bind to proteins; d) the ability to be encapsulated by a delivery system, e) an acceptable therapeutic index, f) an anti amyloid-related disease activity. In order to mimic the effect of an ON, the anti amyloid-related disease polymer may preferably be a polyanion displaying similar space between its units as compared to a PS-ON. Also to mimic the effect of an ON, the anti amyloid-related disease polymer may display a similar hydrophobicity than PS-ON.

[0199] The present invention would be readily understood by referring to the following examples which are given to illustrate the invention rather than to limits its scope.

[0200] ON that can be used in this invention are listed in the following:

Example 1

The binding of PS-ONs to amyloid beta (1-42) is dependent on size and sulfur modification.

[0201] It is shown herein that the binding of various ONs to amyloid beta (1-42) was measured by fluorescence polarization. Briefly, increasing concentrations of peptide were added to 3nM of FITC labelled ON randomers (fully degenerate ONs). The binding of peptide to these ONs was measured by fluorescence polarization at 535nM. The Kd values were determined by the amount of peptide which yielded a polarization 50% of that observed at saturated binding. The results of the binding study are presented in Table 1 : Table 1 Interaction of PS-ON randomers with amyloid beta

FL: refer to a fluorescent linker (FITC label) attached to the ONs.

[0202] These results demonstrate the size dependent binding of amyloid beta by PS- ONs and the requirement of sulfur modification (phosphorothioation). These data suggest that larger PS-ONs (e.g. 40 mers) and sulfur modified ONs may be better therapeutic agents for the treatment of Alzheimer's disease due to their greater affinity for amyloid beta (1-42).

Example 2 Sulfur modified ON randomers bind in a size dependent fashion to amylin

[0203] It is shown herein the binding of IAPP (amylin) by different randomers using the same methodology as described for amyloid beta (1-42) binding above, (see Table 2). It is reported the shift in polarization values (mP) where a higher shift is indicative of greater binding. Table 2 Interaction of PS-ON randomers with amylin.

[0204] The binding interactions observed demonstrate that the interaction of randomers with amylin is size dependent and dependent in sulfur modification (i e phosphorothioation). These results suggest that other sulfur modified ONs will also bind to amylin and that sulfur modified ONs may have uses as a therapy in the prevention of amylin plaques associated with type II diabetes. Example 3

PS-ON randomers have potent, sequence-independent size dependent, anti-prion activity

[0205] It is shown herein the tested anti-PrP activity of PS-ON randomers, in a tissue culture model of PrP conversion. Approximately 20,000 RML sheep or 22L scrapie- infected mouse neuroblastoma cells were added to each well of a 96 well plate in 100 μL of medium prior to the addition of test compounds. 22L-infected cells were developed by re-infection of RML-infected mouse neuroblastoma cells cured by 7 passages in medium containing 1 μg/mL pentosan polysulfate. The cured cells were re-infected by incubation with PrP^Sc purified from mouse brains infected with 22L strain of scrapie. The neuroblastoma cells reinfected with 22L scrapie have stably expressed PrP^Sc for over 70 passages. The cells were allowed to settle for 4 hours before test compounds were added.

[0206] PS-ON randomers were diluted into PBS prior to being introduced to the cell medium. 5 μL of solutions were added to the cell medium. After PS-ON randomers were added, the cells were incubated for 5 days at 37° C in a CO₂ incubator before being lysed.

[0207] Prior to cell lysis, the cells were inspected by light microscopy for toxicity, bacterial contamination, and density compared to controls. After removal of the cell media, 50 μL of lysis buffer was added to each well. Lysis buffer was composed of 0.5% (w/v) Triton X-100™, 0.5% (w/v) sodium deoxycholate, 5 mM tris-HCl, pH 7.4 at 4⁰C, 5 mM EDTA, and 150 mM NaCl. Five minutes after adding lysis buffer, 25 μL of 0.1 mg/mL PK (Calbiochem) in TBS was added to each well and incubated at 37° C for 50 minutes. 225 μL of 1 mM Pefabloc™ (Boehringer Mannheim) was then added to each well to inhibit PK activity. 250 μL of 1 mM Pefabloc was added to samples that were not PK-treated.

[0208] To detect the presence of converted (PK resistant) PrP protein, a 96 well dot blot apparatus (Schleicher and Schuell) was set up with a sheet of 0.45 μm PVDF Immobilon-P™ (Millipore) membrane and each dot rinsed with 500 μL of TBS. Under vacuum, the lysed and PK-treated samples were added to the apparatus over the PVDF membrane and rinsed again with 500 μL of TBS. The PVDF membrane was then removed and covered with 3 M GdnSCN (Fluka) for 10 minutes at ambient temperature. The GdnSCN was removed by 5 PBS rinses and the membrane blocked in 5% (w/v) milk, 0.05% (v/v) Tween 20™ (Sigma) in TBS (TBST-milk) for 30 minutes. An appropriate dilution of a monoclonal antibody 6B10, an IgG 2a reactive against mouse, hamster, elk, and sheep PrP in immunoblots and ELISA assays or 8 μg of purified 6H4 anti-PrP mouse monoclonal antibody (Prionics) in 15 mL TBST-milk was incubated with the membrane for 60 minutes. After rinsing with TBST, a solution of -500 ng of an alkaline phosphatase conjugated goat anti-mouse linked antibody (Zymed) in 15 mL TBST-milk was added for 45 minutes. After additional TBST rinsing, the membrane was treated with enhanced chemofluorescence agent (Amersham) for 10 minutes, allowed to dry, and then scanned using a Storm Scanner™ (Molecular Dynamics). The intensity of the PrP^Sc signal from each well was quantitated using ImageQuant software™ (Molecular Dynamics).

[0209] It was tested to see if PS-ON randomer inhibition of PrP conversion was dependent on randomer size. For this experiment, PS-ON randomers of different sizes were tested (see Table 3).

Table 3 PS-ONs anti-PrP-res activity in vitro is size dependent

a -30% inhibition at 25 μM.

[0210] These data clearly show that PS-ON randomers have a potent anti-PrP conversion activity against both 22L and RML strains of scrapie. Moreover, this activity is dependent on the size of the PS-ON randomer used, with larger randomers being more active. To confirm the sequence independent activity of ONs against scrapie, variety of homo and heteropolymer PS-ONs for anti scrapie activity were tested (see Table 4 ) Table 4 The sequence independent nature of 40 mer PS-ON anti-scrapie activity.

NT = not tested

[0211] This data demonstrates the sequence-independent nature of PS-ONs. In general, the parallel between the pathology and progression of prion and amyloid diseases suggests that sulfur modified ONs will also have applications in the treatment of amyloid-related diseases.

Example 4 The anti scrapie activity of ONs is dependent on sulfur modification

[0212] The requirement for sulfur modification (i.e. phosphorothioation) on scrapie activity in vivo by comparing the anti-scrapie activity of REP 2006 (SEQ ID NO: 6)and REP 2107 (SEQ ID NO: 103) (which are phosphorothioated) in comparison to REP 2086 (SEQ ID NO: 83) (which has no sulfur modification) were tested (Table 5).

Table 5 The in vitro anti-PrP-res activity of randomers is dependent on phosphorothioation

[0213] These data show that sulfur modification (i.e. phosphorothioation) is required for anti-scrapie activity in vitro and suggest that other sulfur modifications on ONs will also confer anti-scrapie activity. In general, the parallel between the pathology and progression of prion and amyloid diseases suggests that sulfur modified ONs will also have applications in the treatment of amyloid-related diseases.

Example 5

PS-ONs are effective prophylactic agents against scrapie in vivo and in vivo activity is dependent on sulfur modification.

[0214] The in vivo activity of REP 2006(SEQ ID NO: 6) in prophylaxis to prevent the progression of scrapie in Tg7 transgenic mice was examined. Tg7 mice were first dosed with 10mg/kg GMP-REP 2006 (SEQ ID NO: 6) in 5% dextrose subcutaneously (sc) or intraperitoneally (ip) daily for 3 days. Also on the third day immediately after the third dose of cGMP-Randomer 1, animals were inoculated ip with 50 μL of 1% 263K hamster scrapie-infected brain homogenate. Afterwards, animals were dosed Monday, Wednesday, and Friday for either the next 4 or 10 weeks with the above mentioned amount of cGMP-REP2006 (SEQ ID NO: 6). Groups of Tg7 mice were also inoculated ip with 50 μL of 1% 263K hamster scrapie-infected brain homogenate and dosed with 5% dextrose either sc or ip as a control.

Table 6

REP 2006 (SEQ ID NO: 6) as a scrapie prophylactic in Tg7 mice inoculated ip with 1%

263K brain homogenate

^aone group died of non-scrapie cause; two groups died of non-scrapie cause.

[0215] These data demonstrate the in vivo activity of PS-ONs as anti scrapie agents. To determine the requirement of sulfur modification for activity, the ability of REP 2006 (SEQ ID NO: 6) and REP 2086 (SEQ ID NO: 83) to disinfect 263K (hamster scrapie) infected hamster brain homogenates was tested. The amount of infectivity in dilutions of hamster 263K scrapie-infected brain homogenate was bioassayed in transgenic mice that over express hamster PrP (Tg7). Untreated 10% (wt/vol) homogenates of 263K hamster scrapie-infected brains were diluted with PBS to 1, 0.1, 0.01, or 0.001% (wt/vol) and incubated at 37° C for one hour. 50 μL of each of these diluted homogenates were then injected intracerebrally into Tg7 mice. Separate 10% 263K-infected brain homogenate solutions were diluted to 1% with PBS and ImM randomer solution to the desired final concentrations. These mixtures of brain homogenate and randomer were also incubated at 37° C for one hour. As with control homogenates, 50 μL of each of these were injected intracerebrally (ic) into Tg7 mice. Table 7

263K brain homogenate scrapie infectivity with REP2006 (SEQ ID NO: 6) or REP 2086

(SEQ ID NO: 83) added.

[0216] These data show that the ability to clear infectivity from brain homogenate was dependent on sulfur modification (i.e. phosphorothioation) suggesting that sulfur modification is required for anti-scrapie activity in vivo and that other sulfur modified ONS will also be active. In general, the parallel between the pathology and progression of prion and amyloid diseases suggests that sulfur modified ONs will also be active against amyloid plaque progression in vivo and may have applications in the treatment of amyloid-related diseases. Example 6

Anti amyloid-related disease ONs with increased pH resistance, lower serum protein binding and superior nuclease resistance.

[0217] It is showed in this example the effect of combining unmodified linkages, phosphorothiate linkages, 2'-0 methyl modified riboses and unmodified ribonucleotides on the serum protein interaction and chemical stability of a 40 base randomer ON, that can be used as a anti amyloid-related disease agent.

[0218] All randomers were prepared using standard solid phase, batch synthesis at the University of Calgary Core DNA Services lab on a 1 or 15 uM synthesis scale, deprotected and desalted on a 50cm Sephadex G-25™ column.

[0219] To determine serum protein interaction, a phosphorothioate randomer labeled at the 3' end with FITC (the bait) is diluted to 2nM in assay buffer (1OmM Tris, pH7.2, 8OmM NaCl, 1OmM EDTA, 10OmM b-mercaptoethanol and 1% tween 20). This oligo is then mixed with the appropriate amount of non heat-inactivated FBS. Following randomer-FBS interaction, the complexes are challenged with various unlabelled randomers to assess their ability to displace the bait from its complex. Displaced bait is measured by fluorescence polarization. The displacement curve was used to determine Kd.

[0220] pH resistance was determined by incubation of randomers in PBS adjusted to the appropriate pH with HCl. 24 hours after incubation, samples were neutralized with IM TRIS, pH 7.4 and run on denaturing acryalmide gels and visualized following EtBr staining.

[0221] For these experiments, behaviours of different modified ON randomers: REP 2006 (SEQ ID NO: 6), REP 2015 (SEQ ID NO: 15), REP 2024 (SEQ ID NO: 24), REP 2107 (SEQ ID NO: 103), REP 2086 (SEQ ID NO: 83) and REP 2060 (SEQ ID NO: 56) were compared (see Table 8 in this example). Table 8 Randomer description

N = unmodified deoxyribonucleotide, unmodified linkage N = unmodified deoxyribonucleotide, phosphorothiate linkage N = 2'-0 methyl modified ribose, unmodified linkage N = 2'-0 methyl modified ribose + phosphorothioate linkage N = unmodified ribonucleotide + phosphorothioate linkage

[0222] The relative affinity of these ON randomers for serum proteins was determined as described above. The results of these experiments showed that REP 2107 has a lower affinity to serum proteins than REP 2006 (SEQ ID NO: 6) or REP 2024 (SEQ ID NO: 24) (see Table 9 in this example) and that there was no interaction detected between REP 2086 (SEQ ID NO: 83) and serum proteins. Moreover, at saturation of competition, REP 2107 (SEQ ID NO: 103) was less effective at displacing bound bait than REP 2006 (SEQ ID NO: 6) or REP 2024 (SEQ ID NO: 24) (see Table 10 in this example).

Table 9 Serum protein affinity of various randomers.

Table 10 Displacement of bait randomer at saturation.

[0223] The pH stability of these randomers in the range of pH 1 to pH 7 over 24 hours of incubation at 37 deg C was tested. While REP 2006 (SEQ ID NO: 6) and REP 2024 (SEQ ID NO: 24) showed significant degradation at pH 3 and complete degradation at pH 2.5, REP 2107 (SEQ ID NO: 103), 2086 (SEQ ID NO: 83) and 2060 (SEQ ID NO: 56) were completely stable at pH 1 after 24h of incubation. [0224] It is demonstrated here that the incorporation of 2'-0 methyl modifications in PS-ON randomers lowers serum protein binding and improve low pH resistance. The fully 2'-0-methylated, fully phosphorothioated randomer (REP 2107; SEQ ID NO: 107) has a weaker interaction with serum proteins and shows a significantly increased resistance to low pH induced hydrolysis.

[0225] 40 mer randomers of various chemistries were assessed for their ability to resist degradation by various nucleases for 4 hours at 37 deg C (see Table 1 1 in this example). While most chemistries exhibited resistance to more than one nuclease, only REP 2107 (SEQ ID NO: 103) was resistant to all four nucleases tested. It is important to note that REP 2024 (SEQ ID NO: 24) (which has 2'-0 methyl modifications at the 4 riboses at each end of the molecule) showed the same resistance profile as its parent molecule REP 2006, being sensitive to Sl nuclease degradation while 2107 (SEQ ID NO: 103) (fully T- O methyl modified) was resistant to this enzyme. These results suggest that fully 2'-0 methyl modified and fully phosphorothioated ON will be the most effective of the tested oligonucleotides in resisting degradation by nucleases.

Table 11 Resistance to various nucleases by different randomer chemistries.

Sensitive (S) or Resistant (R) after 4h incubation at 37 deg C

Phosphodiesterase Sl Nuclease BaI 31 Exonuclease I

Randomer II (Fermentas (NEB (NEB

(Sigma P9041) #EN0321) M0213S) M0293S)

REP2015 (SEQ ID NO: 15) R S S S

REP2107 (SEQ ID NO: 103) R R R R

REP2006 (SEQ ID NO: 6) R S R R

REP2086 (SEQ ID NO: 83) R R S R

REP2024 (SEQ ID NO: 24) R S R R

Example 7

Anti amyloid-related disease phosphorothioated polypyrimidine ONs exhibit acid and nuclease resistance.

[0226] To determine the extent of acid resistance of ONs that can be used as anti amyloid-related disease agents, various 40 base ONs having different chemistries and/or sequences are incubated in PBS buffered to different pH values for 24 hours at 37 deg C. The degradation of these ONs was assessed by urea-polyacryamide gel electrophoresis (see Table 12).

[0227] The results of these studies show that randomer ONs (containing both pyrimidine and purine nucleotides) are resistant to acidic pH only when they were fully 2'-0-methylated. The data indicated that even partially 2'-0-methylated ONs (gapmers, REP 2024; SEQ ID NO: 24) do not display any significant increase in acid resistance compared to fully phosphorothioated ONs. Even fully phosphorothioated randomers show no increased pH resistance compared to unmodified ONs. In contrast, it was noted that the phosphorothioated 40mer ONs containing only the pyrimidine nucleotides cytosine (polyC, REP 2031; SEQ ID NO: 31) or thymidine (polyT, REP 2030; SEQ ID NO: 30) or the polyTC heteropolymer (REP 2056; SEQ ID NO: 52) had equivalent acid resistance compared to the fully 2'-O-methylated randomers whether phosphorothioated (REP 2107; SEQ ID NO: 103) or not (REP 2086; SEQ ID NO: 83). Contrary to the results for the polypyrimidine oligonucleotides, phosphorothioated oligonucleotides containing only the purine nucleotide adenosine (polyA, REP 2029; SEQ ID NO: 29) or any adenosine or guanosine nucleotides (REP 2033, SEQ ID NO: 33; REP 2055, SEQ ID NO: 51; REP 2057, SEQ ID NO: 53) showed no greater acid resistance compared to unmodified DNA.

[0228] These results are significant because the preferred way described in the prior art to achieve greater acid resistance compared to phosphorothioated ONs was to add 2'-O- methyl modifications (or other 2'-ribose modifications) or other modifications. The present data demonstrates that the 2'-O-methyl ribose modification or other 2'-ribose modifications are not required if the ON is a polypyrimidine (i.e. contains only pyrimidine nucleotides [e.g. homopolymers of cytosine or thymidine or a heteropolymer of cytosines and thymidines]) to achieve pH resistance. The presence of purines (A or G) even in the presence of pyrimidines can render ONs acid labile.

Table 12 Acid stability of various 40 mer ONs

PII = phosphodiesterase 11, Sl = Sl nuclease, Exol = Exonuclease 1 , PS = all linkages phosphorothioated, 2'0Me = all riboses are 2'0 methylated. +++ = no degradation, ++ less than 5-% degradation, -/+ = more than 90% degradation, - = completely degraded. [0229] To determine the extent of ON nucleotide composition and modifications on nuclease resistance, various 40 base ONs having different nucleotide compositions and modifications were incubated in the presence of various endo and exonucleases for 4 hours at 37 deg C. The degradation of these ONs was assessed by urea-polyacryamide gel electrophoresis.

[0230] The results of these studies showed that randomer ONs were resistant to all four enzymes tested (phosphodiesterase II [Sigma], Sl nuclease [Fermentas], Bal31 [New England Biolabs] and exonuclease 1 [New England Biolabs]) only when they were fully phosphorothioated and fully 2'-0 -methylated (see table 10). Omission of any of these modifications in randomers resulted in increased sensitivity to one or more of the nucleases tested. It was noted that the fully phosphorothioated, partially 2'-0 -methylated randomer (REP 2024, SEQ ID NO: 24) was equivalent in nuclease resistance to REP 2006 (SEQ ID NO: 6), indicated that 2'-O- methylation may be required on each nucleotide of a phosphorothioated ON to achieve the optimal nuclease resistance. However, it was also noted that the phosphorothioated 40mer polypyrimidine poly cytosine (poly C, REP 2031, SEQ ID NO: 31) had equivalent nuclease resistance compared to the fully phosphorothioated, fully 2'0 methylated randomer (REP 2107, SEQ ID NO: 103).

[0231] These results are significant because the prior art teaches that the preferred way to enhance nuclease resistance of phosphorothioated ONs is to add 2'-0 -methyl modifications, other 2'- ribose modifications, or other modifications. This new data demonstrates that the 2'-O-methyl modification or other 2'-ribose modifications or any other modifications are not required to enhance nuclease resistance if the ON is fully phosphorothioated and consists of a homopolymer of cytosines.

Table 13 Nuclease resistance of various 40 mer ONs

PII = phosphodiesterase 11, Sl = Sl nuclease, Exol = Exonuclease 1, PS = all linkages phosphorothioated, 2'OMe = all riboses are 2'0 methylated. - = complete degredation, ++++ = no degredation, PS = phosphorothioate, 2'0Me = 2'-O-methyl modification of the ribose. [0232] These results demonstrate that phosphorothioated ONs containing only pyrimidine nucleotides, including cytosine and/or thymidine and/or other pyrimidines are resistant to low pH and phosphorothioated ONs containing only cytosine nucleotides exhibit superior nuclease resistance, two important characteristics for oral administration of an ON. Thus, high pyrimidine nucleotide content of an ON is advantageous to provide resistance to low pH resistance and high cytosine content is advantagaeous to provide improved nuclease resistance. For example, in certain embodiments, the pyrimidine content of such an oligonucleotide is more than 50%, more than 60%, or more than 70%, or more than 80%, or more than 90%, or 100%. Furthermore, these results show the potential of a method of treatment using oral administration of a therapeutically effective amount of at least one pharmacologically acceptable ON composed of pyrimidine nucleotides. These results also show the potential of ONs containing high levels of pyrimidine nucleotides as a component of an ON formulation.

Example 8 Tests for Determining if an Oligonucleotide has sequence-independent activity

[0233] It is shown herein that sulfur modified ONs have broad spectrum anti amyloid- related disease activity. Moreover, it is shown that the activity of such ONs are sequence- independent. An ON shall be considered to have sequence-independent activity if it meets the criteria of any one of the 5 tests outlined below. An ON having a reasonable part of its function due to a sequence-independent activity shall be considered to benefit from the inventions described herein. Note that in these tests an in vitro assay of scrapie accumulation is used as a surrogate for in vitro anti-amyloid activity due to the lack of any acceptable cell based assay for amyloidosis and also due to the similar progression and pathology of prion-related and amyloid-related diseases in vivo.

TEST #1 Effect of partial degeneracy of a candidate on its anti amyloid-related disease efficacy

[0234] This test serves to measure the anti amyloid-related disease activity of a candidate ON sequence when part of its sequence is made degenerate. If the degenerate version of the candidate ON having the same chemistry retains its activity as described below, it is deemed to have sequence-independent activity. Candidate ONs will be made degenerate according to the following rule:

L = the number of bases in the candidate ON.

X = the number of bases on each end of the oligo to be made degenerate (but having the same chemistry as the candidate ON).

If L is even, then X=integer (L/4).

If L is odd, then X=integer ((L+l)/4).

X must be equal to or greater than 4.

[0235] The anti-amyloidogenic activity of the candidate and partially degenerate ON shall be determined by the cell free binding activity to amyloid related peptides or in vitro anti-scrapie-activity using the assays described herein. IC 50 values (for binding (Kd) or activity) shall be generated using a minimum of seven concentrations of compound, with three or more points in the linear range of the dose response curve. Using these tests, the IC50 of the candidate ON shall be compared to its degenerate counterpart. If the IC50 of the partially degenerate ON is less than 5-fold greater than the original candidate ON (based on minimum triplicate measurements, standard deviation not to exceed 15% of mean) then the ON shall be deemed to have sequence independent activity.

TEST #2

Comparison ofanti amyloid-related disease activity of a candidate ON with an ON randomer.

[0236] This test serves to compare the anti amyloid-related disease efficacy of a candidate ON with the anti amyloid-related disease efficacy of a randomer ON of equivalent size and chemistry. The anti-amyloidogenic activity of the candidate and partially degenerate ON shall be determined by the cell free binding activity to amyloid related peptides or in vitro anti-scrapie-activity using the assays described herein. IC 50 values (binding (Kd) or activity) shall be generated using a minimum of seven concentrations of compound, with three or more points in the linear range of the dose response curve. Using this test, the IC50 of the candidate ON shall be compared to an ON randomer of equivalent size and chemistry. If the IC50 of the ON randomer is less than 5- fold greater than the candidate ON (based on minimum triplicate measurements, standard deviation not to exceed 15% of mean) then the candidate ON shall be deemed to have sequence-independent activity.

Thresholds used in these tests

[0237] The purpose of these tests are to determine by a reasonable analysis, if ONs benefit from or utilize the sequence-independent anti amyloid-related disease properties of ONs which we have described herein and is acting with sequence-independent activity. Of course anyone skilled in the art will realize that, given the inherent variability of all testing methodologies, especially anti amyloid-related disease testing methods, a determination of differences in anti amyloid-related disease activity between two compounds may not be reliably concluded if the threshold is set at a 2 or 3 fold difference between the activities of said compounds. This is due to the fact that variations from experiment to experiment with the same compound in these assays can yield IC50s which vary in this range. Thus, to be reasonably certain that real differences between the activities of two compounds (e.g. two ONs) exist, we have set a threshold of at least a 5- fold difference between the IC50s of said compounds. This threshold ensures the reliability of the assessment of the above mentioned tests.

[0238] The thresholds described in tests 1 and 2 above are the default thresholds. If specifically indicated, other thresholds can be used in the comparison tests. Thus for example, if specifically indicated, the threshold for determining whether an ON is acting with sequence-independent activity can be any of 10-fold, 8-fold, 6-fold, 5 -fold, 4-fold, 3-fold, 2-fold, 1.5-fold, or equal..

[0239] Similarly, though the default is that satisfying any one of the above 2 tests is sufficient, if specifically indicated, the ON can be required to satisfy both at a default threshold, or if specifically indicated, at another threshold(s) as indicated above. Example 9

Sulfur modified nucleic acids have a therapeutic activity in slowing the progression of plaque formation in a mouse model of Alzheimer's disease.

[0240] The ability of sulfur modified nucleic acids to halt the progression of Alzheimer's disease was assessed by examining the performance of a prototypical sulfur- modified nucleic REP 2006 (SEQ ID NO: 6), a 40mer fully degenerate, fully phosphorothioated oligodeoxynucleotide, in the TgCRND8 transgenic mouse model of Alzheimer's disease. This model mirrors the progression of amyloid beta (a-beta) plaque formation and concomitant impairment in neurological function seen in humans with Alzheimer's disease. Transgenic mice were dosed three times each week with an intraperinoneal injection of REP 2006 (SEQ ID NO: 6) for 4 months. The effects of REP 2006 (SEQ ID NO: 6) on the progression of plaque formation in these animals were assessed by several parameters (see Table 14).

Table 14

Effect of REP 2006 (SEQ ID NO: 6) treatment on plaque progression in the brains of Tg

CRND8 mice.

[0241] These data show a consistent reduction in all four measured parameters of a- beta plaque morphology with REP 2006 (SEQ ID NO: 6). This demonstrates that sulfur modified nucleic acids have therapeutic potential as agents to halt or retard the progression of a-beta plaque formation and concomitant neurological impairment in patients with Alzheimer's disease. Example 10 Size and sulfur-modification dependent interaction with a-beta peptide.

[0242] The interaction with purified amyloid-beta 1-40 with a variety of different sizes of fully degenerate, fully phosphorothioated oligodeoxynucleotides and 40 mer degenerate oligodeoxynucleotides lacking any sulfur (phosphorothioate) modification or having only 2' O-methylation of the ribose or a combination of 2'O-methylation or phosphorothioation was assessed by fluorescence polarization using fluorescently labelled oligonucleotides.

Table 15 Description of fluorescently labelled oligonucleotides

FL: refer to a fluorescent linker (FITC label) attached to the ONs

Table 16

Interaction of oligonucleotides with amyloid beta 1-40 (kD determined by fluorescence polarization

Labelled oligo SEQ ID NO: Kd (ug/ul)

Rep2032-FL SEQ ID NO: 32 No binding

Rep2003-FL SEQ ID NO: 3 No binding

Rep2004-FL SEQ ID NO: 4 Minimal binding

Rep2006-FL SEQ ID NO: 6 0 03390

Rep2007-FL SEQ ID NO' 7 0 03890

Rep2107-FL SEQ ID NO. 103 Moderate binding

Rep2031-FL SEQ ID NO: 3 1 0 03470

Rep2086-FL SEQ ID NO: 83 No binding

FL: refer to a fluorescent linker (FITC label) attached to the ONs

[0243] These data show that the interaction of oligonucleotides with amyloid beta is sequence independent (poly C is as good as a degenerate sequence), and dependent on size and the presence of a sulfur modification (phosphorothioation). Thus any nucleotide sequence could be used to generate an oligonucleotide which could interact with amyloid beta and thus may prevent amyloid fibril (plaque formation) as long it is of sufficient length (greater than 20 bases in length) and consists of at least one sulfur containing modification including but not restricted to phosphorothioation, diphosphorothioation or a sulfur modification to the base moiety at any suitable position.

Claims

WHAT IS CLAIMED IS

1. An anti amyloid-related disease oligonucleotide formulation comprising at least one anti amyloid-related disease oligonucleotide and an excipient, said oligonucleotide having activity against an amyloid-related disease, said activity occurring principally by a sequence independent mode of action.

2. The oligonucleotide formulation of claim 1, wherein said oligonucleotide is at least 15 nucleotides in length.

3. The oligonucleotide formulation of any one of claims 1-2, wherein said oligonucleotide is at least 20 nucleotides in length.

4. The oligonucleotide formulation of any one of claims 1-3, wherein said oligonucleotide is at least 25 nucleotides in length.

5. The oligonucleotide formulation of any one of claims 1-4, wherein said oligonucleotide is at least 30 nucleotides in length.

6. The oligonucleotide formulation of any one of claims 1-5, wherein said oligonucleotide is at least 35 nucleotides in length.

7. The oligonucleotide formulation of any one of claims 1-6, wherein said oligonucleotide is at least 40 nucleotides in length.

8. The oligonucleotide formulation of any one of claims 1-7, wherein said oligonucleotide is at least 45 nucleotides in length.

9. The oligonucleotide formulation of any one of claims 1-8, wherein said oligonucleotide is at least 50 nucleotides in length.

10. The oligonucleotide formulation of any one of claims 1-9, wherein said oligonucleotide is at least 60 nucleotides in length.

11. The oligonucleotide formulation of any one of claims 1-10, wherein said oligonucleotide is at least 80 nucleotides in length.

12. The oligonucleotide formulation of any one of claims 1-3, wherein said oligonucleotide is 20-30 nucleotides in length.

13. The oligonucleotide formulation of any one of claims 1-7, wherein said oligonucleotide is 30-40 nucleotides in length.

14. The oligonucleotide formulation of any one of claims 1-9, wherein said oligonucleotide is 40-50 nucleotides in length.

15. The oligonucleotide formulation of any one of claims 1-10, wherein said oligonucleotide is 50-60 nucleotides in length.

16. The oligonucleotide formulation of any one of claims 1-10, wherein said oligonucleotide is 60-70 nucleotides in length.

17. The oligonucleotide formulation of any one of claims 1-16, wherein said oligonucleotide is 70-80 nucleotides in length.

18. The oligonucleotide formulation of any one of claims 1-17, wherein said oligonucleotide is not complementary to any equal length portion of a genomic sequence.

19. The oligonucleotide formulation of claim 18, wherein said genomic sequence is a genomic sequence of a human.

20. The oligonucleotide formulation of claim 18, wherein said genomic sequence is a genomic sequence of a non human animal.

21. The oligonucleotide formulation of any one of claims 1-20, wherein said oligonucleotide comprises at least 10 contiguous nucleotides of randomer sequence.

22. The oligonucleotide formulation any one of claims 1-21, wherein said oligonucleotide comprises at least 20 nucleotides of randomer sequence.

23. The oligonucleotide formulation of any one of claims 1-22, wherein said oligonucleotide comprises at least 30 nucleotides of randomer sequence.

24. The oligonucleotide formulation of any one of claims 1-23, wherein said oligonucleotide comprises at least 40 nucleotides of randomer sequence.

25. The oligonucleotide formulation of any one of claims 1-24, wherein said oligonucleotide is a randomer oligonucleotide.

26. The oligonucleotide formulation of any one of claims 1-20, wherein said oligonucleotide comprises a homopolymer sequence of at least 10 contiguous A nucleotides.

27. The oligonucleotide formulation of any one of claims 1-20, wherein said oligonucleotide comprises a homopolymer sequence of at least 10 contiguous T nucleotides.

28. The oligonucleotide formulation of any one of claims 1-20, wherein said oligonucleotide comprises a homopolymer sequence of at least 10 contiguous U nucleotides.

29. The oligonucleotide formulation of any one of claims 1-20, wherein said oligonucleotide comprises a homopolymer sequence of at least 10 contiguous G nucleotides.

30. The oligonucleotide of any one of claims 1-20, wherein said oligonucleotide comprises a homopolymer sequence of at least 10 contiguous I nucleotide analogs.

31. The oligonucleotide of any of one claims 1-20, wherein said oligonucleotide comprises a homopolymer sequence of at least 10 contiguous C nucleotides.

32. The oligonucleotide of any of one claims 1-20, wherein said oligonucleotide is a homopolymer of C nucleotides.

33. The oligonucleotide of any one of claims 1-20, wherein said oligonucleotide comprises a polyAT sequence at least 10 nucleotides in length.

34. The oligonucleotide of any one of claims 1-20, wherein said oligonucleotide comprises a polyAC sequence at least 10 nucleotides in length.

35. The oligonucleotide formulation of any one of claims 1-20, wherein said oligonucleotide comprises a polyAG sequence at least 10 nucleotides in length.

36. The oligonucleotide formulation of any one of claims 1-20, wherein said oligonucleotide comprises a poly AU sequence at least 10 nucleotides in length.

37. The oligonucleotide formulation of any one of claims 1-20, wherein said oligonucleotide comprises a poly AI sequence at least 10 nucleotides in length.

38. The oligonucleotide formulation of any one of claims 1-20, wherein said oligonucleotide comprises a polyGC sequence at least 10 nucleotides in length.

39. The oligonucleotide formulation of any one of claims 1-20, wherein said oligonucleotide comprises a polyGT sequence at least 10 nucleotides in length.

40. The oligonucleotide formulation of any one of claims 1-20, wherein said oligonucleotide comprises a polyGU sequence at least 10 nucleotides in length.

41. The oligonucleotide formulation of any one of claims 1 -20, wherein said oligonucleotide comprises a polyGI sequence at least 10 nucleotides in length.

42. The oligonucleotide formulation of any one of claims 1-20, wherein said oligonucleotide comprises a polyCT sequence at least 10 nucleotides in length.

43. The oligonucleotide formulation of any one of claims 1-20, wherein said oligonucleotide comprises a polyCU sequence at least 10 nucleotides in length.

44. The oligonucleotide formulation of any one of claims 1-20, wherein said oligonucleotide comprises a polyCI sequence at least 10 nucleotides in length.

45. The oligonucleotide formulation of any one of claims 1-20, wherein said oligonucleotide comprises a polyTI sequence at least 10 nucleotides in lengthy

46. The oligonucleotide formulation of any one of claims 1-20, wherein said oligonucleotide comprises a polyTU sequence at least 10 nucleotides in length.

47. The oligonucleotide formulation of any one of claims 1-20, wherein said oligonucleotide comprises a polyUI sequence at least 10 nucleotides in length.

48. The oligonucleotide formulation of any one of claims 1-47, wherein said oligonucleotide comprises at least one phosphodiester linkage.

49. The oligonucleotide formulation of any one of claims 1-48, wherein said oligonucleotide comprises at least one ribonucleotide.

50. The oligonucleotide formulation of any one of claims 1-49, wherein said oligonucleotide comprises at least one modification to its chemical structure.

51. The oligonucleotide formulation of any one of claims 1-49, wherein said oligonucleotide comprises at least two different modification to its chemical structure.

52. The oligonucleotide formulation of any of one claims 1-51, wherein said oligonucleotide contains at least one sulfur modification.

53. The oligonucleotide formulation of any one of claims 1-52, wherein said oligonucleotide contains at least one phosphorothioated linkage.

54. The oligonucleotide formulation of any one of claims 1-52, wherein said oligonucleotide contains at least one phosphorodithioated linkage.

55. The oligonucleotide formulation of any one of claims 1-54, wherein said oligonucleotide contains at least one boranophosphate linkage.

56. The oligonucleotide formulation of any one of claims 1-55, wherein said oligonucleotide comprises at least one sulfur modified nucleobase moiety.

57. The oligonucleotide formulation of any one of claims 1-55, wherein said oligonucleotides contains at least one sulfur modified ribose moiety.

58. The oligonucleotide formulation of any one of claims 1-57, wherein said oligonucleotide comprises at least one 2' modification to the ribose moiety.

59. The oligonucleotide formulation of any one of claims 1-58, wherein said oligonucleotide contains at least one 2'-0 alkyl modified ribose moiety.

60. The oligonucleotide formulation of any one of claims 1-59, wherein said oligonucleotide contains at least one 2'-0 methyl modified ribose.

61. The oligonucleotide formulation of any one of claims 1-60, wherein said oligonucleotide contains at least one 2'-methoxyethyl modified ribose.

62. The oligonucleotide formulation of any one of claims 1-61, wherein said oligonucleotide contains at least one 2'-FANA modified ribose.

63. The oligonucleotide formulation of any one of claims 1-62, wherein said oligonucleotide comprises at least one methylphosphonate linkage.

64. The oligonucleotide formulation of any one of claims 1-63, wherein said oligonucleotide comprises at least one portion consisting of glycol nucleic acid (GNA) with an acyclic propylene glycol phosphorothioate backbone.

65. The oligonucleotide formulation of any one of claims 1-64, wherein said oligonucleotide comprises at least one locked nucleic acid moiety.

66. The oligonucleotide formulation of any one of claims 1-65, wherein said oligonucleotide comprises at least one phosphorodiamidate morpholino moiety.

67. The oligonucleotides formulation of any one of claims 1-66, wherein said oligonucleotide comprises at least one abasic nucleic acid.

68. The oligonucleotide formulation of any one of claims 1-67, wherein said oligonucleotide comprises a linker to form a concatemer of two or more oligonucleotide sequences.

69. The oligonucleotide formulation of any one of claims 1-69, wherein said oligonucleotide is linked or conjugated at one or more nucleotide residues, to a molecule modifying the characteristics of the oligonucleotide to obtain one or more characteristics selected from the group consisting of higher stability, lower serum interaction, higher cellular uptake, an improved ability to be formulated, a detectable signal, higher anti amyloid-related disease activity, better pharmacokinetic properties, specific tissue distribution and lower toxicity.

70. The oligonucleotide formulation of claim 69 wherein said oligonucleotide is linked or conjugated to a PEG molecule.

71. The oligonucleotide formulation of claim 79 wherein said oligonucleotide is linked or conjugated to a cholesterol molecule.

72. The oligonucleotide formulation of any of claims 1-20 and 26-71, wherein said oligonucleotide is double stranded.

73. The oligonucleotide formulation of any one of claims 1-72, wherein said oligonucleotide comprises at least one base which is capable of hybridizing via non- Watson-Crick interactions.

74. The oligonucleotide formulation of any one of claims 1-17 and 21-24 and 26-31 and 33-73, wherein said oligonucleotide comprises a portion complementary to a genome.

75. The oligonucleotide formulation of any one of claims 1-17, 21-24, 26-31 and 33-74, wherein said oligonucleotide has at most 90%, preferably 80%, more preferably 75% identity with a genomic sequence.

76. The oligonucleotide formulation of any one of claims 1-17, 21-24, 26-31 and 33-74, wherein said oligonucleotide has at most 60%, preferably 50%, more preferably 40% identity with a genomic sequence.

77. The oligonucleotide formulation of any one of claims 1-76, wherein said oligonucleotide binds to one or more amyloid protein.

78. The oligonucleotide formulation of any one of claims 1-77, wherein said oligonucleotide interacts with one or more cellular components, wherein said interaction resulting in inhibition of a protein activity or expression.

79. The oligonucleotide formulation of any one of claims 1-17 and 21-24 and 26-31 and 33-78, wherein at least a portion of the sequence of said oligonucleotide is derived from a genome.

80. The oligonucleotide formulation of any one of claims 1-79, wherein said amyloid- related disease is Alzeimer's disease.

81. The oligonucleotide formulation of any one of claims 80, wherein Alzheimer's disease is sporadic (non-hereditary) Alzheimer's disease, familial (hereditary) Alzheimer's disease, or early Alzheimer's disease.

82. The oligonucleotide formulation of any one of claims 1-79, wherein said amyloid- related disease is Mild Cognitive Impairment or Mild-to-Moderate Cognitive Impairment.

83. The oligonucleotide formulation of any one of claims 1-79, wherein said amyloid- related disease is Beta 2-microglobulin-related (dialysis-related) amyloidosis.

84. The oligonucleotide formulation of any one of claims 1-79, said amyloid-related disease is Type II diabetes related amyloidosis.

85. The oligonucleotide formulation of any one of claims 1-79, wherein said amyloid- related disease is cerebral amyloid angiopathy or hereditary cerebral hemorrhage.

86. The oligonucleotide formulation of any one of claims 1-79, wherein said amyloid- related disease is senile dementia, Down's syndrome, inclusion body myositis, or age-related macular degeneration.

87. An oligonucleotide mixture, comprising a mixture of at least two different anti amyloid-related disease oligonucleotide formulation of any of one claims 1-86.

88. The oligonucleotide mixture of claim 87, comprising a mixture of at least ten different anti amyloid-related disease oligonucleotide formulation of any one of claims 1-86.

89. The oligonucleotide mixture of claim 88, comprising a mixture of at least 100 different anti amyloid-related disease oligonucleotide formulation of any one of claims 1-86.

90. The oligonucleotide mixture of claim 89, comprising a mixture of at least 1000 different anti amyloid-related disease oligonucleotide formulation of any one of claims 1-86.

91. The oligonucleotide mixture of claim 90, comprising a mixture of at least 10⁶ different anti amyloid-related disease oligonucleotide formulation of any one of claims 1-86.

92. The mixture of any one of claims 87-91, wherein said oligonucleotides are at least 10 nucleotides in length.

93. The mixture of any one of claims 87-92, wherein said oligonucleotides are at least 20 nucleotides in length.

94. The mixture of any one of claims 87-93, wherein said oligonucleotides are at least 30 nucleotides in length.

95. The mixture of any one of claims 87-94, wherein said oligonucleotides are at least 40 nucleotides in length.

96. The mixture of any one of claims 87-95, wherein said oligonucleotides are at least 50 nucleotides in length.

97. The mixture of any one of claims 87-96, wherein said oligonucleotides are at least 60 nucleotides in length.

98. The mixture of any one of claims 87-97, wherein said oligonucleotides are at least 70 nucleotides in length.

99. The mixture of any one of claims 87-98, wherein said oligonucleotides are at least 80 nucleotides in length.

100. The mixture of any one of claims 87-99, wherein said oligonucleotides are at least 120 nucleotides in length.

101. An anti amyloid-related disease pharmaceutical composition comprising a therapeutically effective amount of at least one pharmacologically acceptable, anti amyloid-related disease oligonucleotide formulation according to any one of claims 1 to 86, and a pharmaceutically acceptable carrier.

102. The anti amyloid-related disease pharmaceutical composition of claim 101, adapted for delivery by a mode selected from the group consisting of ocular administration, eye drop administration, oral ingestion, inhalation, subcutaneous injection, intramuscular injection, intrathecal, intracerebral, and intravenous injection.

103. The anti amyloid-related disease pharmaceutical composition of claim 101 or 102, wherein said composition further comprises a delivery system.

104. The anti amyloid-related disease pharmaceutical composition of claim 101, 102 or 103, wherein said composition further comprises at least one other anti amyloid- related disease drug in combination.

105. The anti amyloid-related disease pharmaceutical composition of claim 101, 102 or 103, wherein said composition further comprises a non-nucleotidic anti amyloid- related disease drug in combination.

106. The anti amyloid-related disease pharmaceutical composition of claim 101, 102 or 103, wherein said composition further comprises an anti amyloid-related disease antisense oligonucleotide in combination.

107. The anti amyloid-related disease pharmaceutical composition of claim 101, 102 or 103, wherein said composition further comprises an anti amyloid-related disease RNAi-inducing oligonucleotide.

108. Use of therapeutically effective amount of at least one pharmacologically acceptable anti amyloid-related disease oligonucleotide formulation, according to any of claims 1-86 for treating or preventing amyloid related disease.