(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY OCT)
`
`International Bureau
`
`(19) World Intellectual Property Organization [if1.]
`
` (10) International Publication Number
`(43) International Publication Date
`WO 2010/123369 Al
` 28 October 2010 (28.10.2010) PCT
`
`
`'3
`
`(51)
`
`(21)
`
`International Patent Classification:
`C12N 15/113 (2010.01)
`CIZN 15/11 (2006.01)
`International Application Number:
`PCT/N120 10/050230
`
`(22)
`
`International Filing Date:
`
`(25) Filing Language:
`(26) Publication Language:
`
`26 April 2010 (26.04.2010)
`_
`English
`English
`
`(81) Designated States (unless otherwise indicated, for every
`kind (f national protection available): AE, AG, AL, AM,
`132: 3:1: 1311::’ 3N2: CBS: C131]: CBS: 22’ 113311; 313,131,133:
`DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT,
`HN, HR, HU, 1D, 11, IN, IS, JP, KE, KG, KM, KN, KP,
`KR, KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD,
`ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, N1,
`NO, NZ, OM, PE, PG, PH, PL, PT, R0, RS, RU, SC, SD,
`SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR,
`TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW.
`_
`_
`_
`_
`EP (84) Designated States (unless otherwzse Indicated, for every
`US
`kind (f regional protection available): ARIPO 03W, GH,
`GM, KE, LR, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG,
`ZM, zw), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ,
`(71) Applicant Var all designated States except US): PROS-
`TM), European (AT, BE, BG, CH, CY, CZ, DE, DK, EE,
`ENSA TECHNOLOGIES
`B-V-
`[NITNL]; Wasse—
`ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, Lv,
`naarseweg 72, NL-2333 AL Leiden (NL)-
`MC, MK, MT, NL, NO, PL, PT, R0, SE, SI, SK, SM,
`Inventors; and
`TNIE)’ 1%}?$13121?Ji'SF'i‘g?’ CI’ CM’ GA’ GN’ GQ’ GW’
`VAN
`only):
`US
`fior
`Inventors/Applicants
`’
`’
`’
`’
`’
`'
`DEUTEKOM, Judith Christina Theodora [NL/NL];
`Abeelstraat
`13, NL-3329 AA Dordrecht
`(NL). DE Published:
`£3125” 1111:2212? '3’:aggihwmqgi],leiTE1§Engl§le — with international search report (Art. 21(3))
`Gerardus
`Johannes
`[NL/NL]; Wijngaardenlaan
`56, — with sequence listing part if description mule 5.2(a))
`NL-2252 XR Voorschoten (NL).
`
`(30) Priority Data:
`0915873 1'1
`61/172,506
`
`24 April 2009 (24"042009)
`24 April 2009 (24.04.2009)
`
`(72)
`(75)
`
`
`
`wo2010/123369A1|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
`
`(74) Agent: KETELAARS, Maarten;
`NL-2517 JS The Hague (NL).
`
`J.W. Frisolaan
`
`13,
`
`(54) Title: OLIGONUCLEOTlDE COMPRISING AN INOSINE FOR TREATING DMD
`
`(57) Abstract: The invention provides an oligonucleotide comprising an inosine, and/or a nucleotide containing a base able to
`form a wobble base pair or a functional equivalent thereof, wherein the oligonucleotide, or a functional equivalent thereof, com-
`prises a sequence which is complementary to at least part of a dystrophin pre-m RNA exon or at least part of a non-exon region of
`a dystrophin pre-m RNA said part being a contiguous stretch comprising at least 8 nucleotides. The invention further provides the
`use of said oligonucleotide for preventing or treating DMD or BMD.
`
`

`

`WO 2010/123369
`
`PCT/NL2010/050230
`
`Oligonucleotide comprising an inosine for treating DMD
`
`The invention relates to the fields of molecular biology and medicine.
`
`Field of the invention
`
`Background of the invention
`
`A muscle disorder is a disease that usually has a significant impact on the
`
`life of an individual. A muscle disorder can have either a genetic cause or a
`
`non-genetic cause. An important group of muscle diseases with a genetic
`
`10
`
`cause are Becker Muscular Dystrophy (BMD) and Duchenne Muscular
`
`Dystrophy (DMD). These disorders are caused by defects in a gene for a
`
`muscle protein.
`
`Becker Muscular Dystrophy and Duchenne Muscular Dystrophy are
`
`genetic muscular dystrophies with a relatively high incidence. In both
`
`15
`
`Duchenne and Becker muscular dystrophy the muscle protein dystrophin is
`
`affected. In Duchenne dystrophin is absent, whereas in Becker some
`
`dystrophin is present but its production is most often not sufficient and/or
`
`the dystrophin present is abnormally formed. Both diseases are associated
`
`with recessive X-linked inheritance. DMD results from a frameshift
`
`20
`
`mutation in the DMD gene. The frameshift in the DMD gene's transcript
`
`(mRNA) results in the production of a truncated non-functional dystrophin
`
`protein, resulting in progressive muscle wasting and weakness. BMD
`
`occurs as a mutation does not cause a frame- shift in the DMD transcript
`
`(mRNA). As in BMD some partly to largely functional dystrophin is present
`
`25
`
`in contrast to DMD where dystrophin is absent, BMD has generally less
`
`severe symptoms then DMD. The onset of DMD is earlier than BMD. DMD
`
`usually manifests itself in early childhood, BMD in the teens or in early
`
`adulthood. The progression of BMD is slower and less predictable than
`
`DMD. Patients with BMD can survive into mid to late adulthood. Patients
`
`30
`
`with DMD rarely survive beyond their thirties.
`
`

`

`WO 2010/123369
`
`PCT/NL2010/050230
`
`Dystrophin plays an important structural
`
`role in the muscle fiber,
`
`connecting the extracellular matrix and the cytoskeleton. The N-terminal
`
`region binds actin, whereas the C-terminal end is part of the dystrophin
`
`glycoprotein complex (DGC), which spans the sarcolemma. In the absence of
`
`dystrophin, mechanical stress leads to sarcolemmal ruptures, causing an
`
`uncontrolled influx of calcium into the muscle fiber interior, thereby
`
`triggering calcium-activated proteases and fiber necrosis.
`
`For most genetic muscular dystrophies no clinically applicable and
`
`10
`
`effective therapies are currently available. Exon skipping techniques are
`
`nowadays explored in order to combat genetic muscular dystrophies.
`
`Promising results have recently been reported by us and others on a genetic
`
`therapy aimed at restoring the reading frame of the dystrophin pre-mRNA
`
`in cells from the mdx mouse, the GRMD dog (reference 59) and DMD
`
`15
`
`patients 1-“. By the targeted skipping of a specific exon, a DMD phenotype
`
`(lacking dystrophin) is converted into a milder BMD phenotype (partly to
`
`largely functional dystrophin). The skipping of an exon is preferably
`
`induced by the binding of antisense oligoribonucleotides (AONs) targeting
`
`either one or both of the splice sites, or exon-internal sequences. Since an
`
`20
`
`exon will only be included in the mRNA when both the splice sites are
`
`recognised by the spliceosome complex, splice sites have been considered
`
`obvious targets for AONs. More preferably, one or more AONs are used
`
`which are specific for at least part of one or more exonic sequences involved
`
`in correct splicing of the exon. Using exon-internal AONs specific for an
`
`25
`
`exon 46 sequence, we were previously able to modulate the splicing pattern
`
`in cultured myotubes from two different DMD patients with an exon 45
`
`deletion 11. Following AON treatment, exon 46 was skipped, which resulted
`
`in a restored reading frame and the induction of dystrophin synthesis in at
`
`least 75% of the cells. We have recently shown that exon skipping can also
`
`30
`
`efficiently be induced in human control and patient muscle cells for 39
`
`different DMD exons using exon-internal AONs 1'2 “-15.
`
`

`

`WO 2010/123369
`
`PCT/NL2010/050230
`
`Hence, exon skipping techniques applied on the dystrophin gene
`
`result in the generation of at least partially functional - albeit shorter -
`
`dystrophin protein in DMD patients. Since DMD is caused by a
`
`5
`
`dysfunctional dystrophin protein, it would be expected that the symptoms
`
`of DMD are sufficiently alleviated once a DMD patient has been provided
`
`with functional dystrophin protein. However, the present invention
`
`provides the insight that, even though exon skipping techniques are capable
`
`of inducing dystrophin synthesis, the oligonucleotide used for exon skipping
`
`10
`
`technique can be improved any further by incorporating an inosine and/or a
`
`nucleotide containing a base able to form a wobble base pair in said
`
`oligonucleotide.
`
`15
`
`Oligonucleotide
`
`Description of the invention
`
`In a first aspect, there is provided an oligonucleotide comprising an inosine
`
`and/or a nucleotide containing a base able to form a wobble base pair or a
`
`functional equivalent thereof, wherein the oligonucleotide, or a functional
`
`equivalent thereof, comprises a sequence which is complementary to at
`
`20
`
`least part of a dystrophin pre-mRNA exon or at least part of a non-exon
`
`region of a dystrophin pre-mRNA said part being a contiguous stretch
`
`comprising at least 8 nucleotides.
`
`The use of an inosine and/or a nucleotide containing a base able to form a
`
`wobble base pair in an oligonucleotide of the invention is very attractive as
`
`25
`
`explained below. Inosine for example is a known modified base which can
`
`pair with three bases: uracil, adenine, and cytosine. Inosine is a nucleoside
`
`that is formed when hypoxanthine is attached to a ribose ring (also known
`
`as a ribofuranose) via a B—N9-glycosidic bond. Inosine is commonly found in
`
`tRNAs and is essential for proper translation of the genetic code in wobble
`
`30
`
`base pairs. Awobble base pair is a G-U and I-U / 1-A/ 1-C pair fundamental
`
`in RNA secondary structure.
`
`Its thermodynamic stability is comparable to
`
`

`

`WO 2010/123369
`
`PCT/NL2010/050230
`
`that of the Watson- Crick base pair. Wobble base pairs are critical for the
`
`proper translation of the genetic code. The genetic code makes up for
`
`disparities in the number of amino acids (20) for triplet codons (64), by
`
`using modified base pairs in the first base of the anti-codon. Similarly,
`
`when designing primers for polymerase chain reaction, inosine is useful in
`
`that it will indiscriminately pair with adenine, thymine, or cytosine. Afirst
`
`advantage of using such a base allows one to design a primer that spans a
`
`single nucleotide polymorphism (SNP), without worry that the
`
`polymorphism will disrupt the primer's annealing efficiency. Therefore in
`
`10
`
`the invention, the use of such a base allows to design an oligonucleotide
`
`that may be used for an individual having a SNP within the dystrophin pre-
`
`mRNA stretch which is targeted by an oligonucleotide of the invention.
`
`A second advantage of using an inosine and/or a base able to form a wobble
`
`base pair in an oligonucleotide of the invention is when said oligonucleotide
`
`15
`
`would normally contain a CpG if one would have designed it as being
`
`complementary to at least part of a dystrophin pre-mRNA exon or at least
`
`part of a non-exon region of a dystrophin pre-mRNA said part being a
`
`contiguous stretch comprising at least 8 nucleotides. The presence of a CpG
`
`in an oligonucleotide is usually associated with an increased
`
`20
`
`immunogenicity of said oligonucleotide (reference 60). This increased
`
`immunogenicity is undesired since it may induce the breakdown of muscle
`
`fibers. Replacing one, two or more CpG by the corresponding inosine and/or
`
`a base able to form a wobble base pair in said oligonucleotide is expected to
`
`provide an oligonucleotide with a decreased and/or acceptable level of
`
`25
`
`immunogenicity. Immunogenicity may be assessed in an animal model by
`
`assessing the presence of CD4+ and/or CD8+ cells and/or inflammatory
`
`mononucleocyte infiltration in muscle biopsy of said animal.
`
`Immunogenicity may also be assessed in blood of an animal or of a human
`
`being treated with an oligonucleotide of the invention by detecting the
`
`30
`
`presence of a neutralizing antibody and/or an antibody recognizing said
`
`oligonucleotide using a standard immunoassay known to the skilled person.
`
`

`

`WO 2010/123369
`
`PCT/NL2010/050230
`
`An increase in immunogenicity preferably corresponds to a detectable
`
`increase of at least one of these cell types by comparison to the amount of
`
`each cell type in a corresponding muscle biopsy of an animal before
`
`treatment or treated with a corresponding oligonucleotide having at least
`
`one inosine and/or a base able to form a wobble base pair. Alternatively, an
`
`increase in immunogenicity may be assessed by detecting the presence or
`
`an increasing amount of a neutralizing antibody or an antibody recognizing
`
`said oligonucleotide using a standard immunoassay.
`
`A decrease in immunogenicity preferably corresponds to a detectable
`
`10
`
`decrease of at least one of these cell types by comparison to the amount of
`
`corresponding cell type in a corresponding muscle biopsy of an animal
`
`before treatment or treated with a corresponding oligonucleotide having no
`
`inosine and/or a base able to form a wobble base pair. Alternatively a
`
`decrease in immunogenicity may be assessed by the absence of or a
`
`15
`
`decreasing amount of said compound and/or neutralizing antibodies using a
`
`stande immunoassay.
`
`Athird advantage of using an inosine and/or a base able to form a wobble
`
`base pair in an oligonucleotide of the invention is to avoid or decrease a
`
`20
`
`potential multimerisation
`
`or aggregation of oligonucleotides.
`
`It is for
`
`example known that an oligonucleotide comprising a G-quartet motif has
`
`the tendency to form a quadruplex, a multimer or aggregate formed by the
`
`Hoogsteen base-pairing of four single-stranded oligonucleotides (reference
`
`61), which is of course not desired:
`
`as a result
`
`the efficiency of the
`
`25
`
`oligonucleotide is expected to be decreased. Multimerisation or aggregation
`
`is preferably assessed by standard polyacrylamid non- denaturing
`
`gel
`
`electrophoresis techniques known to the skilled person.
`
`In a preferred
`
`embodiment, less than 20% or 15%, 10%, 7%, 5% or less of a total amount of
`
`an oligonucleotide of the invention has the capacity to multimerise or
`
`30
`
`aggregate assessed using the assay mentioned above.
`
`

`

`WO 2010/123369
`
`PCT/NL2010/050230
`
`A fourth advantage of using an inosine and/or a base able to form a wobble
`
`base pair in an oligonucleotide of the invention is thus also to avoid
`
`quadruplex structures which have been associated with antithrombotic
`
`activity (reference 62) as well as with the binding to, and inhibition of, the
`
`5
`
`macrophage scavenger receptor (reference 63 .).
`
`A fifth advantage of using an inosine and/or a base able to form a wobble
`
`base pair in an oligonucleotide of the invention is to allow to design an
`
`oligonucleotide with improved RNA binding kinetics and/or thermodynamic
`
`10
`
`properties. The RNA binding kinetics and/or thermodynamic properties are
`
`at least in part determined by the melting temperature of an
`
`oligonucleotide (Tm; calculated with the oligonucleotide properties
`
`calculator (http://www.unc.edu/~cail/biotool/oligo/index.html)
`
`for single
`
`stranded RNA using the basic Tm and the nearest neighbour model), and/or
`
`15
`
`the free energy of the AON-target exon complex (using RNA structure
`
`version 4.5). If a Tm is too high, the oligonucleotide is expected to be less
`
`specific. An acceptable Tm and free energy depend on the sequence of the
`
`oligonucleotide. Therefore, it is difficult to give preferred ranges for each of
`
`these parameters. An acceptable Tm may be ranged between 35 and 65°C
`
`20
`
`and an acceptable free energy may be ranged between 15 and 45 kcal/mol.
`
`The skilled person may therefore first choose an oligonucleotide as a
`
`potential
`
`therapeutic compound. In a second step, he may use the invention
`
`to fiirther optimise said oligonucleotide by decreasing its immunogenicity
`
`25
`
`and/or avoiding aggregation and/or quadruplex formation and/or by
`
`optimizing its Tm and/or free energy of the AON-target complex. He may
`
`try to introduce at least one inosine and/or a base able to form a wobble
`
`base pair in said oligonucleotide at a suitable position and assess how the
`
`immunogenicity and/or aggregation and/or quadruplex formation and/or Tm
`
`30
`
`and/or free energy of the AON-target complex have been altered by the
`
`presence of said inosine and/or a base able to form a wobble base pair. If the
`
`

`

`WO 2010/123369
`
`PCT/NL2010/050230
`
`alteration does not provide the desired alteration or decrease of
`
`immunogenicity and/or aggregation and/or quadruplex formation and/or its
`
`Tm and/or free energy of the AON-target complex he may choose to
`
`introduce a further inosine and/or a base able to form a wobble base pair in
`
`said oligonucleotide and/or to introduce a given inosine and/or a base able to
`
`form a wobble base pair at a distinct suitable position within said
`
`oligonucleotide.
`
`An oligonucleotide comprising an inosine and/or a base able to form a
`
`10
`
`wobble base pair may be defined as an oligonucleotide wherein at least one
`
`nucleotide has been substituted with an inosine and/or a base able to form a
`
`wobble base pair. The skilled person knows how to test whether a
`
`nucleotide contains a base able to form a wobble base pair. Since for
`
`example inosine can form a base pair with uracil, adenine, and/or cytosine,
`
`15
`
`it means that at least one nucleotide able to form a base pair with uracil,
`
`adenine and/or cytosine has been substituted with inosine. However, in
`
`order to safeguard specificity, the inosine containing oligonucleotide
`
`preferably comprises the substitution of at least one, two, three, four
`
`nucleotide(s) able to form a base pair with uracil or adenine or cytosine as
`
`20
`
`long as an acceptable level of a functional activity of said oligonucleotide is
`
`retained as defined later herein.
`
`An oligonucleotide comprising an inosine and/or a base able to form a
`
`wobble base pair is preferably an olignucleotide, which is still able to
`
`exhibit an acceptable level of a functional activity of a corresponding
`
`25
`
`oligonucleotide not comprising an inosine and/or a base able to form a
`
`wobble base pair. A fimctional activity of said oligonucleotide is preferably
`
`to provide an individual with a fimctional dystrophin protein and/or mRNA
`
`and/or at least in part decreasing the production of an aberrant dystrophin
`
`protein and/or mRNA. Each of these features are later defined herein. An
`
`30
`
`acceptable level of such a fimctional activity is preferably at least 50%, 60%,
`
`70%, 80%, 90%, 95% or 100% of the fimctional activity of the corresponding
`
`

`

`WO 2010/123369
`
`PCT/NL2010/050230
`
`oligonucleotide which does not comprise an inosine and/or a base able to
`
`form a wobble base pair. Such functional activity may be as measured in a
`
`muscular tissue or in a muscular cell of an individual or in vitro in a cell by
`
`comparison to the functional activity of a corresponding oligonucleotides not
`
`comprising an inosine and/or a base able to form a wobble base pair. The
`
`assessment of the functionality may be carried out at the mRNA level,
`
`preferably using RT-PCR. The assessment of the functionality may be
`
`carried out at the protein level, preferably using western blot analysis or
`
`immunofluorescence analysis of cross-sections.
`
`10
`
`Within the context of the invention, an inosine and/or a base able to form a
`
`wobble base pair as present in an oligonucleotide is/are present in a part of
`
`said oligonucleotide which is complementary to at least part of a dystrophin
`
`pre-mRNA exon or at least part of a non-exon region of a dystrophin pre-
`
`15
`
`mRNA said part being a contiguous stretch comprising at least 8
`
`nucleotides. Therefore, in a preferred embodiment, an oligonucleotide
`
`comprising an inosine and/or a nucleotide containing a base able to form a
`
`wobble base pair or a functional equivalent thereof, wherein the
`
`oligonucleotide, or a functional equivalent thereof, comprises a sequence
`
`20
`
`which is complementary to at least part of a dystrophin pre-mRNA exon or
`
`at least part of a non-exon region of a dystrophin pre-mRNA said part being
`
`a contiguous stretch comprising at least 8 nucleotides and wherein said
`
`inosine and/or a nucleotide containing a base able is/are present within the
`
`oligonucleotide sequence which is complementary to at least part of a
`
`25
`
`dystrophin pre-mRNA as defined in previous sentence.
`
`However, as later defined herein such inosine and/or a base able to form a
`
`wobble base pair may also be present in a linking moiety present in an
`
`oligonucleotide of the invention. Preferred linking moieties are later defined
`
`30
`
`herein.
`
`

`

`WO 2010/123369
`
`PCT/NL2010/050230
`
`In a preferred embodiment, such oligonucleotide is preferably a
`
`medicament. More preferably, said medicament is for preventing or
`
`treating Duchenne Muscular Dystrophy or Becker Muscular Dystrophy in
`
`an individual or a patient. As defined herein a DMD pre-mRNA preferably
`
`means the pre-mRNA of a DMD gene of a DMD or BMD patient. A patient
`
`is preferably intended to mean a patient having DMD or BMD or a patient
`
`susceptible to develop DMD or BMD due to his or her genetic background.
`
`In the case of a DMD patient, an oligonucleotide used will preferably
`
`correct at least one of the DMD mutations as present in the DMD gene of
`
`10
`
`said patient and therefore will preferably create a dystrophin that will look
`
`like a BMD dystrophin: said dystropin will preferably be a functional
`
`dystrophin as later defined herein.
`
`In the case of a BMD patient, an oligonucleotide as used will preferably
`
`correct at least one of the BMD mutations as present in the DMD gene of
`
`15
`
`said patient and therefore will preferably create a, or more of a, dystrophin,
`
`which will be more functional than the dystrophin which was originally
`
`present in said BMD patient. Even more preferably, said medicament
`
`provides an individual with a functional or more (of a) functional
`
`dystrophin protein and/or mRNA and/or at least in part decreases the
`
`20
`
`production of an aberrant dystrophin protein and/or mRNA.
`
`Preferably, a method of the invention by inducing and/or promoting
`
`skipping of at least one exon of the DMD pre-mRNA as identified herein in
`
`one or more cells, preferably muscle cells of a patient, provides said patient
`
`with an increased production of a more (of a functional dystrophin protein
`
`25
`
`and/or mRNA and/or decreases the production of an aberrant or less
`
`functional dystrophin protein and/or mRNA in said patient.
`
`Providing a patient with a more functional dystrophin protein and/or
`
`mRNA and/or decreasing the production of an aberrant dystrophin protein
`
`and/or mRNA in said patient is typically applied in a DMD patient.
`
`30
`
`Increasing the production of a more functional or functional dystrophin
`
`and/or mRNA is typically applied in a BMD patient.
`
`

`

`WO 2010/123369
`
`PCT/NL2010/050230
`
`10
`
`Therefore a preferred method is a method, wherein a patient or one or more
`
`cells of said patient
`
`is provided with an increased production of a more
`
`fimctional or fimctional dystrophin protein and/or mRNA and/or wherein
`
`the production of an aberrant dystrophin protein and/or mRNA in said
`
`patient
`
`is decreased, wherein the level of said aberrant or more fimctional
`
`dystrophin protein and/or mRNA is assessed by comparison to the level of
`
`said dystrophin and/or mRNA in said patient at the onset of the method.
`
`As defined herein, a functional dystrophin is preferably a wild type
`
`10
`
`dystrophin corresponding to a protein having the amino acid sequence as
`
`identified in SEQ ID NO: 1.Afunctional dystrophin is preferably a
`
`dystrophin, which has an actin binding domain in its N terminal part (first
`
`240 amino acids at the N terminus), a cystein-rich domain (amino acid 3361
`
`till 3685) and a Cterminal domain (last 325 amino acids at the Cterminus)
`
`15
`
`each of these domains being present in a wild type dystrophin as known to
`
`the skilled person. The amino acids indicated herein correspond to amino
`
`acids of the wild type dystrophin being represented by SEQ ID NO: 1. In
`
`another embodiment, a fimctional dystrophin is a dystrophin, which
`
`exhibits at least to some extent an activity of a wild type dystrophin. "At
`
`20
`
`least to some exten " preferably means at least 30%, 40%, 50%, 60%, 70%,
`
`80%, 90%, 95% or 100% of a corresponding activity of a wild type functional
`
`dystrophin. In this context, an activity of a wild type dystrophin is
`
`preferably binding to actin and to the dystrophin-associated glycoprotein
`
`complex (DGC)56. Binding of dystrophin to actin and to the DGC complex
`
`25
`
`may be visualized by either co-immunoprecipitation using total protein
`
`extracts or immunofluorescence analysis of cross-sections, from a biopsy of
`
`a muscle suspected to be dystrophic, as known to the skilled person.
`
`Individuals suffering from Duchenne muscular dystrophy typically
`
`have a mutation in the gene encoding dystrophin that prevents synthesis of
`
`30
`
`the complete protein, i.e a premature stop prevents the synthesis of the C-
`
`terminus of the protein. In Becker muscular dystrophy the dystrophin gene
`
`

`

`WO 2010/123369
`
`PCT/NL2010/050230
`
`11
`
`also comprises a mutation compared to the wild type but the mutation does
`
`typically not include a premature stop and the C-terminus of the protein is
`
`typically synthesized. As a result a functional dystrophin protein is
`
`synthesized that has at least the same activity in kind as a wild type
`
`5
`
`protein, although not necessarily the same amount of activity.
`
`In a
`
`preferred embodiment, a functional dystrophin protein means an in frame
`
`dystrophin gene.The genome of a BMD individual typically encodes a
`
`dystrophin protein comprising the N terminal part (first 240 amino acids at
`
`the N terminus), a cystein-rich domain (amino acid 3361 till 3685) and a C
`
`10
`
`terminal domain (last 325 amino acids at the Cterminus) but its central
`
`rod shaped domain may be shorter than the one of a wild type dystrophin 56.
`
`Exon -skipping for the treatment of DMD is preferably but not exclusively
`
`directed to overcome a premature stop in the pre-mRNA by skipping an
`
`exon in the rod- domain shaped domain to correct the reading frame and
`
`15
`
`allow synthesis of remainder of the dystrophin protein including the C-
`
`terminus, albeit that the protein is somewhat smaller as a result of a
`
`smaller rod domain. In a preferred embodiment, an individual having DMD
`
`and being treated using an oligonucleotide as defined herein will be
`
`provided a dystrophin, which exhibits at least to some extent an activity of
`
`20
`
`a wild type dystrophin. More preferably, if said individual is a Duchenne
`
`patient or is suspected to be a Duchenne patient, a functional dystrophin is
`
`a dystrophin of an individual having BMD: preferably said dystrophin is
`
`able to interact with both actin and the DGC, but its central rod shaped
`
`domain may be shorter than the one of a wild type dystrophin (Aartsma-
`
`25
`
`Rus et al (2006, ref 56). The central rod domain of wild type dystrophin
`
`comprises 24 spectrin-like repeats 56. For example, a central rod shaped
`
`domain of a dystrophin as provided herein may comprise 5 to 23, 10 to 22 or
`
`12 to 18 spectrin-like repeats as long as it can bind to actin and to DGC.
`
`Decreasing the production of an aberrant dystrophin in said patient or in a
`
`30
`
`cell of said patient may be assessed at the mRNA level and preferably
`
`means that 99%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5% 01‘ less
`
`

`

`WO 2010/123369
`
`PCT/NL2010/050230
`
`12
`
`of the initial amount of aberrant dystrophin mRNA, is still detectable by RT
`
`PCR. An aberrant dystrophin mRNA or protein is also referred to herein as
`
`a non-functional or less to non-functional or semi-functional dystrophin
`
`mRNA or protein. A non-functional pre-mRNA dystrophin is preferably
`
`leads to an out of frame dystrophin protein, which means that no
`
`dystrophin protein will be produced and/or detected. A non functional
`
`dystrophin protein is preferably a dystrophin protein which is not able to
`
`bind actin and/or members of the DGC protein complex. Anon-functional
`
`dystrophin protein or dystrophin mRNA does typically not have, or does not
`
`10
`
`encode a dystrophin protein with an intact C-terminus of the protein.
`
`Increasing the production of a functional dystrophin in said patient or in a
`
`cell of said patient may be assessed at the mRNA level (by RT-PCR
`
`15
`
`analysis) and preferably means that a detectable amount of a functional or
`
`in frame dystrophin mRNA is detectable by RT PCR. In another
`
`embodiment, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or
`
`more of the detectable dystrophin mRNA is a functional or in frame
`
`dystrophin mRNA.
`
`20
`
`Increasing the production of a functional dystrophin in said patient or in a
`
`cell of said patient may be assessed at the protein level (by
`
`immunofluorescence and western blot analyses) and preferably means that
`
`a detectable amount of a functional dystrophin protein is detectable by
`
`immunofluorescence or western blot analysis. In another embodiment, 1%,
`
`25
`
`5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the
`
`detectable dystrophin protein is a functional dystrophin protein.
`
`An increase or a decrease is preferably assessed in a muscular tissue or in a
`
`muscular cell of an individual or a patient by comparison to the amount
`
`30
`
`present in said individual or patient before treatment with said molecule or
`
`composition of the invention. Alternatively, the comparison can be made
`
`

`

`WO 2010/123369
`
`PCT/NL2010/050230
`
`13
`
`with a muscular tissue or cell of said individual or patient, which has not
`
`yet been treated with said oligonucleotide or composition in case the
`
`treatment
`
`is local.
`
`In a preferred method, one or more symptom(s) from a DMD or a BMD
`
`patient
`
`is/are alleviated and/or one or more characteristic(s) of a muscle
`
`cell or tissue from a DMD or a BMD patient
`
`is/are alleviated using a
`
`molecule or a composition of the invention. Such symptoms may be assessed
`
`on the patient self. Such characteristics may be assessed at the cellular,
`
`10
`
`tissue level of a given patient. An alleviation of one or more characteristics
`
`may be assessed by any of the following assays on a myogenic cell or
`
`muscle cell from a patient: reduced calcium uptake by muscle cells,
`
`decreased collagen synthesis, altered morphology, altered lipid
`
`biosynthesis, decreased oxidative stress, and/or improved muscle fiber
`
`15
`
`function, integrity, and/or survival. These parameters are usually assessed
`
`using immunofluorescence and/or histochemical analyses of cross sections
`
`of muscle biopsies.
`
`Alleviating one or more symptom(s) of Duchenne Muscular Dystrophy or
`
`20
`
`Becker Muscular Dystrophy in an individual using a molecule or a
`
`compositionof the invention may be assessed by any of the following assays:
`
`prolongation of time to loss of walking, improvement of muscle strength,
`
`improvement of the ability to lift weight, improvement of the time taken to
`
`rise from the floor, improvement in the nine-meter walking time,
`
`25
`
`improvement in the time taken for four- stairs climbing, improvement of the
`
`leg function grade, improvement of the pulmonary function, improvement of
`
`cardiac function, improvement of the quality of life. Each of these assays is
`
`known to the skilled person. As an example, the publication of Manzur at al
`
`(2008, ref 58) gives an extensive explanation of each of these assays. For
`
`30
`
`each of these assays, as soon as a detectable improvement or prolongation
`
`of a parameter measured in an assay has been found, it will preferably
`
`

`

`WO 2010/123369
`
`PCT/NL2010/050230
`
`14
`
`mean that one or more symptoms of Duchenne Muscular Dystrophy or
`
`Becker Muscular Dystrophy has been alleviated in an individual using a
`
`molecule or composition of the invention. Detectable improvement or
`
`prolongation is preferably a statistically significant improvement or
`
`prolongation as described in Hodgetts et al (2006, ref 57). Alternatively,
`
`the
`
`alleviation of one or more symptom(s) of Duchenne Muscular Dystrophy or
`
`Becker Muscular Dystrophy may be assessed by measuring an
`
`improvement of a muscle fiber function, integrity and/or survival as later
`
`defined herein.
`
`10
`
`An oligonucleotide as used herein preferably comprises an antisense
`
`oligonucleotide or antisense oligoribonucleotide. In a preferred embodiment
`
`an exon skipping technique is applied. Exon skipping interferes with the
`
`natural splicing processes occurring within a eukaryotic cell. In higher
`
`15
`
`eukaryotes the genetic information for proteins in the DNA of the cell is
`
`encoded in exons which are separated from each other by intronic
`
`sequences. These introns are in some cases very long. The transcription
`
`machinery of eukaryotes generates a pre-mRNA which contains both exons
`
`and introns, while the splicing machinery, often already during the
`
`20
`
`production of the pre-mRNA, generates the actual coding region for the
`
`protein by splicing together the exons present in the pre-mRNA.
`
`Exon- skipping results in mature mRNA that lacks at least one
`
`skipped exon. Thus, when said exon codes for amino acids, exon skipping
`
`leads to the expression of an altered product. Technology for exon-skipping
`
`25
`
`is currently directed towards the use of antisense oligonucleotides (AONs).
`
`Much of this work is done in the mdx mouse model for Duchenne muscular
`
`dystrophy. The mdx mouse carries a nonsense mutation in