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`PCT /NL 2013
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`2 9 JAN. 2013
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`'ffl>A'1ID,'!n)Wff:QM£'11Uias.� �BJE·SlE�� �. �McEm
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`UNITED STATES DEPARTMENT OF COMMERCE
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`Title of Invention:
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`OLIGONUCLEOTIDES WITH IMPROVED CHARACTERISTICS
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`RNA MODULATING
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`FOR THE TREATMENT OF DUCHENNE AND BECKER MUSCULAR DYSTROPHY
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`First Named Inventor/Applicant Name: Peter Christian DE VISSER
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`p eter Christian
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`DE VISSER Leiden
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`Title of Invention
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`RNA MODULATING OLIGONUCLEOTIDES WITH IMPROVED
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`CHARACTERISTICS FOR THE TREATMENT OF DUCHENNE AND BECKER
`MUSCULAR DYSTROPHY
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`No. 2001-1956
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`Attorney Docket
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`RNA modulating oligonucleotides with improved characteristics for the treatment of
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`Duchenne and Becker muscular dystrophy.
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`neuromuscular more specifically 5 The invention relates to the field of human genetics,
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`Field
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`disorders. The invention in particular relates to the use of an oligonucleotide with
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`improved characteristics enhancing clinical applicability as further defined herein.
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`10
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`Background of the invention
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`Neuromuscular diseases are characterized by impaired functioning of the
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`muscles due to either muscle or nerve pathology (myopathies and neuropathies). The
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`myopathies include genetic muscular dystrophies that are characterized by progressive
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`weakness and degeneration of skeletal, heart and/or smooth muscle. Duchenne
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`most dystrophy (BMD) are the 15 muscular dystrophy (DMD) and Becker muscular
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`common childhood forms of muscular dystrophy. DMD is a severe, lethal
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`neuromuscular disorder resulting in a dependency on wheelchair support before the
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`age of 12 and patients often die before the age of thirty due to respiratory-or heart
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`failure. It is caused by caused by reading frame-shifting deletions (~67%) or
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`20 duplications (~7%) of one or more exons, or by point mutations (~25%) in the 2.24
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`is also caused dystrophin. BMD Mb DMD gene, resulting in the absence of functional
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`frame, yield the open reading by mutations in the DMD gene, but these maintain
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`semi-functional dystrophin proteins, and result in a typically much milder phenotype
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`and longer lifespan. During the last decade, specific modification of splicing in order
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`25 to restore the disrupted reading frame of the transcript has emerged as a promising
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`therapy for DMD (van Ommen et al., 2008; Yokota et al., 2007; van Deutekom et al.,
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`2007; Goemans et al., 2011; Cirak et al., 2011).Using highly sequence-specific
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`antisense oligonucleotides (AONs) which bind to the exon flanking or containing the
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`mutation and which interfere with its splicing signals, the skipping of that exon can be
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`Despite the resulting of the DMD pre-mRNA. 30 induced during the processing
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`truncated transcript, the open reading frame is restored and a protein is introduced
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`which is similar to those found in BMD patients. AON-induced exon skipping
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`provides a mutation-specific, and thus personalized, therapeutic approach for DMD
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`patients. As the majority of the mutations cluster around exons 45 to 55, the skipping
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`of one specific exon may be therapeutic for many patients with different mutations.
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`Attorney Docket No. 2001-1956
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`The skipping of exon 51 applies to the largest subset of patients (~13%), including
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`those with deletions of exons 45 to 50, 48 to 50, 50, or 52. The AONs applied are
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`chemically modified to resist endonucleases, exonucleases and RNaseH, and to
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`are AON chemistries stability .. Two different and duplex 5 promote RNA binding
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`currently being developed for exon 51 skipping in DMD: 2' -O-methyl
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`phosphorothioate RNA AONs (2OMePS, GSK2402968/PRO051) and
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`phosphorodiamidate morpholino oligomers (PMO, AVI-4658) (Goemans et al., 2011;
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`Cirak et al., 2011). In two independent phase I/II studies, both were shown to
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`dystrophin expression and at least partly restore 10 specifically induce exon 51 skipping
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`at the muscle fiber membranes after systemic administration. Although AONs are
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`typically not well taken up by healthy muscle fibers, the dystrophin deficiency in
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`DMD, resulting in damaged and thus more permeable fiber membranes, actually
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`promotes uptake. In studies in the dystrophin-deficient mdx mouse model, 2' -0-
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`an up to 10 times RNA oligonucleotides have demonstrated 15 methyl phosphorothioate
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`higher uptake in different muscle groups when compared to that in wild type mice
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`(Heemskerk et al., 2010). Although the recent phase I/II results with both 2'-O-methyl
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`phosphorothioate RNA and phosphorodiamidate morpholino AONs in DMD patients
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`confirm this enhanced uptake in dystrophic muscle, the different chemical
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`through uptake by and distribution 20 modifications seemed to result in a differential
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`muscle. The levels of novel dystrophin in both studies after 3 months of treatment
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`were promising but still moderate and challenges the field to investigate next
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`generation oligochemistry.
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`the chemistry at least in part affects 25 The particular characteristics of a chosen
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`delivery of an AON to the target transcript: administration route, biostability,
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`biodistribution, intra-tissue distribution, and cellular uptake and trafficking. In
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`addition, further optimization of oligonucleotide chemistry is conceived to enhance
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`binding affinity and stability, enhance activity, improve safety, and/or to reduce cost
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`30 of goods by reducing
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`length or improving synthesis and/or purification procedures.
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`Multiple chemical modifications have become generally and/or commercially
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`available to the research community (such as 2' -O-methyl RNA and 5-substituted
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`pyrimidines and 2,6-diaminopurines), whereas most others still present significant
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`synthetic
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`effort to obtain. Especially preliminary encouraging results have been
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`2
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`Copy provided by USPTO from the IFW lmai:ie Database on �n1n�,.,rw>
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`Attorney Docket No. 2001-1956
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`on the RNA containing modifications phosphorothioate obtained using 2' -O-:methyl
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`pyrimidine and adenine bases as identified herein ..
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`In conclusion, to enhance the therapeutic applicability of AONs for DMD, there
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`5 is a need for AONs with further improved characteristics.
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`Description of the invention
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`Oligonucleotide
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`10 In a first aspect, the invention provides an oligonucleotide comprising a 2'-O-methyl
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`RNA monomer and a phosphorothioate backbone or consisting of 2' -O-methyl RNA
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`monomers
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`linked by phosphorothioate backbones, and comprising a 5-
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`methylpyrimidine and/or a 2,6-diaminopurine base preferably for use as a medicament
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`for treating Duchenne Muscular Dystrophy or Becker Muscular Dystrophy.
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`15
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`Preferably, an oligonucleotide is an oligonucleotide with less than 34 nucleotides.
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`Said oligonucleotide may have 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
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`24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides. Such oligonucleotide may also be
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`identified as an oligonucleotide having from 10 to 33 nucleotides.
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`20
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`An oligonucleotide of the invention comprises or consists of a 2' -O-methyl
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`phosphorothioate RNA. Such oligonucleotide comprises a 2' -O-methyl RNA
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`monomer connected through or linked by a phosphorothioate backbone or consists of
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`2' -O-methyl phosphorothioate RNA. Preferably, such oligonucleotide consists of a 2'-
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`25 O-methyl
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`phosphorothioate RNA. Such chemistry is known to the skilled person.
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`Throughout the application, an oligonucleotide comprising a 2'-O-methyl RNA
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`monomer and a phosphorothioate backbone may be replaced by an oligonucleotide
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`comprising a 2' -O-methyl phosphorothioate RNA. Throughout the application, an
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`oligonucleotide consisting of 2' -O-methyl RNA monomers linked by or connected
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`30 through phosphorothioate backbones may be replaced by an oligonucleotide
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`consisting of 2'-O-methyl phosphorothioate RNA.
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`a base modification that of the invention may comprise In addition, an oligonucleotide
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`increases binding affinity to target strands, increases melting temperature of the
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`resulting
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`duplex of said oligonucleotide with its target, and/or decreases
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`3
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`Copy provided by USPTO from the IFW lmaoe l'la,,..,,..,,. "" 101011?01?
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`immunostimulatory effects, and/or increases biostability, and/or improves
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`Attorney Docket No. 2001-1956
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`biodistribution and/or intra-tissue distribution, and/or cellular uptake and trafficking.
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`In a more preferred embodiment, an oligonucleotide of the invention comprises a 5-
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`methylpyrimidine and/or a 2,6-diaminopurine base. A 5-methylpyrimidine is selected
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`5 from a 5-methylcytosine and/or a 5-methyluracil and/or a thymine, in which thymine
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`is identical to 5-methyluracil.
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`'Thymine' and ' 5-methyluracil' may be interchanged throughout the document. In
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`analogy, 2,6-diaminopurine is identical to 2-aminoadenine and these terms may be
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`interchanged throughout the document.
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`10
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`An oligonucleotide of the invention comprising a 5-methylcytosine and/or a 5-
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`methyluracil and/or a 2,6-diaminopurine base means that at least one of the cytosine
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`nucleobases of said oligonucleotide has been modified by substitution of the proton at
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`ring with a methyl group , i.e. a 5-substituted the 5-position of the pyrimidine
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`said oligonucleotide has one of the uracil nucleobases of 15 cytosine, and/or that at least
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`been modified by substitution of the proton at the 5-position of the pyrimidine ring
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`with a methyl group (i.e. a 5-methyluracil), and/or that at least one of the adenine
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`nucleobases of said oligonucleotide has been modified by substitution of the proton at
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`the 2-position with an amino group (i.e. a 2,6-diaminopurine), respectively. Within
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`a methyl of a proton with the expression "the substitution 20 the context of the invention,
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`group in position 5 of the pyrimidine ring" may be replaced by the expression "the
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`substitution of a pyrimidine with a 5-methylpyrimidine," with pyrimidine referring to
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`only uracil, only cytosine or both. Likewise, within the context of the invention, the
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`expression "the substitution of a proton with an amino group in position 2 of adenine"
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`of an adenine with a 2,6-''the substitution 25 may be replaced by the expression
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`diaminopurine." If said oligonucleotide comprises 1, 2, 3, 4 , 5, 6, 7, 8, 9 or more
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`cytosines, uracils, and/or adenines, at least one, 2, 3, 4, 5, 6, 7, 8 9 or more cytosines,
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`uracils and/or adenines respectively have been modified this way. Preferably all
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`cytosines, uracils and/or adenines have been modified this way or substituted by 5-
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`respectively. No need to 30 methylcytosine, 5-methyluracil and/or 2,6-diaminopurine,
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`say that the invention could only be applied to oligonucleotides comprising at least
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`one cytosine, uracil, or adenine, respectively, in their sequence.
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`4
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`Copy provided by USPTO from the IFW lmaqe Database on 10/01/2012
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`Attorney Docket No. 2001-1956
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`We discovered that the presence of a 5-methylcytosine, 5-methyluracil and/or a 2,6-
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`diaminopurine in an olignucleotide of the invention has a positive effect on at least
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`one of the parameters of said oligonucleotides. In this context, parameters may
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`include: binding affinity and/or kinetics, exon skipping activity, biostability, (intra-
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`5 tissue) distribution, cellular uptake and/or trafficking, and/or immunogenicity of
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`said oligonucleotide, as explained below.
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`Binding affinity and kinetics depend on the AON's thermodynamic properties. These
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`are at least in part determined by the melting temperature of said oligonucleotide (Tm;
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`10 calculated with e.g. the oligonucleotide properties calculator
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`(http://www.unc.edu/~cail/biotool/oligo/index.html
`or
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`http://eu.idtdna.com/analyzer/Applications/OligoAnalyzer/) for single stranded RNA
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`using the basic Tm and the nearest neighbor model), and/or the free energy of the
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`oligonucleotide-target exon complex (using RNA structure version 4.5 or RNA mfold
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`but increases, activity typically the exon skipping 15 version 3.5). If a Tm is increased,
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`when a Tm is too high, the AON is expected to become less sequence-specific. An
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`acceptable Tm and free energy depend on the sequence of the oligonucleotide.
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`Therefore, it is difficult to give preferred ranges for each of these parameters.
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`from RNA isolated total 20 Exon skipping activity is preferably measured by analysing
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`AON-treated muscle cell cultures or muscle tissue by reverse transcriptase
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`polymerase chain reaction (RT-PCR) using DMD gene-specific primers flanking the
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`targeted exon as described (Aartsma-Rus et al., 2003). RT-PCRproducts are analyzed
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`on 1-2% agarose gels or with the Agilent 2100 bioanalyzer (Agilent Technologies,
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`25 The Netherlands). The ratio of shorter transcript fragments, representing transcripts in
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`which the targeted exon is skipped, to the total of transcript products is assessed
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`(calculated as percentage of exon skipping induced by an AON). Shorter fragments
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`may also be sequenced to determine the correctness and specificity of the targeted
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`exon skipping.
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`30
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`Biodistribution and biostability are preferably at least in part determined by a
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`validated hybridization ligation assay adapted from Yu et al., 2002. In an
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`embodiment, plasma or homogenized tissue samples are incubated with a specific
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`capture oligonucleotide probe. After separation, a DIG-labeled oligonucleotide is
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`5
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`Copy provided by USPTO from the IFW lmane Databiiis,.. o� Ar11r1A ,.,r,
`.,
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`A
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`ligated to the complex and detection followed using an anti-DIG antibody-linked
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`AttomeyDocketNo. 2001-1956
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`peroxidase. Non-compartmental pharmacokinetic analysis is performed using
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`WINNONLIN software package (model 200, version 5.2, Pharsight, Mountainview,
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`CA). Levels of AON (ug) per mL plasma or mg tissue are monitored over time to
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`5 assess area under the curve (AUC), peak concentration
`(Cmax), time to peak
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`lag time (t1ag). Such a preferred
`concentration half life and absorption (T max), terminal
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`assay has been disclosed in the experimental part.
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`receptors the Toll-like response by activating 10 AONs may stimulate an innate immune
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`(TLR), including TLR9 and TLR7 (Krieg et al., 1995). The activation ofTLR9
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`typically occurs due to the presence of non-methylated CG sequences present in
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`oligodeoxynucleotides (ODNs), by mimicking bacterial DNA which activates the
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`innate immune system through TLR9-mediated cytokine release. The 2' -O-methyl
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`effect. TLR 7 has such possible reduce to markedly 15 modification is however suggested
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`been described to recognize uracil repeats in RNA (Diebold et al., 2006).
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`Activation of TLR9 and TLR 7 result in a set of coordinated immune responses that
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`include innate immunity (macrophages, dendritic cells (DC), and NK cells)(Krieg et
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`al., 1995; Krieg, 2000). Several chemo-and cytokines, such as IP-10, TNFa, IL-6,
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`d in this et al., 2006) have been implicate20 MCP-1 and IFNa (Wagner, 1999; Popovic
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`process. The inflammatory cytokines attract additional defensive cells from the blood,
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`such as T and B cells. The levels of these cytokines can be investigated by in vitro
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`testing. In short, human whole blood is incubated with increasing concentrations of
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`AONs after which the levels of the cytokines are determined by standard
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`in the a preferred assay has been described 25 commercially available ELISA kits. Such
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`experimental part. A decrease in immunogenicity preferably corresponds to a
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`detectable decrease of concentration of at least one of the cytokines mentioned above
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`by comparison to the concentration of corresponding cytokine in an assay in a cell
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`treated with an oligonucleotide comprising at least one 5-methylcytosine compared to
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`30 a cell treated
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`with a corresponding oligonucleotide having no 5-methylcytosines.
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`Accordingly, a preferred oligonucleotide of the invention has an improved parameter,
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`such as an acceptable or a decreased immunogenicity and/or a better biodistribution
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`and/or acceptable or improved RNA binding kinetics and/or thermodynamic
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`6
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`Copy provided
`by USPTO from the IFW IMaf"P n,.t ... h ....... "" 1n,n1
`,.,n1.,
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`Attorney Docket No. 2001-1956
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`properties by comparison to a corresponding oligonucleotide consisting of a 2'-O­
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`methyl phosphorothioate RNA without a 5-methylcytosine, a 5-methyluracil and/or a
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`2,6-diaminopurine. Each of these parameters could be assessed using