WO2005042777A2 - Antisens anti-app/ena - Google Patents

Antisens anti-app/ena Download PDF

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WO2005042777A2
WO2005042777A2 PCT/GB2004/004483 GB2004004483W WO2005042777A2 WO 2005042777 A2 WO2005042777 A2 WO 2005042777A2 GB 2004004483 W GB2004004483 W GB 2004004483W WO 2005042777 A2 WO2005042777 A2 WO 2005042777A2
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reagents
antisense
silencing
app
sequences
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PCT/GB2004/004483
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WO2005042777A3 (fr
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Peter Estibeiro
David Page
Jenny Godfray
Eleanor Barnard
Makoto Koizumi
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Expresson Biosystems Limited
Sankyo Company Limited
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/323—Chemical structure of the sugar modified ring structure
    • C12N2310/3231—Chemical structure of the sugar modified ring structure having an additional ring, e.g. LNA, ENA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/34—Spatial arrangement of the modifications
    • C12N2310/341—Gapmers, i.e. of the type ===---===
    • C—CHEMISTRY; METALLURGY
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    • C12N2320/00—Applications; Uses
    • C12N2320/10—Applications; Uses in screening processes
    • C12N2320/12—Applications; Uses in screening processes in functional genomics, i.e. for the determination of gene function

Definitions

  • the present invention relates to strategies to address the deposition of APP which is associated with early etiology of disease states, such as Alzheimer's disease, by reducing the early deposition of amyloid ⁇ peptide (A ⁇ ) in the brain.
  • a ⁇ amyloid ⁇ peptide
  • amyloid ⁇ peptide derived by proteolytic processing of the amyloid precursor protein is thought to be a crucial event in the etiology of Alzheimer's disease.
  • a ⁇ amyloid ⁇ pept ⁇ de
  • a ⁇ _ 42 forms fibrils at elevated concentration that can aggregate and lead directly to neuronal death.
  • Both A ⁇ - 42 and its precursor APP have been considered as therapeutic targets against Alzheimer's disease. It is thought that preventing the disease-associated increase in A ⁇ . 2 either directly, or by reducing the concentration of APP, will slow or even prevent the onset of Alzheimer's disease. There have been recent antisense experiments in both mice and rats to.validate the concept that reducing the levels of APP in the brain can reverse deficits in learning and memory that are often found in patients suffering from Alzheimer's disease.
  • Antisense technology offers a precise and specific means of knocking down expression of a target gene and is a major focus for research in both neuroscience and in a number of other areas. Antisense technology is also emerging as a therapeutic technology of immense potential, with the antisense concept being both simple and elegant .
  • Gene expression relies on genomic DNA sequences being transcribed into messenger R As (mRNA) which in turn are themselves translated into proteins .
  • An antisense oligonucleotide (AS-ODN) is a short nucleic acid sequence or analogue of such sequence that is able to hybridise specifically to complementary sequences within its mRNA target. This prevents its translation by either or both of the two postulated mechanisms.
  • Any existing protein encoded by the target gene depletes as a function of its half-life, and thus a rapid and highly specific knockdown is achieved.
  • siRNA or small interfering RNA
  • silencing reagents can be taken to cover both antisense oligonucleotides and siRNAs .
  • Anti-Alzheimer drugs such as AriceptTM and CognexTM cause a net increase in the amount of acetylcholine available in the brain, and thus slow down the loss of cognitive ability.
  • Other strategies that have been put forward rely on antioxidant compounds to minimise free radical damage, for example Eldepryl, and Estrogen therapy may also confer protection.
  • silencing reagents that are able to bind to the accessible region of APP, for use in the modulation of APP, selected from the following list of antisense sequences of APP; CATTCTGGACATTCATGTGCATGTTCAGTCTGCCACAGAACATGG AACATGGCAATCTGGGGTTCAGCCAGCAGGCCA GTGGGGATGGGTCTTGCACTGCTTGCGGCCC GATCTGCAGTTCAGGGTAGACTTCTTGGCAA TTCCACCTCAGCCACTTCTTCCTCCTCTGCTACTTCTACTACTTT GACATCCGAGTCATCCTCCTCCGCATCAGCAGAAT TGCTCGGCACGGCCCCGTCTCGGCTTGTTCAGA GCAATGCTGGTGGTTCTCTCTCTGTGGCTTCTTCGTAGGGTTC CCTCATCACCATCCTCATCGTCCTCGTCATC CATGCAGTACTCTTCTGTGTC AAAGTTGTTCCGGTTGCCGCCACA GTTGCCGCCACATCCGCCGTA
  • silencing reagents are fragments of sequences from the abovementioned list.
  • silencing reagents show >90% homology to the antisense sequences from the abovementioned list.
  • antisense sequences are combined with sense strands to form siRNA.
  • the T bases are replaced with U bases.
  • silencing reagents that are able to bind to accessible parts of an APP gene, wherein the antisense reagents are modified by the incorporation of ethylene bridged nucleic acids (ENAs) .
  • the silencing reagents are antisense reagents or small interfering RNA.
  • the silencing reagents comprise a mixture of ethylene bridged nucleic acids (ENAs) , and unmodified nucleic acids, such that the unmodified nucleotides promote enzymatic cleavage of the RNA strand of a hetero duplex formed between the antisense reagent and a target RNA transcript by means of recruiting RNase H or a similar endogenous or ectopic enzyme.
  • ENAs ethylene bridged nucleic acids
  • silencing reagents comprise a mixture of ethylene bridged nucleic acids and any type of modified nucleotide that is able mediate cleavage of a target transcript by RNase H.
  • the antisense reagents are chosen from the following sequences: CATTCTGGACATTCATGTGCATGTTCAGTCTGCCACAGAACATGG AACATGGCAATCTGGGGTTCAGCCAGCAGGCCA GTGGGGATGGGTCTTGCACTGCTTGCGGCCC GATCTGCAGTTCAGGGTAGACTTCTTGGCAA TTCCACCTCAGCCACTTCTTCCTCCTCTGCTACTTCTACTACTTT GACATCCGAGTCATCCTCCTCCGCATCAGCAGAAT TGCTCGGCACGGCCCCGTCTCGGCTTGTTCAGA GCAATGCTGGTGGTTCTCTCTCTGTGGCTTCTTCGTAGGGTTC CCTCATCACCATCCTCATCGTCCTCGTCATC CATGCAGTACTCTTCTGTGTC AAAGTTGTTCCGGTTGCCGCCACA GTTGCCGCCACATCCGCCGTAAAA AAAGAATGGGGCACACTTCCCTTCAGTCACATCAAA CAAGTTCTTTGCTTGACGTTCTGCCTCTCTTCCCATTCTCTCT ATAAA
  • the siRNA are chosen from the following antisense strands combined with corresponding sense strands; ACAGGUGGCGCUCCUCUGG UCUGGGGUGACAGCGGCGU CAAACAUCCAUCCUCUCCU CCUCUCCUGGUGUAAGAAU GUUCCGGUUGCCGCCACAU UGCCGCCACAUCCGCCGUA UCCUUCUGUUCUGCGCGGA CGCGGACAUACUUCUUUAG GACGUUCUGCCUCUUCCCA CCUCUUCCCAUUCUCUCAU GGAUCCACCAGCGCACAUG UCGGAACUUGUCAAUUCCG GAUGGGUAGUGAAGCAAUG CAUGAUUGUGAUCGUGGUGUGAUCGUGGUGUGAUCGUGGUGUGAUCGUGGUGCAGUGGUGCAGUGGUG
  • the siRNA are prepared such that the sense and antisense strands are formed with an overhang.
  • the antisense sequences are flanked by between one and ten modified synthetic nucleotides that may themselves form part of the antisense sequence, or may not .
  • the modified synthetic nucleotides are ethylene bridged nucleic acids .
  • Figures 1 to 5 show the results of a series of structure mapping experiments to determine accessible regions on the APP gene
  • Figure 6 shows the relative level of knockdown of the human APP gene transcript achieved by the indicated antisense oligonucleotide
  • Figure 7 shows the comparison between selected antisense reagents comprising either ENA, LNA or phosphorothioate chemistries
  • Figure 8 shows the structure of A p ;
  • Figure 9 shows the structure of G p ;
  • Figure 10 shows the structure of C p ;
  • FIG 11 shows the structure of T p ;
  • Figure 12 shows the structure of A e2t ;
  • Figure 13 shows the structure of A e2p ;
  • Figure 14 shows the structure of G e2t ;
  • Figure 15 shows the structure of G e2p ;
  • Figure 16 shows the structure of C e2t ;
  • Figure 17 shows the structure of C e2p ;
  • Figure 18 shows the structure of T e2t ;
  • Figure 19 shows the structure of ⁇ e2p ;
  • Figure 20 is a bar graph showing the results of experiments to determine the ability of siRNA sequences to knockdown APP mRNA
  • the present invention is based around using a method for empirically mapping the structure of the APP gene transcript using an ACCESSarray ® .
  • An ACCESSarray ® is a microarray based on a structure mapping system, as described in O02/072886.
  • the microarray may be fully degenerate or may be molecule specific.
  • RNA structures are mapped empirically as opposed to by algorithm, which confers both time and cost benefits.
  • a human, or any other species, APP transcript can be reacted with the ACCESSarray ® and the interaction pattern can then be interpreted into an access map of the target transcript, which in this case is APP.
  • Figure 1 shows an example of an APP access map.
  • the antisense reagents or siRNAs can be designed based on this and, in the case of siRNA, other factors for choosing appropriate sequences can be taken into account as has been described in the art.
  • Antisense reagents designed to be complementary to accessible regions as determined by interpreting the ACCESSarray ® map will be effective modulators of gene expression, and one embodiment of the present invention features ACCESSarray ® facilitated design of antisense reagents effective at modulating the expression of APP.
  • Oligonucleotides containing novel 2'-0, 4' -C-methylene nucleosides (LNA) whose sugar puckering is fixed in the N conformation have a higher level of affinity towards their complementary RNA and other modified oligonucleotides.
  • LNA 2'-0, 4'-C-methylene nucleosides
  • ENAs novel 2'-0, 4'-C- ethylene nucleosides
  • oligonucleotides containing these ENA residues exhibit equivalent binding affinity to the corresponding oligonucleotides that contain LNA residues. However, they also exhibit much greater nuclease resistance than the corresponding oligonucleotides that contained LNA residues.
  • ENAs are incorporated into the antisense reagents in order to provide improved binding affinity, along with greater nuclease resistance.
  • the invention features the ACCESSarray ® -facilitated design of small interfering RNA (siRNA) that are effective at modulating expression of APP.
  • siRNA small interfering RNA
  • a molecule specific array comprises overlapping oligonucleotide elements each complementary to the APP transcript .
  • Each oligonucleotide on the array has between 12 and 15 bases of complementarity to APP and each overlaps the next by 2 nucleotides .
  • Each molecule specific array has a set of oligonucleotide probes that cover either a portion of the full APP transcript or the full APP transcript. The position of each oligonucleotide on the array is known.
  • siRNA An example relating to siRNA is that an APP cDNA clone was transcribed in vitro and was applied to a fully degenerate array.
  • a fully degenerate array of this type comprises 4096 elements, the final six nucleotides on each element being one of every combination of bases for a 6-base sequence. Thus every possible 6-base sequence is represented on the array. The specific individual 6- base sequence at each of the 4096 positions on the array is known.
  • ACCESSmapperTM interprets the array and builds up an access map by interpreting each element with reference to the known target. It is possible to determine which of the elements on the array are binding to the APP. Antisense reagents can then be built up on the basis of the access map that is generated for the target.
  • Antisense reagents may be chemically synthesised nucleic acids modified by the incorporation of ethylene bridged nucleic acids (ENA) .
  • Antisense reagents may also comprise a mixture of ENA and unmodified nucleotides such that the unmodified nucleotides promote cleavage of the RNA strand of a heteroduplex formed between the antisense reagent and the target RNA transcript .
  • Antisense reagents may also comprise a mixture of ENA and any other modified nucleotide that is able to mediate cleavage of a target transcript by RNase H.
  • Antisense reagents comprising an ENA component confer advantages over the prior art because they confer very high affinity interactions between the antisense reagent and the target transcript with minimal toxicity. These properties confer therapeutic advantage over the prior art .
  • a combination of ENA based antisense reagents comprising a mixture of several individual oligonucleotides directed against different accessible regions on APP target transcript is also envisaged here. Mixtures of oligonucleotides may interact with multiple accessible regions on the same transcript or may interact with the same or different accessible regions on multiple transcripts.
  • Example 1 APP1- 666 ; HO-G e p -T e2p -G e2p -C e2p -A e2p -T e2p -G p -T p -T p -C p -A p -G p -T e2p - Synthesis of an oligonucleotide derivative was carried out using a DNA/RNA synthesiser (ABI model 394: a product of Perkin-Elmer Corporation) on 1.0 ⁇ mole program. The solvents, reagents and concentrations of phosphoramidite in every synthetic cycle are the same as those in the synthesis of natural oligonucleotides.
  • Solvents, reagents and phosphoramidites of the natural type nucleosides are products of PE Biosystems Corporation. Every modified oligonucleotide derivative sequence was synthesised by condensation of the ENA phosphoramidites obtained in Examples 9, 14, 22 and 27 described in O0107455. Universal-Q 500 (1.2 ⁇ mol, Glen Research) was used as the CPG.
  • the synthetic cycle is as follows : 1) detritylation trichloroacetic acid/dichloromethane; 35sec 2) coupling phosphoramidite (about 20eq) , 1H- tetrazole/acetonitrile; 25sec or 15min 3) capping 1-methylimidazole/tetrahydrofuran, acetic anhydride/pyridine/tetrahydrofuran; 15sec 4) oxidation iodine/water/pyridine/tetrahydrofuran; 15sec
  • the carrier containing the desired product was conventionally treated with concentrated aqueous ammonia solution in order to detach the oligomer from the carrier and to deprotect the cyanoethyl group that is protecting the phosphate.
  • the amino protecting groups in adenine, guanine and cytosine were also removed from the oligomer.
  • the crude oligonucleotide was purified by reverse-phase HPLC (HPLC: LC-VP: a product of Shimazu Corp.; column : Wakopak WS-DNA (10 x 250 mm): a product of ako Pure Chemical Industry Ltd.; solvent A: 5% acetonitrile, 0.1 M triethylammonium acetate (TEAA, pH 7.0); solvent B: acetonitrile; B% : 10%- 50% (linear gradient, 10 min) , 50% (10 min) , and then 50%-70% (linear gradient, 5 min), temperature : 60°C ; flow rate: 2 mL/min; detection: 254 nm) .
  • HPLC LC-VP: a product of Shimazu Corp.
  • column Wakopak WS-DNA (10 x 250 mm): a product of ako Pure Chemical Industry Ltd.
  • solvent A 5% acetonitrile, 0.1 M triethylammonium acetate (TE
  • the collected fraction was co-evaporated with H 2 0 to remove TEAA.
  • the residues were treated with 80% acetic acid for 20 min to detach dimethoxytrityl group from the oligonucleotide, and then the mixture was concentrated in vacuo .
  • the oligonucleotide was purified by reverse-phase HPLC (HPLC: LC-VP: a product of Shimazu Corp.; column : Wakopak WS- DNA (10 x 250 mm) : a product of Wako Pure Chemical Industry Ltd.; solvent A: 5% acetonitrile, 0.1 M TEAA (pH 7.0); solvent B: acetonitrile; B% : 0%-10% (linear gradient, 15 min), 10% (5 min), and then 10%-15% (linear gradient, 5 min), temperature: 60°C ; flow rate: 2 mL/min; detection: 254 nm) .
  • the oligonucleotide, APP1-666 was obtained (14.7 A 26 o units) .
  • This sequences is complementary to the nucleotide number 262-279 of APP cDNA (GenBank accession No. NM_000484) .
  • Negative ion ESI mass spectroscopy calcd: 6042.15, found: 6042.98.
  • ENA oligonucleotide, APP2-666, was synthesised according to the similar method of Example 1, APP1-666. According to this synthetic method, the oligonucleotide, APP2-666, was obtained (32.3 A 260 units). This sequences is complementary to the nucleotide number 266-283 of APP cDNA (GenBank accession No. NM_000484) .
  • Negative ion ESI mass spectroscopy calcd: 6027.14, found: 6026.94.
  • ENA oligonucleotide, APP3/4-666 was synthesised according to the similar method of Example 1, APPl-666. According to this synthetic method, the oligonucleotide, APP3/4-666, was obtained (19.1 A 260 units). This sequences is complementary to the nucleotide number 451-468 of APP cDNA (GenBank accession No. NM_000484) .
  • Negative ion ESI mass spectroscopy calcd: 6044.13, found: 6044.04.
  • ENA oligonucleotide, APPll-666 was synthesised according to the similar method of Example 1, APPl-666. According to this synthetic method, the oligonucleotide, APPll-666, was obtained (34.9 A 26o units). This sequences is complementary to the nucleotide number 1105-1122 of APP cDNA (GenBank accession No. NM_000484) .
  • Negative ion ESI mass spectroscopy calcd: 5984.13, found: 5984.43.
  • ENA oligonucleotide, APP13-666 was synthesised according to the similar method of Example 1, APPl-666. According to this synthetic method, the oligonucleotide, APP13-666, was obtained (21.8 A 26 o units). This sequences is complementary to the nucleotide number 1078-1094 of APP cDNA (GenBank accession No. NM_000484) .
  • Negative ion ESI mass spectroscopy calcd: 5915.09, found: 5915.00.
  • Control APPAS2-666 HO-C e2p -C e2p -A e2p -G e2p -T e2p -G e2p -A p -A p -G p -A p -
  • ENA oligonucleotide, Control APPAS2-666, was synthesised according to the similar method of Example 1, APPl-666. According to this synthetic method, the oligonucleotide, Control APPAS2-666, was obtained (22.9 A 26 o units). This sequences is complementary to the nucleotide number 580- 597 of APP cDNA (GenBank accession No. NM_000484) .
  • Negative ion ESI mass spectroscopy calcd: 6085.19, found: 6085.09.
  • Example 7 Scrambled APPAS2-666: HO-A e2 -T e2p -G e2p -A e2p -T e2p -T e2p -G p -T p -G p - A p -T p -G p -C e2p -T e2p -C e2p -C e2p -T e2p -C e2t -H
  • ENA oligonucleotide, Scrambled APPAS2-666, was synthesised according to the similar method of Example 1, APPl-666. According to this synthetic method, the oligonucleotide, Scrambled APPAS2-666, was obtained (42.5 A 260 units) .
  • Negative ion ESI mass spectroscopy calcd: 6041.17, found: 6041.18.
  • ENA oligonucleotide, APPl-585 was synthesised according to the similar method of Example 1, APPl-666. According to this synthetic method, the oligonucleotide, APPl-585, was obtained (30.2 A 260 units). This sequences is complementary to the nucleotide number 262-279 of APP cDNA (GenBank accession No. NM_000484) .
  • ENA oligonucleotide, APP2-585, was synthesised according to the similar method of Example 1, APPl-666. According to this synthetic method, the oligonucleotide, APP2-585, was obtained (69.5 A 26 o units). This sequences is complementary to the nucleotide number 266-283 of APP cDNA (GenBank accession No. NM_000484) .
  • Negative ion ESI mass spectroscopy calcd: 5943.06, found: 5942.93.
  • ENA oligonucleotide, APP3/4-585 was synthesised according to the similar method of Example 1, APPl-666. According to this synthetic method, the oligonucleotide, APP3/4-585, was obtained (63.8 A 2S0 units). This sequences is complementary to the nucleotide number 451-468 of APP cDNA (GenBank accession No. NM_000484) .
  • Retention time 8.71 min (reverse-phase HPLC: LC-VP: a product of Shimazu Corp.; column : Chromolith Performance RP-18e (4.6 x 100 mm, Merck); solvent A: 5% acetonitrile, 0.1 M TEAA (pH 7.0); solvent B: acetonitrile; B% : 0%-10% (linear gradient, 10 min); temperature: 60°C ; flow rate: 2 mL/min; detection: 254 nm) .
  • Negative ion ESI mass spectroscopy calcd: 5946.02, found: 5945.92.
  • Example 11 APP5-585; HO-G e2p -T e p -T e2p -G e2p -G e2p -T p -A p -C p -T p -C p -T p -T p -C p -j,e2p_ ⁇ e2p_js L e2p_ ⁇ e2 _ ⁇ j,e2t_jj
  • ENA oligonucleotide, APP5-585 was synthesised according to the similar method of Example 1, APPl-666. According to this synthetic method, the oligonucleotide, APP5-585, was obtained (39.8 A 260 units). This sequences is complementary to the nucleotide number 622-639 of APP cDNA (GenBank accession No. NM_000484) .
  • Negative ion ESI mass spectroscopy calcd: 5880.00, found: 5879.59.
  • Example 12 APP6-585 ; HO-G e2p -C e2p -A e2p -A e2p -G e2p -T p -T p -G p -G p -T p -A p -C p -T p - C e p -T e p -T e2p -C o2p -T e '"-H ENA oligonucleotide, APP6-585, was synthesised according to the similar method of Example 1, APPl-666.
  • the oligonucleotide, APP6-585 was obtained (27.6 A 2 6o units). This sequences is complementary to the nucleotide number 626-643 of APP cDNA (GenBank accession No. NM_000484) .
  • Negative ion ESI mass spectroscopy calcd: 5943.06, found: 5943.13.
  • Example 13 APP7-585: HO-C e2p -A e2p -C e2p -T e2p -T e2p -C p -T p -T p -C p -C p -T p -C p -C p - rr e2p -C e2p -T e2p -G e2p -C e2t -H
  • ENA oligonucleotide, APP7-585 was synthesised according to the similar method of Example 1, APPl-666. According to this synthetic method, the oligonucleotide, APP7-585, was obtained (43.9 A 26 o units). This sequences is complementary to the nucleotide number 832-849 of APP cDNA (GenBank accession No. NM_000484) .
  • Negative ion ESI mass spectroscopy calcd: 5788.97, found: 5788.80.
  • Example 14 APP8-585; HO-C e2p -A e2p -G e2p -C e2p -C e2p -A p -C p -T p -T p -C p -T p -T p -C p -j e2 P _ e2 _ pe2 _ e2p _ ⁇ 2t _ ⁇
  • ENA oligonucleotide, APP8-585 was synthesised according to the similar method of Example 1, APPl-666. According to this synthetic method, the oligonucleotide, APP8-585, was obtained (56.6 A 260 units). This sequences is complementary to the nucleotide number 836-853 of APP cDNA (GenBank accession No. NM_000484) .
  • Negative ion ESI mass spectroscopy calcd: 5826.04, found: 5825.96.
  • Example 15 APP11-585; HO-G e2p -C e2p -C e2p -A e2p -C e2p -A p -T p -C p -C p -G p -C p -C p -G p - ⁇ e2p -A e2p -A e2p -A e2p -A e2t -H
  • ENA oligonucleotide, APP11-585 was synthesised according to the similar method of Example 1, APPl-666.
  • the oligonucleotide, APP11-585 was obtained (48.9 A 260 units). This sequences is complementary to the nucleotide number 1105-1122 of APP cDNA (GenBank accession No. NM_000484) .
  • Negative ion ESI mass spectroscopy calcd: 5900.06, found: 5899.95.
  • Example 16 APP12-585; HO-G e2p -T e2p -T e2p -G e2p -C e2p -C p -G p -C p -C p -A p -C p -A p -T p - C e2p -C e2p -G e2p -C e2p -C e2t -H
  • ENA oligonucleotide, APP12-585 was synthesised according to the similar method of Example 1, APPl-666. According to this synthetic method, the oligonucleotide, APP12-585, was obtained (49.4 A 260 units). This sequences is complementary to the nucleotide number 1111-1128 of APP cDNA (GenBank accession No. NM_000484) .
  • Negative ion ESI mass spectroscopy calcd: 5887.05, found: 5886.51.
  • ENA oligonucleotide, APP13-585 was synthesised according to the similar method of Example 1, APPl-666. According to this synthetic method, the oligonucleotide, APP13-585, was obtained (32.4 A 26 o units). This sequences is complementary to the nucleotide number 1078-1094 of APP cDNA (GenBank accession No. NM_000484) .
  • Negative ion ESI mass spectroscopy calcd: 5831.02, found: 5830.69.
  • ENA oligonucleotide, APP14-585 was synthesised according to the similar method of Example 1, APPl-666. According to this synthetic method, the oligonucleotide, APP14-585, was obtained (34.1 A 26 o units). This sequences is complementary to the nucleotide number 1081-1098 of APP cDNA (GenBank accession No. NM_000484) .
  • Example 19 APP15-585; HO-C e2p -T o2p -C e2p -T e2p -T e2p -C p -T p -G p -T p -G p -T p -C p -A p - ⁇ e2 P_a ⁇ 2 P_(3 e2 P_ ⁇ e2 P_'P e2t _t
  • ENA oligonucleotide, APP15-585 was synthesised according to the similar method of Example 1, APPl-666. According to this synthetic method, the oligonucleotide, APP15-585, was obtained (35.7 A 26 o units). This sequences is complementary to the nucleotide number 1135-1152 of APP cDNA (GenBank accession No. NM_000484) .
  • Negative ion ESI mass spectroscopy calcd: 5904.02, found: 5904.28.
  • Control APPAS2-585 HO-C e2p -C e2p -A e2p -G e2p -T e2p -G p -A p -A p -G p -A p -
  • ENA oligonucleotide, Control APPAS2-585 was synthesised according to the similar method of Example 1, APPl-666. According to this synthetic method, the oligonucleotide, Control APPAS2-585, was obtained (39.4 A 260 units). This sequences is complementary to the nucleotide number 580- 597 of APP cDNA (GenBank accession No. NM_000484) .
  • Negative ion ESI mass spectroscopy calcd: 6001.12, found: 6000.81.
  • ENA oligonucleotide, Scrambled APPl-585 was synthesised according to the similar method of Example 1, APPl-666. According to this synthetic method, the oligonucleotide, Scrambled APPl-585, was obtained (20.2 A 26 o units).
  • Negative ion ESI mass spectroscopy calcd: 5858.08, found: 5858.61.
  • Example 22 Scrambled APP2-585; HO-A e2p -T e p -G e2p -A e2p -T e2p -T p -G p -T p -G p -A p - T p - G p - C p - T e2p - C e2p - C o2p - T e2p - C e21 - H
  • ENA oligonucleotide, Scrambled APP2-585 was synthesised according to the similar method of Example 1, APPl-666. According to this synthetic method, the oligonucleotide, Scrambled APP2-585, was obtained (36.7 A 26 o units).
  • silencing reagents There are a large number of delivery mechanisms that can be used depending on how the silencing reagents are intended to be used. For example, the following methods are useful when using reagents as a research tool into cells in culture Transfection by liposome Transfection by polymer By scrape-loading By electroportation By expression cassette By conjugant-mediatated transport e.g. penetratin ® (given here only as an example) .
  • the silencing reagent when being used as a therapeutic into animals or humans the silencing reagent must be delivered across the blood brain barrier either by trans-BBB transport or by direct injection.
  • conjugated oligonucleotides to facilitate transport is envisaged.
  • the following methods can be used: - Use of osmotic pumps, mechanical pumps, electric pumps direct into brain - Use of liposomes or polymers to facilitate transport either or both across the blood brain barrier or across the plasma membrane - Use of conjugants that target the antisense reagent to specific intracellular compartments or to specific tissues within the body of the organism to which the compounds are administered.
  • siRNA sequences that 3 are suitable. Again, it is anticipated that siRNA 4 reagents incorporating all or part of the sequences would 5 also be appropriate. 6 7 Table 2 8 siRNA Sense Sequence of Antisense sequence of % remaining (start siRNA (all synthesised siRNA (in siRNA U is APP position) and annealed as used in place of T) expression double-stranded RNA adj for 90% with TT overhangs on transfection each end) efficiency 2291P CCAGAGGAGCGCCACCTGT ACAGGUGGCGCUCCUCUGG 64.4477 2277D ACGCCGCTGTCACCCCAGA UCUGGGGUGACAGCGGCGU 43.5368 531P AGGAGAGGATGGATGTTTG CAAACAUCCAUCCUCUCCU 52.0878 520D ATTCTTACACCAGGAGAGG CCUCUCCUGGUGUAAGAAU 5.39431 1087P ATGTGGCGGCAACCGGAAC GUUCCGGUUGCCGCCACAU 21
  • Oligonucleotides can be transfected into HeLa cells by any available method including but not limited to liposome-mediated (e.g. lipofectamine ® , Oligofectamine ® from Life-Technologies and similar reagents from various commercial sources) or polymer-mediated (e.g. PEI, EPEI from various suppliers) or any other transfection method known to the art.
  • liposome-mediated e.g. lipofectamine ® , Oligofectamine ® from Life-Technologies and similar reagents from various commercial sources
  • polymer-mediated e.g. PEI, EPEI from various suppliers
  • Antisense oligonucleotides are either Phosphorothioate, or comprise flanks of ENA of between 1 and 10 modified synthetic nucleotides at either end of a central "gap" or “window” of un-modified nucleotide or of a nucleotide chemistry that supports RNase H activity.
  • ENA gapmer conformations are: 5 ' -EEEEEENNNNNNEEEEEE-3' 5'-EEEEENNNNNNNNEEEEE-3' 5 ' -EEEENNNNNNNNEEEEE-3' 5 ' -EEEENNNNNNNNNNEEEE-3 ' 5'-EEENNNNNNNNNNEEE-3' Where E is an ENA and N is an unmodified nucleotide or other nucleotide that can support RNase H activity. Total oligo length is normally between 15 and 25 bases in length but it is anticipated that this may vary.
  • RNA is harvested from cells by any method known to the art and is quantitated by fluorescence-linked quantitative polymerase chain reaction (QPCR) standardised against a similar QPCR reaction against a house-keeping gene such as HPRT or GAP-DH.
  • QPCR fluorescence-linked quantitative polymerase chain reaction
  • This disclosure describes for the first time the use of ENA and ENA/DNA antisense reagents against the amyloid precursor protein APP and for the first time the use of siRNA reagents against APP. For the first time it uses a fully-degenerate microarray-based tool to map accessible regions on a transcript and design silencing reagents effective against them. Antisense sequences revealed by this method are not anticipated in the prior art .
  • This invention has commercial application as a research tool and has potential in the development of a therapeutic agent against Alzheimer's disease.
  • Figures 1,2,3,4 and 5 show the result of a series of structure mapping experiments to determine accessible regions on the APP gene.
  • Figure 6 shows the relative level of knockdown of the human APP gene transcript achieved by the indicated antisense oligonucleotide.
  • Oligonucleotides in these examples are either ENA gapmers with a generic composition of (ENA) 5 - (DNA) 8 - (ENA) 5 indicated as ENA585, or (ENA) 6 - (DNA) 6 - (ENA) 6 , indicated as ENA666, or complete phosphorothioate oligonucleotides.
  • Figure 7 Shows a comparison between selected antisense reagents comprising either ENA, LNA or phosphorothioate chemistries and indicated that ENA based gapmers are the superior chemistry for antisense reagent design.
  • siRNA sequences have also been tested to determine their ability to knockdown APP mRNA.
  • the siRNAs were made up from 2X19 base strands of RNA, each with 2 DNA bases on the 3' end (both Ts) .
  • the 19 RNA bases of the sense and antisense strands were annealed together by complementary base-pairing such that the TT on each strand acts as a 2 base overhang. This is one of a number of ways to design an siRNA - but they all contain a core double stranded section with overhangs.
  • Figure 20 shows the results of these experiments (with the oligonucleotides corresponding to those in table 2) .

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Abstract

Cette invention porte sur des stratégies visant à lutter contre le dépôt de protéine précurseur amyloïde (APP) qui est associé à l'étiologie précoce d'états pathologiques, tels que la maladie d'Alzheimer, au moyen de la réduction du dépôt précoce de peptide amyloïde β (Aβ) dans le cerveau. Les stratégies peuvent aussi être utilisées comme outils de recherche. Cette invention fait appel à des réactifs de silençage se présentant sous la forme de molécules antisens spécifiques ou d'ARNsi pour moduler la protéine précurseur amyloïde (APP).
PCT/GB2004/004483 2003-10-24 2004-10-22 Antisens anti-app/ena WO2005042777A2 (fr)

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WO2013019954A1 (fr) 2011-08-04 2013-02-07 Amgen Inc. Procédé de traitement de défauts d'espace osseux
WO2013040429A1 (fr) 2011-09-14 2013-03-21 Rana Therapeutics Inc. Composés oligonucléotidiques multimères
WO2013101451A1 (fr) 2011-12-28 2013-07-04 Amgen Inc. Méthode de traitement d'une perte osseuse alvéolaire au moyen d'anticorps anti-sclérostine
WO2013173638A1 (fr) 2012-05-16 2013-11-21 Rana Therapeutics, Inc. Compositions et procédés de modulation de l'expression de la famille génique smn
WO2013184209A1 (fr) 2012-06-04 2013-12-12 Ludwig Institute For Cancer Research Ltd. Mif destiné à être utilisé dans des méthodes de traitement de sujets atteints d'une maladie neurodégénérative
US8809289B2 (en) 2008-03-11 2014-08-19 Saitama Medical University Double-stranded nucleic acid molecule, cancer cell proliferation inhibitor and pharmaceutical agent suitable for prevention or treatment of cancer
WO2015200697A1 (fr) 2014-06-25 2015-12-30 The General Hospital Corporation Ciblage de hsatii (human satellite ii)
EP3260540A1 (fr) 2010-11-12 2017-12-27 The General Hospital Corporation Arn non codants associés à polycomb
EP3511416A1 (fr) 2012-05-16 2019-07-17 Translate Bio MA, Inc. Composés et procédés pour moduler l'expression génique
WO2020132227A3 (fr) * 2018-12-19 2020-07-30 Alnylam Pharmaceuticals, Inc. Compositions d'agent d'arni de précurseur de la protéine amyloïde (app) et leurs méthodes d'utilisation
US11253601B2 (en) 2016-07-11 2022-02-22 Translate Bio Ma, Inc. Nucleic acid conjugates and uses thereof
US11732260B2 (en) 2018-03-02 2023-08-22 Ionis Pharmaceuticals, Inc. Compounds and methods for the modulation of amyloid-β precursor protein
EP3918073A4 (fr) * 2019-01-29 2023-11-22 Ionis Pharmaceuticals, Inc. Composés et méthodes permettant de réduire l'expression de l'app
WO2024026474A1 (fr) 2022-07-29 2024-02-01 Regeneron Pharmaceuticals, Inc. Compositions et méthodes d'administration médiée par le récepteur de la transferrine (tfr) au cerveau et au muscle
WO2024098002A1 (fr) 2022-11-04 2024-05-10 Regeneron Pharmaceuticals, Inc. Protéines de liaison de sous-unité auxiliaire gamma 1 du canal calcique dépendant de la tension (cacng1) et administration médiée par cacng1 au muscle squelettique
WO2024107765A2 (fr) 2022-11-14 2024-05-23 Regeneron Pharmaceuticals, Inc. Compositions et procédés d'administration médiée par le récepteur 3 du facteur de croissance des fibroblastes à des astrocytes

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US8809289B2 (en) 2008-03-11 2014-08-19 Saitama Medical University Double-stranded nucleic acid molecule, cancer cell proliferation inhibitor and pharmaceutical agent suitable for prevention or treatment of cancer
JP5683261B2 (ja) * 2008-03-11 2015-03-11 学校法人 埼玉医科大学 癌の予防乃至治療に好適な二本鎖核酸分子、癌細胞増殖抑制剤、並びに医薬
US9284557B2 (en) 2008-03-11 2016-03-15 Saitama Medical University Double-stranded nucleic acid molecule, cancer cell proliferation inhibitor and pharmaceutical agent suitable for prevention or treatment of cancer
EP3260540A1 (fr) 2010-11-12 2017-12-27 The General Hospital Corporation Arn non codants associés à polycomb
WO2013019954A1 (fr) 2011-08-04 2013-02-07 Amgen Inc. Procédé de traitement de défauts d'espace osseux
WO2013040429A1 (fr) 2011-09-14 2013-03-21 Rana Therapeutics Inc. Composés oligonucléotidiques multimères
EP3533873A1 (fr) 2011-09-14 2019-09-04 Translate Bio MA, Inc. Composés d'oligonucléotides multimères
WO2013101451A1 (fr) 2011-12-28 2013-07-04 Amgen Inc. Méthode de traitement d'une perte osseuse alvéolaire au moyen d'anticorps anti-sclérostine
EP3712168A2 (fr) 2011-12-28 2020-09-23 Amgen Inc. Procédé de traitement d'une perte osseuse alvéolaire au moyen d'anticorps anti-sclérostine
EP3511416A1 (fr) 2012-05-16 2019-07-17 Translate Bio MA, Inc. Composés et procédés pour moduler l'expression génique
WO2013173638A1 (fr) 2012-05-16 2013-11-21 Rana Therapeutics, Inc. Compositions et procédés de modulation de l'expression de la famille génique smn
WO2013184209A1 (fr) 2012-06-04 2013-12-12 Ludwig Institute For Cancer Research Ltd. Mif destiné à être utilisé dans des méthodes de traitement de sujets atteints d'une maladie neurodégénérative
WO2015200697A1 (fr) 2014-06-25 2015-12-30 The General Hospital Corporation Ciblage de hsatii (human satellite ii)
EP3760208A1 (fr) 2014-06-25 2021-01-06 The General Hospital Corporation Ciblage de hsatii (human satellite ii)
US11253601B2 (en) 2016-07-11 2022-02-22 Translate Bio Ma, Inc. Nucleic acid conjugates and uses thereof
US11732260B2 (en) 2018-03-02 2023-08-22 Ionis Pharmaceuticals, Inc. Compounds and methods for the modulation of amyloid-β precursor protein
WO2020132227A3 (fr) * 2018-12-19 2020-07-30 Alnylam Pharmaceuticals, Inc. Compositions d'agent d'arni de précurseur de la protéine amyloïde (app) et leurs méthodes d'utilisation
US11034957B2 (en) 2018-12-19 2021-06-15 Alnylam Pharmaceuticals, Inc. Amyloid precursor protein (APP) RNAi agent compositions and methods of use thereof
EP3918073A4 (fr) * 2019-01-29 2023-11-22 Ionis Pharmaceuticals, Inc. Composés et méthodes permettant de réduire l'expression de l'app
WO2024026474A1 (fr) 2022-07-29 2024-02-01 Regeneron Pharmaceuticals, Inc. Compositions et méthodes d'administration médiée par le récepteur de la transferrine (tfr) au cerveau et au muscle
WO2024098002A1 (fr) 2022-11-04 2024-05-10 Regeneron Pharmaceuticals, Inc. Protéines de liaison de sous-unité auxiliaire gamma 1 du canal calcique dépendant de la tension (cacng1) et administration médiée par cacng1 au muscle squelettique
WO2024107765A2 (fr) 2022-11-14 2024-05-23 Regeneron Pharmaceuticals, Inc. Compositions et procédés d'administration médiée par le récepteur 3 du facteur de croissance des fibroblastes à des astrocytes

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