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WO2012131365A1 - Molécules thérapeutiques destinées à être utilisées dans la suppression de la maladie de parkinson - Google Patents

Molécules thérapeutiques destinées à être utilisées dans la suppression de la maladie de parkinson Download PDF

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Publication number
WO2012131365A1
WO2012131365A1 PCT/GB2012/050692 GB2012050692W WO2012131365A1 WO 2012131365 A1 WO2012131365 A1 WO 2012131365A1 GB 2012050692 W GB2012050692 W GB 2012050692W WO 2012131365 A1 WO2012131365 A1 WO 2012131365A1
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nucleotide
nucleic acid
rna
antisense strand
seq
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PCT/GB2012/050692
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English (en)
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Christopher Sibley
Matthew Wood
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Isis Innovation Limited
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N15/1137—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 against enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/11—Protein-serine/threonine kinases (2.7.11)
    • C12Y207/11001—Non-specific serine/threonine protein kinase (2.7.11.1), i.e. casein kinase or checkpoint kinase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/34—Spatial arrangement of the modifications
    • C12N2310/344—Position-specific modifications, e.g. on every purine, at the 3'-end
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/50—Physical structure
    • C12N2310/53—Physical structure partially self-complementary or closed
    • C12N2310/531—Stem-loop; Hairpin

Definitions

  • the present invention relates to therapeutic molecules and the use of such therapeutic molecules in the suppression or prevention of Parkinson's disease.
  • Parkinson's disease is a progressive neurological condition that is associated with the loss of dopamine-producing neurons in the substantia nigra area of the brain.
  • PD affects approximately 120,000 people in the United Kingdom, with an average age of onset of 60 years.
  • Clinical symptoms of PD include bradykinesia (slowness of movement), tremor, muscle rigidity and postural instability. Due to the progressive nature of the disease, the symptoms may worsen over time and have a significant impact on the patient's quality of life. In addition, cognitive impairment may develop during the later stages of the disease. The need for high levels of support and care for PD patients can also use significant resources of health care systems.
  • dsNA double stranded nucleic acid
  • the invention provides a double stranded nucleic acid (dsNA) molecule comprising a nucleic acid sense strand and an RNA antisense strand; wherein the RNA antisense strand binds to position 6176 on a target RNA nucleotide sequence that comprises the nucleotide sequence of SEQ ID NO: 10; wherein at least a portion of the RNA antisense strand and the nucleic acid sense strand together define a base-paired nucleic acid duplex having the structure of Formula (I):
  • nucleotides of the antisense RNA strand define consecutively numbered antisense nucleotide positions, said numbers increasing in a 5' to 3' direction on the antisense strand, with position 1 (p1 ) defined as the extreme 5' nucleotide present on the RNA antisense strand of the nucleic acid duplex that is base paired with a corresponding nucleotide present on the nucleic acid sense strand of the nucleic acid duplex;
  • RNA antisense strand has a uracil nucleotide located at any one of positions p1 to p9 that binds to an adenine nucleotide located at position 6176 of an RNA nucleotide sequence comprising the nucleotide sequence of SEQ ID NO: 10.
  • hereditary PD While the precise causes of PD are yet to be fully elucidated, it is believed that certain cases of PD have a hereditary component.
  • Research into the genetics of hereditary PD has identified 16 chromosomal "PARK" loci with linkage to the disease. Subsequently, a group of ten genes have been identified which are implicated in molecular pathways leading to PD pathogenesis. In some cases, a mutant form of a gene is created by the presence of a single nucleotide polymorphism (SNP).
  • SNP single nucleotide polymorphism
  • the LRRK2-G2019S mutation is the most common PD-linked mutation at present and represents the most attractive PD-mutation for allele-specific silencing.
  • the mutation leads to a G:A conversion in the LRRK2 mRNA.
  • the mutant LRRK2 gene may be present in heterozygous form, such that an affected individual carries one mutant LRRK2 allele and one wildtype LRRK2 allele.
  • the nucleotide sequence of SEQ ID NO: 10 is an mRNA sequence encoding the product of a human LRRK2 gene that has a G2019S single nucleotide polymorphism (also referred to herein as mutant LRRK2 or LRRK2-G2019S).
  • the G2019S SNP causes the presence in the mutant LRRK2 mRNA (SEQ ID NO: 10) of an adenine nucleotide (mutant residue) at position 6176, whereas the wildtype LRRK2 mRNA sequence (SEQ ID NO: 1 1 ) has a guanine nucleotide at position 6176.
  • the dsNA molecule of the invention targets and/or binds to the LRRK2 gene, in particular to the LRRK2-G2019S mutant.
  • RNAi RNA interference
  • RNA interference has emerged as a highly credible strategy with which to sequence-specifically silence genes-of-interest by preventing the translation of targeted mRNA transcripts into proteins.
  • the endogenous RNAi pathway is now well characterised and involves the processing of non-coding RNA sequences with characteristic stem-loop secondary structures, termed primary-microRNAs (pri-miRNAs), into short 21 -23nt single-stranded mature miRNAs that are antisense to targeted transcripts - see Fig. 1 .
  • pri-miRNAs primary-microRNAs
  • Complete complementarity of the mature miRNA sequence to the target mRNA leads to target cleavage and inhibition of protein synthesis, whereas incomplete pairing leads to translational repression either through mRNA destabilization or removal of the 5' cap or 3' poly-A termination signal.
  • RNAi pathway precursors termed short hairpin RNAs (shRNAs) or primary-miRNA (pri-miRNA) mimics. These precursors are recognised and processed by the enzyme Dicer.
  • shRNAs short hairpin RNAs
  • pri-miRNA primary-miRNA
  • Dicer The resulting short lengths of double stranded RNA are termed short interfering RNAs (siRNA).
  • siRNA short interfering RNAs
  • Dicer In addition to cleaving RNA, Dicer also promotes incorporation of siRNA into the RNA-induced silencing complex (RISC).
  • RISC RNA-induced silencing complex
  • RNA strands termed the guide strand and the passenger strand.
  • the guide strand binds to an exonuclease enzyme present in the RISC complex termed Argonaute, while the passenger strand is degraded.
  • Argonaute an exonuclease enzyme present in the RISC complex
  • the strand with the most thermodynamically unstable 5' end is favoured.
  • Argonaute targets and cleaves mRNA molecules complementary to the guide strand.
  • the guide strand must contain the antisense sequence of the target mRNA.
  • the dsNA molecules of the present invention reduce the expression of the mutant LRRK2 allele in preference to reducing the expression of the wildtype LRRK2 allele. This provides an advantage in that the negative effects of the mutant LRRK2 allele are reduced, while necessary wildtype LRRK2 activity is retained.
  • the antisense strand of a dsNA molecule of the invention binds to a region of the mutant LRRK2 mRNA sequence that encodes the mutant residue defining the G2019S mutation.
  • the antisense strand is then capable of activating the RNAi mechanisms that will lead to degradation of the mutant LRRK2 mRNA and thus decrease mutant LRRK2 expression.
  • the antisense strand is complementary to, and binds to, a region of the mutant LRRK2 mRNA sequence that includes the mutant adenine (A) residue defining the G2019S mutation.
  • the wildtype LRRK2 mRNA sequence does not possess the mutant adenine (A) residue defining the G2019S SNP, and thus the antisense strand of the dsNA molecule of the invention is not complementary to the wildtype LRRK2 mRNA strand at the site of the G2019S polymorphism.
  • the wildtype possesses a guanine (G) residue at the same position.
  • the antisense strand binds more weakly to the wildtype LRRK2 mRNA sequence than it does to the mutant LRRK2 mRNA sequence - i.e.
  • the antisense strand has a greater affinity for the mutant LRRK2 mRNA sequence than for the wildtype LRRK2 sequence.
  • the antisense nucleotide located immediately 5' to position p1 (as defined above), to the extent that any such 5' nucleotide is present, is not base-paired with a corresponding nucleotide on the sense strand.
  • complementary refers to a nucleic acid molecule that forms hydrogen bonds with another nucleic acid molecule, or with itself, with Watson-Crick base pairing.
  • Watson-Crick base pairing refers to the following hydrogen bonded nucleotide pairings: A:T and C:G (for DNA); and A:U and C:G (for RNA).
  • two or more complementary nucleic acid molecule strands can have the same number of nucleotides (i.e. have the same length and form one double-stranded region, with or without an overhang) or have a different number of nucleotides (e.g. one strand may be shorter than but fully contained within another strand or one strand may overhang the other strand).
  • the double stranded nucleic acid (dsNA) molecule is any type of double stranded nucleic acid molecule that is able to mediate sequence- specific RNA interference against the target mutant LRRK2 gene.
  • the dsNA molecule may be a double stranded RNA, an siRNA, a short interfering nucleic acid, an shRNA, or a pri-miRNA.
  • One or both of the sense strand and antisense strand of the dsNA molecule may comprise additional nucleotides that do not form part of the double stranded duplex portion.
  • one or both of the sense strand and antisense strand may have a 3' and/ or a 5' overhang region.
  • the uracil nucleotide located at any one of positions p1 to p9 on the antisense strand is located at a position selected from p1 , p2, p3, p4, p5, p6, p7 p8 or p9.
  • the uracil nucleotide located at any one of positions p1 to p9 on the antisense strand is located at a position selected from p1 , p2, p3, p4, p5, p6 or p7.
  • the uracil nucleotide located at any one of positions p1 to p9 on the antisense strand is located at a position selected from p2, p3, p4, p5, or p6.
  • the uracil nucleotide located at any one of positions p1 to p9 on the antisense strand is located at a position selected from p3, p4, or p5.
  • the uracil nucleotide located at any one of positions p1 to p9 on the antisense strand is located at a position selected from p3 or p4, or from p4 or p5. In another embodiment, the uracil nucleotide located at any one of positions p1 to p9 on the antisense strand is located at position p4.
  • the antisense strand of the dsNA molecule comprises or consists of a nucleic acid sequence selected from any one of SEQ ID NOs: 1 -9, wherein the first nucleotide position of said SEQ ID NO occupies position p1 on the antisense strand.
  • the antisense strand of the dsNA molecule comprises or consists of SEQ ID NO: 4, wherein the first nucleotide position of said SEQ ID NO occupies position p1 on the antisense strand.
  • the underlined uracil nucleotide in said nucleic acid sequence binds to the adenine nucleotide located at position 6176 of a nucleotide sequence having the sequence of SEQ ID NO: 10.
  • the antisense strand binds to a mutant LRRK2 mRNA sequence at the site of the G2019S mutation.
  • the antisense strand of the dsNA molecule comprises a nucleic acid sequence selected from any of SEQ ID NOs: 1 -9, wherein the first nucleotide position of said SEQ ID NO occupies position p1 on the antisense strand, and wherein said antisense strand is up to 21 nucleotides in length (for example, 20 or 21 nucleotides).
  • the antisense strand of the dsNA molecule comprises a nucleic acid sequence selected from any of SEQ ID NOs: 1 -9, wherein the first nucleotide position of said SEQ ID NO occupies position p1 on the antisense strand, and wherein said antisense strand is up to 27 nucleotides in length (for example, 20, 21 , 22, 23, 24, 25, 26 or 27 nucleotides).
  • the antisense strand of the dsNA molecule comprises or consists of a nucleic acid sequence that differs from SEQ ID NO: 3 by a single nucleotide, wherein the first nucleotide position of said SEQ ID NO occupies position p1 on the antisense strand, and wherein said difference is that the final 3' uracil (U) nucleotide in said SEQ ID NO has been replaced with a cytosine (C) nucleotide.
  • the antisense strand of the dsNA molecule comprises or consists of a nucleic acid sequence that differs from SEQ ID NO: 4 by a single nucleotide, wherein the first nucleotide position of said SEQ ID NO occupies position p1 on the antisense strand, and wherein said difference is that the final 3' uracil (U) nucleotide in said SEQ ID NO has been replaced with a cytosine (C) nucleotide.
  • the antisense strand of the dsNA molecule comprises or consists of a nucleic acid sequence that differs (by at most 3, or 4 nucleotides) from a nucleotide sequence selected from any one of SEQ ID NOs: 1 -9, wherein the first nucleotide position of said SEQ ID NO occupies position p1 on the antisense strand, and with the proviso that said difference does not occur at the underlined uracil nucleotide identified in any of SEQ ID NOs: 1 -9.
  • the antisense strand of the dsNA molecule comprises or consists of a nucleic acid sequence that differs (by at most 1 , or 2 nucleotides) from a nucleotide sequence selected from any one of SEQ ID NOs: 1 -9, wherein the first nucleotide position of said SEQ ID NO occupies position p1 on the antisense strand, and with the proviso that said difference does not occur at the underlined uracil nucleotide identified in any of SEQ ID NOs: 1 -9.
  • the dsNA molecule is a dsNA molecule that is capable of being processed in the cytoplasm of a target cell by the enzyme Dicer.
  • Dicer is an endoribonuclease enzyme present in eukaryotic cells that catalyses the breakdown of double stranded RNA molecules (including pre-microRNA molecules and short hairpin RNA molecules) into short double stranded RNA fragments approximately 20-25 nucleotides in length.
  • the fragments produced by Dicer may be incorporated into the RNA-induced Silencing Complex (RISC).
  • RISC RNA-induced Silencing Complex
  • the dsNA molecule is a short hairpin RNA (shRNA).
  • shRNA has a length of from about 40 nucleotides to about 50 nucleotides (for example, 40, 41 , 42, 43, 43, 45, 46, 47, 48, 49 or 50 nucleotides). In one embodiment, the shRNA has a maximum length of 120 nucleotides.
  • the antisense strand and the sense strand comprise part of a single strand of ribonucleic acid that is folded upon itself to form a short hairpin RNA.
  • the shRNA molecule is typically transcribed as a single length of ribonucleic acid comprising self-complementary nucleotide sequences that enable the ribonucleic acid to fold upon itself to form the short hairpin RNA that is recognised by the RNAi pathway elements.
  • a short hairpin RNA of the invention is delivered into target cells using a nucleic acid vector as described in more detail below.
  • the dsNA molecule comprises or consists of a nucleic acid sequence selected from any one of SEQ ID NOs: 12-20.
  • the dsNA molecule is an shRNA comprising, or consisting of, a nucleic acid sequence that differs (by at most 1 , 2, 3, 4, or 5 nucleotides) from a nucleotide sequence selected from any one of SEQ ID NOs: 12-20.
  • the antisense strand and the sense strand comprise part of a single strand of ribonucleic acid that is folded upon itself to form a mimic of precursors of the miRNA pathway, referred to as pri-miRNAs or pre-miRNAs.
  • the pri-miRNA or pre-miRNA molecule is typically transcribed as a single length of ribonucleic acid comprising self-complementary nucleotide sequences that enable the ribonucleic acid to fold upon itself to form the pri-miRNA or pre-miRNA that is recognised by the RNAi pathway elements.
  • the dsNA molecule is a sequencing mimicking a primary microRNA (pri-miRNA) or a pre-miRNA.
  • the dsNA molecule is a small internally segmented interfering RNA (sisiRNA), an asymmetric interfering RNA (aiRNA), or a DNA- RNA chimeric interfering RNA.
  • miRNA small internally segmented interfering RNA
  • aiRNA asymmetric interfering RNA
  • DNA- RNA chimeric interfering RNA a DNA- RNA chimeric interfering RNA.
  • the dsNA molecule is an siRNA molecule. Such molecules are typically 21 -23 base pairs in length, and may include 3' and/or 5' overhangs (typically 1 -4, 1 -3 or 1 -2 base overhangs). siRNA molecules typically have unphosphorylated hydroxyl groups at the 2' and 3' positions.
  • the dsNA molecule is a dicer substrate siRNA (D-siRNA) molecule.
  • D-siRNA dicer substrate siRNA
  • the dsNA molecule is 25-27 base pairs in length (for example, 25, 26 or 27 base pairs).
  • the dsNA molecule that is a dicer substrate siRNA molecule includes 3' and/or 5' overhangs (typically 1 -4, 1 -3 or 1 -2 base overhangs).
  • D-siRNAs are recognised and processed by Dicer into siRNAs of 21 -23 base pairs and facilitate loading into the RNA induced silencing complex.
  • D-siRNA molecules typically have unphosphorylated hydroxyl groups at the 2' and 3' positions.
  • the dsNA molecule that is a dicer substrate siRNA molecule has unphosphorylated hydroxyl groups at the 2' and 3' positions
  • the antisense strand of a dsNA molecule of the present invention may further comprise one or more nucleotides, wherein, when the antisense strand binds to position 6176 on the target RNA nucleotide sequence (e.g. SEQ ID NO: 10), said one or more nucleotides forms mismatch base-pairing with the corresponding nucleotide(s) located on the target RNA nucleotide sequence.
  • the target RNA nucleotide sequence e.g. SEQ ID NO: 10
  • the antisense strand contains a nucleotide that does not form a standard Watson- Crick base pair when the antisense strand is bound to the target mutant LRRK2 mRNA sequence.
  • the present inventors have found that the deliberate incorporation of a nucleotide that introduces a mismatch base pairing with the target mutant LRRK2 mRNA sequence surprisingly improves the discrimination of dsNA molecules of the invention with regard to mutant (i.e. target) and corresponding wildtype LRRK2 mRNA.
  • mutant i.e. target
  • mutant i.e. target
  • mutant i.e. target
  • the antisense nucleotide that forms the mismatch pairing as described above may be any nucleotide capable of forming such a mismatch pairing.
  • the presence of a mismatch pairing disrupts the surrounding nucleic acid duplex formed between the antisense strand of a dsNA molecule of the invention and an LRRK2 mRNA molecule.
  • purine bases A and G
  • pyrimidine bases U and C
  • a purine:purine mismatch occupies more space than a standard C:G or A:U pairing and so disrupts the surrounding nucleic acid duplex to a greater extent than a pyrimidine:purine or pyrimidine:pyrimidine mismatch - see Fig. 4B & 5.
  • the extent of the disruption to the nucleic acid duplex surrounding the mismatch is believed to influence the ability of the antisense strand to direct cleavage (via RISC) of the LRRK2 mRNA sequence.
  • the presence (in the antisense strand) of a nucleotide that gives rise to a mismatch pairing occupying a large amount of space is understood to result in a maximum decrease in the ability of said antisense strand to direct cleavage of the LRRK2 mRNA sequence.
  • the present inventor believes that the presence of mismatch pairing decreases the affinity with which the antisense strand binds to both the mutant LRRK2 mRNA sequence and the wildtype LRRK2 mRNA sequence.
  • the antisense strand already has a first mismatch nucleotide for the wildtype LRRK2 mRNA sequence (i.e. the uracil (U) nucleotide that binds to the mutant G2019S SNP)
  • U uracil
  • the presence of a second mismatch nucleotide provides a much greater mismatch effect between the antisense strand and the wildtype LRRK2 mRNA sequence.
  • the effect of a second (or subsequent) mismatch on the affinity of the antisense strand for the wildtype LRRK2 sequence is more pronounced compared to LRRK2-G2019S.
  • a hierarchy of mismatch pairs showing the extent to which the formation of a given mismatch increases the ability of the antisense strand to discriminate between target LRRK2-G2019S and wildtype LRRK2 is shown in Figure 5.
  • the above-described mismatch is provided at a position adjacent to the uracil nucleotide on the antisense strand that binds to position 6176 on SEQ ID NO: 10.
  • the mismatch nucleotide is located immediately 5' and/or 3' to the uracil nucleotide (i.e. the uracil that binds to position 6176 on SEQ ID NO: 10).
  • the mismatch nucleotide may be located 2, 3, 4, 5 or 6 nucleotide positions 3' and/or 5' away from the uracil nucleotide (i.e. the uracil that binds to position 6176 on SEQ ID NO: 10).
  • the mismatch nucleotide may be located up to 18 nucleotide positions (for example, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, or 18 nucleotide positions) 3' and/or 5' away from the uracil nucleotide (i.e. the uracil that binds to position 6176 on SEQ ID NO: 10).
  • the antisense strand of a dsNA molecule of the invention comprises a variant sequence of any one of SEQ ID NOs: 1 -9.
  • the antisense strand of a dsNA molecule of the invention comprises or consists of a variant of any one of SEQ ID NOs: 1 -9, wherein one or more (e.g. at most 1 , at most 2, at most 3, at most 4, or at most 5) mismatch nucleotides other than the underlined uracil nucleotide is replaced by a different nucleotide.
  • the variant retains the underlined uracil nucleotide as depicted in SEQ ID NOs: 1 -9 as a uracil nucleotide.
  • the one or more mismatch nucleotide(s) forms a mismatch pairing with the corresponding nucleotide located on the target mRNA, wherein said mismatch pairing is selected from a purine:purine, a purine:pyrimidine, or a pyrimidine:pyrimidine mismatch pairing.
  • At least one strand of the dsNA molecule of the invention may comprise one or more chemical modifications. Said chemical modification may be introduced to the antisense strand and/ or to the sense strand.
  • the at least one chemical modification improves the stability of the dsNA molecule.
  • a chemical modification increases the half-life of a dsNA molecule of the invention (e.g. in an aqueous solution).
  • a chemical modification increases the half-life of a dsNA molecule of the invention when introduced into a target cell.
  • the chemical modification may comprise a substitution or modification in which the substitution or modification may be in a phosphate backbone bond, a sugar, a base, or a nucleoside.
  • nucleoside substitutions can include natural nonstandard nucleosides (e.g., 5-methyluridine or 5-methylcytidine or a 2- thioribothymidine), and such backbone, sugar, or nucleoside modifications can include an alkyl or heteroatom substitution or addition, such as a methyl, alkoxyalkyl, halogen, nitrogen or sulphur, or other modifications known in the art.
  • Reference to nucleic acid(s) and/or nucleotide(s) embraces modified nucleic acid(s).
  • a nucleic acid or nucleotide may be modified to increase or decrease the stability of said nucleic acid or nucleotide.
  • a modified nucleic acid comprises a locked nucleotide (LNA).
  • LNA locked nucleotide
  • the ribose moiety of an LNA nucleotide is modified with an extra bridge connecting the 2' oxygen and 4' carbon. The bridge "locks" the ribose in the 3'-endo (North) conformation, which is often found in the A-form duplexes.
  • LNA nucleotides can be mixed with DNA or RNA residues in the oligonucleotide whenever desired. Such oligomers are commercially available.
  • the locked ribose conformation enhances base stacking and backbone pre-organization. This significantly increases the hybridization properties (melting temperature) of oligonucleotides.
  • nucleic acid analogues are composed of three parts: a phosphate backbone, a pucker-shaped pentose sugar, either ribose or deoxyribose, and one of four nucleobases.
  • An analogue may have any of these altered.
  • the analogue nucleobases confer, among other things, different base pairing and base stacking proprieties. Examples include universal bases, which can pair with all four canon bases, and phosphate-sugar backbone analogues such as PNA, which affect the properties of the chain (PNA can even form a triple helix).
  • Artificial nucleic acids include peptide nucleic acid (PNA), Morpholino and LNA, as well as glycol nucleic acid (GNA) and threose nucleic acid (TNA). Each of these is distinguished from naturally-occurring DNA or RNA by changes to the backbone of the molecule.
  • the dsNA molecules of the present invention may be made using any suitable process known in the art.
  • the dsNA molecules of the present invention may be made using chemical synthesis techniques.
  • the dsNA molecules of the present invention may be made using molecular biology techniques (for example, as reported in Yu et al. 2002, PMID: 1 1972060, which is hereby incorporated by reference in its entirety).
  • the dsNA molecules of the present invention may be synthesized by a commercial supplier.
  • the invention provides a nucleic acid vector comprising a nucleic acid sequence encoding a dsNA molecule as described herein.
  • the resultant RNA may be produced in vivo.
  • the dsNA molecules of the present invention may be made by conventional expression of a nucleic acid vector encoding said dsNA molecule, followed by conventional RNA recovery.
  • the RNA is produced in vitro.
  • the nucleic acid vector is a plasmid.
  • the nucleic acid vector is a viral vector.
  • the dsNA molecules of the present invention may be delivered into a target cell using a viral vector.
  • the viral vector may be any virus which can serve as a viral vector. Suitable viruses are those which infect the target cells, can be propagated in vitro, and can be modified by recombinant nucleotide technology known in the art.
  • the viral vector is selected from the group consisting of: an adenovirus vector; an adeno-associated virus vector; a pox virus vector, such as a fowlpox virus vector; an alpha virus vector; a bacloviral vector; a herpes virus vector; a retrovirus vector, such as a lentivirus vector; a Modified Vaccinia virus Ankara vector; a Ross River virus vector; a Sindbis virus vector; a Semliki Forest virus vector; and a Venezuelan Equine Encephalitis virus vector.
  • the invention provides a method for reducing the expression in a cell of a human LRRK2 gene having the G2019S SNP, comprising administering a therapeutically effective amount of a dsNA molecule as described above to a patient in need thereof, wherein the antisense strand of the dsNA molecule is capable of binding to position 6176 on an RNA nucleotide sequence having the sequence of SEQ ID NO: 10
  • the invention provides a dsNA molecule as described herein, or a nucleic acid vector as described herein, for use in the treatment of Parkinson's disease.
  • the treatment of Parkinson's disease comprises the treatment of a human subject having Parkinson's disease.
  • the invention provides a therapeutic and/or prophylactic formulation, comprising a dsNA molecule as described above.
  • the therapeutic and/or prophylactic formulation may be used in the therapeutic and/or prophylactic treatment of Parkinson's Disease.
  • the invention provides a pharmaceutical composition, comprising a dsNA molecule as described above; and a pharmaceutically acceptable carrier.
  • the dsNA molecule of the invention may be formulated into a pharmaceutical composition as neutral or salt forms.
  • Pharmaceutically acceptable salts include acid addition salts formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or with organic acids such as acetic, oxalic, tartaric, maleic, and the like. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.
  • compositions of the invention are generally by conventional routes e.g. intravenous, subcutaneous, intraperitoneal, or mucosal routes.
  • the administration may be by parenteral administration; for example, a subcutaneous or intramuscular injection.
  • the pharmaceutical compositions of the invention may be prepared as injectables, either as liquid solutions or suspensions. Solid forms suitable for solution in, or suspension in, liquid prior to injection may alternatively be prepared.
  • the preparation may also be emulsified, or the peptide encapsulated in liposomes or microcapsules.
  • the active ingredients are often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient.
  • excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof.
  • the pharmaceutical compositions may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, and/or pH buffering agents.
  • Non-limiting examples of pharmaceutically acceptable carriers include water, saline, and phosphate-buffered saline.
  • the composition is in lyophilized form, in which case it may include a stabilizer, such as bovine serum albumin (BSA).
  • BSA bovine serum albumin
  • buffering agents include, but are not limited to, sodium succinate (pH 6.5), and phosphate buffered saline (PBS; pH 6.5 and 7.5).
  • Additional formulations which are suitable for other modes of administration include suppositories and, in some cases, oral formulations or formulations suitable for distribution as aerosols.
  • traditional binders and carriers may include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably 1 %-2%.
  • Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders.
  • dsNA molecules of the present invention may be desired to direct the dsNA molecules of the present invention (as described above) to the respiratory system of a subject. Efficient transmission of a therapeutic/prophylactic formulation or medicament to the site of infection in the lungs may be achieved by oral or intra-nasal administration.
  • Formulations for intranasal administration may be in the form of nasal droplets or a nasal spray.
  • An intranasal formulation may comprise droplets having approximate diameters in the range of 100-5000 ⁇ , such as 500-4000 ⁇ , 1000-3000 ⁇ or 100-1000 ⁇ .
  • the droplets may be in the range of about 0.001 -100 ⁇ , such as 0.1 -50 ⁇ or 1 .0-25 ⁇ , or such as 0.001 -1 ⁇ .
  • the therapeutic/prophylactic formulation or medicament may be an aerosol formulation.
  • the aerosol formulation may take the form of a powder, suspension or solution.
  • the size of aerosol particles is relevant to the delivery capability of an aerosol . Smaller particles may travel further down the respiratory airway towards the alveoli than would larger particles.
  • the aerosol particles have a diameter distribution to facilitate delivery along the entire length of the bronchi, bronchioles, and alveoli.
  • the particle size distribution may be selected to target a particular section of the respiratory airway, for example the alveoli.
  • the particles may have diameters in the approximate range of 0.1 - 50 ⁇ , preferably 1 -25 ⁇ , more preferably 1 -5 ⁇ .
  • Aerosol particles may be for delivery using a nebulizer (e.g. via the mouth) or nasal spray.
  • An aerosol formulation may optionally contain a propellant and/or surfactant.
  • the therapeutic formulations and pharmaceutical compositions of the invention comprise a pharmaceutically acceptable carrier, and optionally one or more of a salt, excipient, diluent and/ or adjuvant.
  • the therapeutic formulations and pharmaceutical compositions of the invention may comprise one or more immunoregulatory agents selected from, for example, immunoglobulins, antibiotics, interleukins (e.g. IL-2, IL-12), and/or cytokines (e.g. IFNy).
  • immunoregulatory agents selected from, for example, immunoglobulins, antibiotics, interleukins (e.g. IL-2, IL-12), and/or cytokines (e.g. IFNy).
  • the therapeutic formulations and pharmaceutical compositions of the invention may comprise one or more antimicrobial compounds, (for example, conventional anti-tuberculosis drugs such as rifampicin, isoniazid, ethambutol or pyrizinamide).
  • antimicrobial compounds for example, conventional anti-tuberculosis drugs such as rifampicin, isoniazid, ethambutol or pyrizinamide.
  • the therapeutic formulations and pharmaceutical compositions of the invention may be given in a single dose schedule (i.e. the full dose is given at substantially one time).
  • the immunogenic compositions, therapeutic formulations, medicaments, pharmaceutical compositions, and prophylactic formulations of the invention may be given in a multiple dose schedule.
  • a multiple dose schedule is one in which a primary course of treatment (e.g. vaccination) may be with 1 -6 separate doses, followed by other doses given at subsequent time intervals required to maintain and or reinforce the immune response, for example (for human subjects), at 1 -4 months for a second dose, and if needed, a subsequent dose(s) after a further 1 -4 months.
  • the dosage regimen will be determined, at least in part, by the need of the individual and be dependent upon the judgment of the practitioner (e.g. doctor)
  • Simultaneous administration means administration at (substantially) the same time.
  • Sequential administration of two or more compositions/therapeutic agents means that the compositions/therapeutic agents are administered at (substantially) different times, one after the other.
  • the therapeutic formulations and pharmaceutical compositions may contain 5% to 95% of active ingredient, such as at least 10% or 25% of active ingredient, or at least 40% of active ingredient or at least 50, 55, 60, 70 or 75% active ingredient.
  • the therapeutic formulations and pharmaceutical compositions are administered in a manner compatible with the dosage formulation, and in such amount as will be prophylactically and/or therapeutically effective.
  • an “effective amount” is a dosage or amount that is sufficient to achieve a desired biological outcome.
  • a “therapeutically effective amount” is an amount which is effective, upon single or multiple dose administration to a subject (e.g. a human) for treating, preventing, curing, delaying, reducing the severity of, ameliorating at least one symptom of a disorder or recurring disorder, or prolonging the survival of the subject beyond that expected in the absence of such treatment.
  • the quantity of active ingredient to be administered depends on the subject to be treated, capacity of the subject's immune system to generate a protective immune response, and the degree of protection desired. Precise amounts of active ingredient required to be administered may depend on the judgment of the practitioner and may be particular to each subject.
  • SEQ ID NOs 1 -9 Nucleotide sequences of an RNA antisense strand complementary to LRRK2-G2019S wherein the uracil nucleotide at position p1 -9 (underlined), respectively, binds to the site of the G2019S SNP on the LRRK2-G2019S mRNA.
  • SEQ ID NO: 10 mRNA sequence of LRRK2 gene having G2019S SNP.
  • SEQ ID NO: 1 1 mRNA sequence of wildtype LRRK2 gene.
  • SEQ ID NO: 12-20 shRNA molecules directed against LRRK2-G2019S, having an antisense strand corresponding to SEQ ID NOs 1 -9, respectively (i.e. wherein the nucleotide that binds the G2019S SNP location on the target mRNA is located on the shRNA antisense strand at positions p1 -p9, respectively (as defined above)).
  • RNA interference Mechanisms of RNA interference that reduce expression of a target gene.
  • Figures 2a-b show a comparison of multiple shRNA molecules directed against the LRRK2-G2019S mRNA.
  • the position of the nucleotide in the shRNA antisense strand that binds the G2019S SNP location on the target mRNA varies from positions p1 to p9 (wherein the positions are defined as above). Changes to the sense strand in order to bias correct processing of the shRNA are indicated in bold.
  • FIGS 3a-c show the effect on LRRK2 mutant and wildtype expression of the shRNA molecules depicted in Figure 2.
  • Figures 4a-b show examples of how the presence of a mismatch nucleotide decreases targeting of non-target wildtype sequence.
  • a hierarchy of mismatch pairs showing the extent to which the formation of a given mismatch increases the ability of the antisense strand to discriminate between a given target nucleotide is shown.
  • the mismatch of a G:U that results between an antisense strand targeting the LRRK2-G2019S RNA and the wildtype LRRK2 RNA sequence is shown with a box.
  • siRNAs with alignment of the mutations at p3, p4 and p5 were screened against the luciferase targets.
  • siRNA p4 displayed a 7.7-fold ( ⁇ , ⁇ . ⁇ ) discrimination that was improved upon that seen with shRNA p4 at this time point ( Figure 6a).
  • siRNAs p3 and p5 displayed less discrimination between the two alleles, agreeing with the trends from previous shRNA data which showed alignment at p4 to be superior to these two constructs.

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Abstract

L'invention concerne une molécule d'acide nucléique bicaténaire (ANds) comprenant un brin sens d'acide nucléique et un brin antisens d'ARN, le brin antisens d'ARN se liant à la position 6176 sur une séquence nucléotidique d'ARN cible qui comprend la séquence nucléotidique de SEQ ID NO: 10 ; au moins une portion du brin antisens d'ARN et du brin sens d'acide nucléique définissent ensemble un duplex d'acide nucléique apparié par des bases, les nucléotides du brin d'ARN antisens définissant des positions de nucléotides antisens consécutivement numérotées, lesdits nombres augmentant dans une direction 5' vers 3' sur le brin antisens, la position 1 (p1) étant définie comme le nucléotide 5' d'extrémité présent sur le brin antisens d'ARN du duplex d'acide nucléique qui forme une paire de bases avec un nucléotide correspondant présent sur le brin sens d'acide nucléique du duplex d'acide nucléique ; et le brin antisens d'ARN comprenant un nucléotide uracile situé en l'une quelconque des positions p1 à p9 qui se lie à un nucléotide adénine situé en position 6176 d'une séquence nucléotidique d'ARN comprenant la séquence nucléotidique de SEQ ID NO: 10. L'invention concerne également des vecteurs codant lesdits ANds, et des utilisations thérapeutiques de la molécule d'ANds pour supprimer/prévenir la maladie de Parkinson.
PCT/GB2012/050692 2011-03-28 2012-03-28 Molécules thérapeutiques destinées à être utilisées dans la suppression de la maladie de parkinson WO2012131365A1 (fr)

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US9695171B2 (en) 2013-12-17 2017-07-04 Pfizer Inc. 3,4-disubstituted-1 H-pyrrolo[2,3-b]pyridines and 4,5-disubstituted-7H-pyrrolo[2,3-c]pyridazines as LRRK2 inhibitors
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US9156845B2 (en) 2012-06-29 2015-10-13 Pfizer Inc. 4-(substituted amino)-7H-pyrrolo[2,3-d] pyrimidines as LRRK2 inhibitors
US9642855B2 (en) 2012-06-29 2017-05-09 Pfizer Inc. Substituted pyrrolo[2,3-d]pyrimidines as LRRK2 inhibitors
US9695171B2 (en) 2013-12-17 2017-07-04 Pfizer Inc. 3,4-disubstituted-1 H-pyrrolo[2,3-b]pyridines and 4,5-disubstituted-7H-pyrrolo[2,3-c]pyridazines as LRRK2 inhibitors
US12173286B2 (en) 2015-04-03 2024-12-24 University Of Massachusetts Fully stabilized asymmetric siRNA
US12077755B2 (en) 2015-08-14 2024-09-03 University Of Massachusetts Bioactive conjugates for oligonucleotide delivery
US10039753B2 (en) 2015-09-14 2018-08-07 Pfizer Inc. Imidazo[4,5-c]quinoline and imidazo[4,5-c][1,5]naphthyridine derivatives as LRRK2 inhibitors
US9840710B2 (en) 2015-11-18 2017-12-12 Rosalind Franklin University Of Medicine And Science Antisense compounds targeting leucine-rich repeat kinase 2 (LRRK2) for the treatment of parkinsons disease
US10370667B2 (en) 2015-11-18 2019-08-06 Rosalind Franklin University Of Medicine And Science Antisense compounds targeting leucine-rich repeat kinase 2 (LRRK2) for the treatment of parkinsons disease
US10787669B2 (en) 2015-11-18 2020-09-29 Rosalind Franklin University Of Medicine And Science Antisense compounds targeting Leucine-Rich repeat kinase 2(LRRK2) for the treatment of Parkinsons disease
US20180362988A1 (en) * 2016-01-05 2018-12-20 Ionis Pharmaceuticals, Inc. Methods for reducing lrrk2 expression
EP3400300A4 (fr) * 2016-01-05 2019-08-07 Ionis Pharmaceuticals, Inc. Procédés pour réduire l'expression de lrrk2
US10907160B2 (en) 2016-01-05 2021-02-02 Ionis Pharmaceuticals, Inc. Methods for reducing LRRK2 expression
US11530411B2 (en) 2016-01-05 2022-12-20 Ionis Pharmaceuticals, Inc. Methods for reducing LRRK2 expression
WO2017120365A1 (fr) * 2016-01-05 2017-07-13 Ionis Pharmaceuticals, Inc. Procédés pour réduire l'expression de lrrk2
US11896669B2 (en) 2016-01-31 2024-02-13 University Of Massachusetts Branched oligonucleotides
US12049627B2 (en) 2017-06-23 2024-07-30 University Of Massachusetts Two-tailed self-delivering siRNA
WO2019118325A1 (fr) * 2017-12-11 2019-06-20 Rosalind Franklin University Of Medicine And Science Composés antisens ciblant une kinase 2 à répétition riche en leucine (lrrk2) pour le traitement de la maladie de parkinson
US11873495B2 (en) 2018-06-27 2024-01-16 Ionis Pharmaceuticals, Inc. Compounds and methods for reducing LRRK2 expression
US11332746B1 (en) 2018-06-27 2022-05-17 Ionis Pharmaceuticals, Inc. Compounds and methods for reducing LRRK2 expression
US12241067B2 (en) 2018-06-27 2025-03-04 Ionis Pharmaceuticals, Inc. Compounds and methods for reducing LRRK2 expression
US11827882B2 (en) 2018-08-10 2023-11-28 University Of Massachusetts Modified oligonucleotides targeting SNPs
EP3833763A4 (fr) * 2018-08-10 2023-07-19 University of Massachusetts Oligonucléotides modifiés ciblant des snp
US12297430B2 (en) 2018-08-23 2025-05-13 University Of Massachusetts O-methyl rich fully stabilized oligonucleotides
US12180477B2 (en) 2019-01-18 2024-12-31 University Of Massachusetts Dynamic pharmacokinetic-modifying anchors
US11820985B2 (en) 2019-03-26 2023-11-21 University Of Massachusetts Modified oligonucleotides with increased stability
EP4010476A4 (fr) * 2019-08-09 2023-12-27 University Of Massachusetts Oligonucléotides modifiés chimiquement ciblant des snp
US12024706B2 (en) 2019-08-09 2024-07-02 University Of Massachusetts Modified oligonucleotides targeting SNPs
US12365894B2 (en) 2019-09-16 2025-07-22 University Of Massachusetts Branched lipid conjugates of siRNA for specific tissue delivery
US12146136B2 (en) 2020-03-26 2024-11-19 University Of Massachusetts Synthesis of modified oligonucleotides with increased stability

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