WO2011078797A2

WO2011078797A2 - Antisense oligonucleotides and uses threreof

Info

Publication number: WO2011078797A2
Application number: PCT/SG2010/000479
Authority: WO
Inventors: Dwi Pramono Sarengat; Woon Chee Yee; Poh San Lai; Keng Boon Wee
Original assignee: Singapore Health Services Pte. Ltd; Agency For Science, Technology And Research
Priority date: 2009-12-22
Filing date: 2010-12-22
Publication date: 2011-06-30
Also published as: WO2011078797A3

Abstract

The present invention refers to an antisense oligonucleotide having a L3 score of about < 0.1; a L4_OR(5´) score of about < 0.15; a L4_OR(3´) score of about < 0.12; a P_ESE score of about > 70 %; and the antisense oligonucleotide binds to a target exon pre-mRNA starting in the first one third of the target exon pre-mRNA, counting from 5´ to 3' as well as method of a method of selecting at least one antisense oligonucleotide from a plurality of antisense oligonucleotides targeting a target sequence on one of a plurality of exons on a pre-mRNA.

Description

ANTISENSE OLIGONUCLEOTIDES AND USES THEREOF

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority of US provisional application No. 61/288,877, filed December 22, 2009, the contents of it being hereby incorporated by reference in its entirety for all purposes.

FIELD OF THE INVENTION

[0002] The present invention refers to the field of molecular genetics and bioinformatics. In particular the present invention refers to a method of selecting at least one antisense oligonucleotide from a plurality of antisense oligonucleotides targeting a target sequence on an exon on a pre-mRNA and to specific antisense oligonucleotides.

BACKGROUND OF THE INVENTION

[0003] Antisense oligonucleotides (AONs) are synthetic single-stranded DNA or RNA molecules, typically consisting of 15 to 30 nucleotides that are complementary to a specific sequence in the targeted RNA. After introduction into a cell, the "antisense" oligonucleotide acts either in the cytoplasm or nucleus by hybridizing to the corresponding RNA sequence through Watson-Crick binding. By binding to the RNA sequence, it can initiate mRNA degradation, inhibit protein translation, or modulate pre-mRNA splicing, depending on the targeted site of the pre-mRNA or mRNA, on whether the molecule is DNA or RNA, and on structural modifications made to the antisense oligonucleotide molecule. AON agents that have received FDA approval or are undergoing review for clinical use include Fpmivirsen (Vitravene) for CMV- retinitis in AIDS and Genasense in chronic lymphocytic leukemia.

[0004] As compared to conventional gene therapy, AON has significant advantages in terms of delivery and safety. Use of a viral vector, commonly practiced for gene therapy, is not necessary for delivery. This avoids major problems associated with viral vectors, such as adverse immunologic reactions and; other side effects. With regards to the dosage, because AONs are synthetic molecules, control of AON dosage and concentration is efficient, unlike viral vector packed cDNA for gene therapy. AONs also work via the innate gene transcription and translation processes of the cell. Compared to conventional drugs, AONs may have some potential advantages, They may potentially show stronger binding affinity for their targets, induce longer therapeutic effect with action at RNA level, and their therapeutic effects may be readily measured, AONs are also more readily manufactured. On the other hand, one major drawback of AONs comes from the fact that they are often highly specific for the molecular defect targeted. As a result, for example, different AONs will have to be designed for different mutations of the genetic disease to be treated, and requiring repeated administration.

[0005] ; Apart from their well-documented applications to suppress gene expression, AONs have been used to modulate pre-inessenger RNA (pre-mRNA) splicing as potential therapeutic strategy for genetic diseases such as Duchenne muscular dystrophy or DMD, thalassemia, spinal muscular atrophy (SMA), Hutchinson-Gilford progeria syndrome, frontotemporal dementia with parkinsonism, ocular albinism and cancer.

[0006] For example, for Duchenne muscular dystrophy or DMD, a fatal X-linked disorder affecting 1 in 3300 newborn males caused by mutations in the dystrophin gene, there is currently no effective therapy. In one third of patients, the mutation arose spontaneously. The severity and frequency of DMD have urgently driven research to find effective therapy of this devastating disease. Among the therapeutic strategies proposed are gene transfer therapy, over-expression ^'. of utrophin (a protein functionally related to dystrophin), targeted gene repair, "readthrough" of stop codon and AON mediated pre-mRNA splicing modulation to restore dystrophin expression.

[0007] In 65% of DMD patients, the dystrophin gene mutation is a deletion, while in the remaining patients, it is a point mutation, micro-deletion or duplication. Whatever the specific mutation, it almost invariably results in a loss of reading frame with premature stop codon, or in a nonsense mutation causing premature termination. The strategy of AON therapy for DMD involves induction of selective exon skipping to restore the mRNA reading frame for frame-shift; mutations caused by deletions or duplications, of less commonly, to remove the in-frame exon carrying ; a nonsense mutation. The AON is designed to induce exon skipping by competitive binding to its target site to block splicing factors during transcription. During the splicing process involving pre-mRNA, splicing factors associated with the 60S splicing machinery called the spliceosome removes the introns while retaining the exons. These splicing factors bind to important sequences in pre-mRNA (cis-elements), which include donor and acceptor splice sites, branch points (BP), pyrimidine tracts and exon splicing enhancersi (ESEs). Blocking these pre-mRNA sites with AONs prevents the spliceosome from identifying the targeted region as an exon, and thereby causes the exon to be removed along with the introns.

[0008] In case of DMD, AON induced exon skipping will result in a shorter dystrophin protein which, nevertheless, frequently remains functional, because the very large dystrophin gene carries large non-critical sequences. It is expected to reverse the DMD phenptype or to reduce disease severity. AONs have been demonstrated to restore widespread dystrophin expression in DMD mouse and dog models when given locally . as well as systemically. Currently, the first phase I/I I clinical trials of AON in amenable DMD patients have just been completed i by two centers,; one in the UK and the other in the Netherlands.

[0009] i For example, to be efficacious in inducing exon skipping, AONs have to bind to the correct target site before splicing factors. Thus, besides accurate identification of cis- elements in the target site, the accessibility of the site for the AON to bind is also vital. While biomformatics tools for identifying czs-elements have been developed and are available for example, ESEFinder (Cartegni L ct al, (2003) ESEfinder; A web resource to identify exonic splicing enhancers Nucleic Acids Res 31 : 3568-3571) and Rescue-ESE (Fairbrother WG, Yeh RF, Sharp P A, Burge CB (2002) Predictive identification of exonic splicing enhancers in human genes. Science 297: 1007-1013), the prediction of accessibility of the target site for AON binding remains an issue in designing efficient AONs. Target site accessibility is theoretically dependent oh secondary structure formation caused by complementary base pairing of pre-mRNA nucleotides. Paired nucleotides will not be available for AON binding. The conventional application of software for prediction of pre-mRNA secondary structures, e.g. Mfoid (Zuker, M., 2003, Nucleic Acid Res, vol.31 , pp.3406), to select AON target sites is unsatisfactory. This is . because; Mfold] and other; energy-minimizing algorithms predict secondary structures from a wide range of similarly energetically favored configurations based purely on a static segment of input sequence: Hence, such prediction of absence of secondary structures does not always correlate to efficacy of an AON. For example, Aartsma- Rus et al. (Aartsma-Rus, A., et al., 2005, Oligonucleotides,; vol.15, pp.284) concluded that they still have no clear insight into the accessibility of the targeted sequence within the folded pre-mRNA structure.

[0010] Current approaches in designing AONs for exon skipping are generally semi- empirical which entail a combination of trial-and-error ahd usage of design variables. Wilton et al. (Wilton, S.D., 2007, Molecular Therapy, vol.15, pp.1288) designed 470 preliminary AONs to locate motifs involved in processing the dystrophin pre-mRNA and followed by a second round of overlapping AONs targeting the motifs identified. Aartsma-Rus et al. (Aartsma-Rus, A., 2005, Oligonucleotides, vol.15, pp.284 & Aartsma-Rus, A., et al., 2009, Therapy, vol.17, pp.548) designed AONs primarily based on targeting an open region in the secondary structure of the target exon as predicted by Mfokl. As a primary design variable was used, a trial-and-error approach is inevitable due to many possible target sites. Popplewell et al. (Popplewell, LJ., et al., 2009, Molecular Therapy, vol.17, pp.554) employed four design variables (hybridization array data, target length, presence of ESE and Exonic Splicing Silencers motifs) and is the most systematic among them although the strategy of designing a series of AONs targeting overlapping sites is used. It is perhaps not surprising that Popplewell et al. (supra) obtain higher hit rates for both efficient and effective AONs than Wilton et al. (supra) and Aartsma-Rus et al. (supra).

[001 1 ] A prerequisite for rational design of AONs is the selection of a set of key design variables. Several retrospective studies have attempted to identify factors that could correlate with AON efficiencies observed in experiments. In general, the inferred variables differ from the different studies, and their effectiveness to design efficient AONs has not been experimentally validated. Some of these design variables even impinge on each other.

[0012] Design variables inferred are as follows: distance from exon 5', target length, number of ESE motifs (SF2/ASF, SC35, SRp40 and Tra2/3), binding energies (AON-target, AON-AON complex and AON-AON), (static) binding accessibility of target site, number of target site GC content, AON melting temperature, proportion of overlaps with hexamer-array hybridization peaks and co-transcriptional binding accessibility of pre-mRNA. Note that not all factors were subjected to statistical tests. Moreover, Aartsma-Rus et al. (supra) and Popplewell et al. (supra) performed linear discriminant analysis to identify a set of factors that could classify 80% of AONs as effective or ineffective correctly; Aartsma-Rus et al. (supra) derived four design variables of which three of them are based on ESEs (number of RESCUE-ESE hexamers, ESE-Tra2jS value, ESE-SC35 value, and the binding energy of AON-target exon) whereas Popplewell et al. (supra) derived three (proportion of ESE- SF2/ASF motif, AON length, and proportion of overlap with hexamer-array hybridization peaks).

[0013] Clearly, retrospective analyses from different labs using their own AON datasets do not reveal identical factors. Nonetheless, three most consistently reported factors arc distance from exon 5 ', target length, and presence of ESE motifs (if specific motifs are ignored). However, they were not reported in combination for all AON datasets, and thus confounding their significarice. Furthermore, not all of them are present in each set of design variables ^'. derived from linear discriminant analysis of two independent datasets. Besides, these analyses discriminate effective rather than efficient AONs, and for this reason the derived sets of design variables may not be optimized for designing efficient AONs. And most importantly, their; effectiveiiess has not been validated experimentally. If consistency of a factor; is a reasonable; indication ; of its importance on AON efficiency, its statistical significance to correlate AON efficiency must be tested using datasets from independent labs [0014] It is therefore an object of the present invention to provide an improved method which allows designing antisense oligonucleotides which can be efficiently used for the treatment of genetic diseases.

SUMMARY OF THE INVENTION

[0015] In a first aspect, a method for selecting at least one antisense oligonucleotide from a plurality of antisense oligonucleotides targeting a target sequence on an exon on a pre- mRNA may be provided. The method may include: determining for each antisense oligonucleotide of the plurality of antisense oligonucleotides a first evaluation value according to a first evaluation criterioh; determining a first subset of the plurality of antisense oligonucleotides based on the first evaluation values; determining for each antisense oligonucleotide of the first subset a second evaluation value according to a second evaluation criterion; and determining a second subset of the first subset of antisense oligonucleotides based on the second evaluation values.

[0016] According to various embodiments, at least one of the first evaluation criterion and the second evaluation criterion rriay be related to binding of the antisense oligonucleotide with the exon. ^; · ; ;

[0017] According to various embodiments, determining the first subset may include: ordering the antisense oligonucleotides of the plurality of antisense oligonucleotides according to the first evaluation values; and selecting as the first subset a pre-determined number or a pre-determined percentage of antisense oligonucleotides of the plurality of antisense oligonucleotides based on the ordering of the plurality of antisense oligonucleotides. ;· [0018] According to various embodiments, determining the second subset may include: ordering the antisense oligonucleotides of the first subset according to the second evaluation values; and selecting as the second subset a pre-determined number or a pre-determined percentage of antisense oligonucleotides of the first subset based on the ordering of the antisense oligonucleotides of the first subset.

[0019] According to various embodiments, at least one of the first evaluation criterion and the second evaluation criterion may include or may be at least one of an L3 score for the target sequence, a L4_OR(5 ^') score for the target sequence, a L4_OR(3 ') score for the target sequence, a cumulative position score (ACP) for the antisense oligonucleotide, a PE_SE score for the target sequence, and a length of the antisense oligonucleotide.

[0020] According to various embodiments, the method may further include: determining for each antisense oligonucleotide of the second subset a third evaluation value according to a third evaluation criterion; and determining a third subset of the second subset of antisense oligonucleotides based on the third evaluation values.

[0021 ] According to various embodiments, the third evaluation criterion may be related to binding of the antisense oligonucleotide with the exon.

[0022] According to various embodiments, determining the third subset may include: ordering the antisense oligonucleotides of the second subset according to the third evaluation values; and selecting as the third subset a pre-determined number or a pre-determined percentage of antisense oligonucleotides of the second subset based on the ordering of the antisense oligonucleotides of the second subset.

[0023] According to various embodiments, the third evaluation criterion may include or may be at least one of an L3 score for the target sequence, a L4_OR(5 ^') score for the target sequence, a L4_OR(3 ') score for the target sequence, a cumulative position score (ACP) for the antisense oligonucleotide, a PESE score for the target sequence, and a length of the antisense oligonucleotide.

[0024] According to various embodiments, the method may further include: determining for each antisense oligonucleotide of the third subset a fourth evaluation value according to a fourth evaluation criterion; and determining a fourth subset of the third subset of antisense oligonucleotides based on the fourth evaluation values.

[0025] According to various embodiments, the fourth evaluation criterion may be related to binding of the antisense oligonucleotide with the exon.

[0034] According to various embodiments, determining the sixth subset may include: ordering the antisense oligonucleotides of the fifth subset according to the sixth evaluation values; and selecting as the sixth subset a pre-determined number or a pre-determined percentage of antisense oligonucleotides of the fifth; subset based on the ordering of the antisense oligonucleotides of the fifth subset.

[0035] According to various embodiments, the sixth evaluation criterion may include or may be at least one of an L3 score for the target sequence, a L4_OR(5 ^') score for; the target sequence, a L4_OR(3 ') score for the target sequence, a cumulative position score (ACP) for the: antisense oligonucleotide a PESE score for the target sequence, and a length of the antisense oligonucleotide.

[0036] According to various embodiments, the first selection criterion may include or may be an L3 score for the target sequence, the second selection criterion may include or may be a L4_ OR(5 ') score for the target sequence, the third selection criterion may include or may be a L4_OR(3 ^') score for the target sequence, the fourth selection criterion may include or may be a cumulative position score (ACP) for the antisense oligonucleotide, the fifth selection criterion may include or may be a P_ESE score for the target sequence, and the sixth selection criterion may include or may be a length of the antisense oligonucleotide.

[0037] According to various embodiments, the first selection criterion may include or may be an L3 score for the target sequence, the second selection criterion may include or may be a L4_OR(5 ^') score for the target sequence, the third selection criterion may include or may be a L4_OR(3 ') score for the target sequence, the fourth selection criterion may include or may be a cumulative position score (AGP) for the antisense oligonucleotide, the fifth selection criterion may include or may be a length of the antisense oligonucleotide, and ; the sixth selection criterion may include or may be a PE_SE score for the target sequence.

[0038] According to various embodiments, the first selection criterion may include or may be an L3 score for the target sequence, the second selection criterion may include or may be a L4_OR(3 ') score for the target sequence, the third selection criterion may include or may be a L4_OR(5 ') score for the target sequence, the fourth selection criterion may include or may be a cumulative position score (ACP) for the antisense oligonucleotide, the fifth ; selection criterion may include or may be a PESE score for the target sequence, and the sixth selection criterion may include or may be a length of the antisense oligonucleotide. [0039] : According to various embodiments, the first selection criterion may include or may be an L3 score for the target sequence, the second selection criterion may include or may be a L4_OR(3 ') score for the target sequence, the third selection; criterion may include or may be a L4_OR(5 ^') score for the target sequence, the fourth selection criterion may include or may be a cumulative position score (ACP) for the antisense oligonucleotide, the fifth selection criterion may include or may be a length of the antisense oligonucleotide, and the sixth selection criterion may include or may be a PESE spore for this target . sequence.

[0040] According to various embodiments, the first selection criterion may include or may be a cumulative position score (ACP) for the antisense oligonucleotide, the second selection criterion may include or may be an L3 score for the target sequence, the third selection criterion may include or may be : a L4_OR(5 ') score for the target sequence, the fourth selection criterion may include or may be a L4_OR(3 ') score for the target sequence, the fifth selection criterion may include or may be a PESE score for the target sequence, and the sixth selection criterion may include or may be a length of the antisense oligonucleotide.

[0041] According to various embodiments, the first selection criterion may include or may be a cumulative position score (ACP) for the antisense oligonucleotide, the second selection criterion may include or may be ; an L3 score for the target sequence, the third selection criterion may include or may be a L4_OR(3 ') score for the target sequence, the fourth selection criterion may include or may be a L4_OR(5') score for the target sequence, the fifth selection criterion may include or may be a PESE score for the target sequence, and the sixth selection criterion may include or may be a length of the antisense oligonucleotide.

[0042] According to various embodiments, the first selection criterion may include or may be a cumulative position score (ACP) for. the antisense oligonucleotide, the second selection criterion; may include or may be an L3 score for the target sequence, the third selection criterion may include or may be a L4_OR(5 ') score for the target sequence, the fourth selection criterion may include or may be a L4_OR(3 ') score for the target sequence, the fifth selection criterion may include or may be a length of the antisense oligonucleotide, and the sixth selection criterion may include or may be a PESE score for the target sequence.

[0043] According to various embodiments, the first selection criterion may include or may be a cumulative position score (ACP) for the antisense oligonucleotide, the second selection criterion may include or may be an L3 score for the target sequence, the third selection criterion may include or may be a L4_OR(3 ') score for the target sequence, the fourth selection criterion may include or may be a L4_OR(5 ^') score for the target sequence, the fifth selection criterion may include or may be a length of the antisense oligonucleotide, the sixth selection criterion may include or may be a P_ESE score for the target sequence.

[0044] According to various embodiments, the first selection criterion may include or may be a L4_OR(5 ') score for the target sequence, the second selection criterion may include or may be a L4_OR(3 ') score for the target sequence, the third selection criterion may include or may be an L3 score for the target^' sequence, the fourth selection criterion may include or may be a cumulative position ; score (ACP) for the antisense oligonucleotide, the fifth selection criterion may include or may be a PE_SE score for the target sequence, the sixth selection criterion may include or may be a length of the antisense oligonucleotide.

[0045] According to various embodiments, the first selection criterion may include or may be a L4_OR(3 ') score for the target sequence, the second selection criterion may include or may be a L4_OR(5 ') score for the target sequence, the third selection criterion may include or may be an L3 score for the target sequence, the fourth selection criterion may include or may be a cumulative position score (ACP) for the antisense oligonucleotide, the fifth selection criterion may include or may be a PESE score for the target sequence, and the sixth selection criterion may include or may be a length of the antisense oligonucleotide.

[0046] According to various embodiments, the first selection criterion may include or may be a L4_OR(5 ') score for the target sequence, the second selection criterion may include or may be a L4_OR(3 ') score for the target sequence, the third selection criterion may include or may be an L3 score for the target sequence, the fourth selection criterion may include or may be a cumulative position score (ACP) for the antisense oligonucleotide, the fifth selection criterion may include or may be a length of the antisense oligonucleotide, and the sixth selection criterion may include or may be a P_ESE score for the target sequence.

[0047] According to various embodiments, the first selection criterion may include or may be a L4_OR(3 ') score for the target sequence, the second selection criterion may include or may be a L4_OR(5 ') score for the target sequence, the third selection criterion may include or may be an L3 score for the target sequence, the fourth selection criterion may include or may be a cumulative position score (ACP) for the antisense oligonucleotide, the fifth selection criterion may include or may be a length of the antisense oligonucleotide, and the sixth selection criterion may include or may be a P_ESE score for the target sequence. [0048] In asecond aspect, there is provided a method of selecting at least one antisense oligonucleotide from a plurality of antisense oligonucleotides targeting a target sequence on one of a plurality of exons on a pre-mRNA. The method includes:

selecting an exon on the pre-mRNA;

■ generating a plurality of antisense oligonucleotides for target sequences on the exon pre- mRNA, wherein each of the antisense oligonucleotides has a length of between 15 to 35 nucleotides;

determining a L3 score for a target sequence, a L4_OR(5 ') score for a target sequence, a L4_OR(3 ^') score for; a target sequence, a cumulative position score (ACP score) for an antisense oligonucleotide, a PE_SE score for a target sequence

ordering the antisense oligonucleotides at first according to the lowest L3 score in a first group;

selecting a specified number of antisense oligonucleotides starting with the antisense oligonucleotide with the lowest L3 score from the first group and ordering them secondly according to the lowest L4_OR(5 ') score in a second group;

selecting a specified number of antisense oligonucleotides starting with the antisense oligonucleotide with the lowest L4_OR(5 ') from the second group and ordering them thirdly according to the lowest L4_OR(3 ') score in a third group;

selecting a specified number of antisense oligonucleotides starting with the antisense oligonucleotide with the lowest L4_OR(3 ') from the third group and ordering them fourthly according to the lowest ACP score in a fourth group;

selecting a specified number of antisense oligonucleotides starting with the antisense oligonucleotide with the lowest ACP score from the fourth group and ordering them fifthly according to the highest P_ESE score in a fifth group;

selecting a specified number of antisense oligonucleotides starting with the antisense oligonucleotide with the highest PESE score from the fifth group and ordering them sixthly according to the length starting with the longest antisense oligonucleotides in a sixth group;

selecting at least one antisense oligonucleotides starting with the longest antisense oligonucleotide from the sixth group.

[0049] In other words, a first group of AONs may be provided, and the AONs in the first group may be ordered according to the L3 score, then the AONs in the first group with a lowest L3 score may be selected and may make up a second group. Then, the AONs in the second group may be ordered according to the L4_OR(5') score, and the AONs in the second group with a lowest L4_OR(5') score may be selected and may make up a third group. Then, the AONs in the third group may be ordered according to the L4_OR(3') score, and the AONs in the third group with a lowest L4_OR(3') score may be selected and may make up a fourth group. Then, the AONs in the fourth group may be ordered according to the ACP score, and the AONs in the fourth group with a lowest ACP score may be selected and may make up a fifth group. Then, the AONs in the fifth group may be ordered according to the PESE score, and the AONs in the fifth group with a highest PESE score may be selected and may make up a sixth group. Then, the AONs in the sixth group may be ordered according to the length, and the AONs in the sixth group with the highest length may be selected.

[0050] According to various embodiments, the method may include determining a L3 score for each target sequence, a L4_OR(5 ') score for each target sequence, a L4_OR(3 ') score for each target sequence, a cumulative position score (ACP score) for each antisense oligonucleotide, a PE_SE score for each target sequence.

[0051] According to various embodiments, the L3 score may be determined for each target sequence corresponding to an antisense oligonucleotide in the first group.

[0052] According to various embodiments, the L4_OR(5^?) score may be determined for each target sequence corresponding to an antisense oligonucleotide in the second group.

[0053] According to various embodiments, the L4_OR(3') score may be determined for each target sequence corresponding to an antisense oligonucleotide in the third group.

[0054] According to various embodiments, the ACP score may be determined for each antisense oligonucleotide in the fourth group.

[0055] According to various embodiments, the PE_SE score may be determined for each target sequence corresponding to an antisense oligonucleotide in the fifth group.

[0056] In a third aspect, an antisense oligonucleotide selector for selecting at least one antisense oligonucleotide from a plurality of antisense oligonucleotides targeting a target sequence on an exoh on a pre-mRNA may be provided. The antisense oligonucleotide selector may include: a first evaluation values determiner configured to determine for each antisense oligonucleotide of the plurality of antisense oligonucleotides a first evaluation value according to a first evaluation criterion; a first subset determiner configured to determine a first subset of the plurality of antisense oligonucleotides based on the first evaluation values; a second evaluation values determiner configured to determine for each antisense oligonucleotide of the first subset a second evaluation value according to a second evaluation criterion; and a second subset determiner configured to determine a second subset of the first subset of antisense oligonucleotides based on the second evaluation values.

[0057] According to various embodiments, at least one of the first evaluation criterion and the second evaluation criterion may be related to binding of the antisense oligonucleotide with the exon.

[0058] According to various embodiments, the first subset determiner may include: a first ordering circuit configured to order the antisense oligonucleotides of the plurality of antisense oligonucleotides according to the first evaluation values; and a first selecting circuit configured to select as the first subset a pre-determined number or a pre-determined percentage of antisense oligonucleotides of the plurality of antisense oligonucleotides based on the ordering of the plurality of antisense oligonucleotides.

[0059] According to various embodiments, the second subset determiner may include: a second ordering circuit configured to order the antisense oligonucleotides of the first subset according to the second evaluation values; and a second selecting circuit configured to select as the second subset a pre-determined number or a pre-determined percentage of antisense oligonucleotides of the first subset based on the ordering of the antisense oligonucleotides of the first subset.

[0060] According to various embodiments, at least one of the first evaluation criterion and the second evaluation criterion may include or may be at least one of an L3 score for the target sequence, a L4_OR(5 ^') score for the target sequence, a L4_OR(3 ^') score for the target sequence, a cumulative position score (ACP) for the antisense oligonucleotide, a PE_SE score for the target sequence, and a length of the antisense oligonucleotide.

[0061 ] According to various embodiments, the antisense oligonucleotide selector may further include: a third evaluation values determiner configured to determine for each antisense oligonucleotide of the second subset a third evaluation value according to a third evaluation criterion; and a third subset determiner configured to determine a third subset of the second subset of antisense oligonucleotides based on the third evaluation values.

[0062] According to various embodiments, the third evaluation criterion may be related to binding of the antisense oligonucleotide with the exon. [0063] According to various embodiments, the third subset determiner may include: a third ordering circuit configured to order the antisense oligonucleotides of the second subset according to the third evaluation values; and a third selecting circuit configured to select as the third subset a pre-determined number or a pre-determined percentage of antisense oligonucleotides of the second subset based on the ordering of the antisense oligonucleotides of the second subset.

[0064] According to various embodiments, the third evaluation- criterion may include or may be at least one of an L3 score for the target sequence, a L4_OR(5 ') score for the target sequence, a L4_OR(3 ^') score for the target sequence, a cumulative position score (ACP) for the antisense oligonucleotide, a P_ESE score for the target sequence, and a length of the antisense oligonucleotide.

[0065] According to various embodiments, the antisense oligonucleotide selector may further include: a fourth evaluation values determiner configured to determine for each antisense oligonucleotide of the third subset a fourth evaluation value according to a fourth evaluation criterion; and a fourth subset determiner configured to determine a fourth subset of the third subset of antisense oligonucleotides based on the fourth evaluation values.

[0066] According to various embodiments, the fourth evaluation criterion may be related to binding of the antisense oligonucleotide with the exon.

[0067] According to various embodiments, the fourth subset determiner may include: a fourth ordering circuit configured to order the antisense oligonucleotides of the third subset according to the fourth evaluation values; and a fourth selecting circuit configured to select as the fourth subset a pre-determined number or a pre-determined percentage of antisense oligonucleotides of the third subset based on the ordering of the antisense oligonucleotides of the third subset.

[0068] According to various embodiments, the fourth evaluation criterion may include or may be at least one of an L3 score for the target sequence, a L4_OR(5 ^') score for the target sequence, a L4_OR(3 ^') score for the target sequence, a cumulative position score (ACP) for the antisense oligonucleotide, a PESE score for the target sequence, and a length of the antisense oligonucleotide.

[0069] According to various embodiments, the antisense oligonucleotide selector may further include: a fifth evaluation values determiner configured to determine for each antisense oligonucleotide of the fourth subset a fifth evaluation value according to a fifth ^■' i s

evaluation criterion; and a fifth subset determiner configured to determine a fifth subset of the fourth subset of antisense oligonucleotides based on the fifth evaluation values.

[0070] According to various embodiments, the fifth evaluation criterion may be related to binding of the antisense oligonucleotide with the exon.

[0071 ] According to various embodiments, the fifth subset determiner may include: a fifth ordering circuit configured to order the antisense- oligonucleotides of the fourth subset according to the fifth evaluation values; and a fifth selecting circuit configured to select as the fifth subset a pre-determined number or a pre-determined percentage of antisense oligonucleotides of the fourth subset based on the ordering of the antisense oligonucleotides of the fourth subset.

[0072] According to various embodiments, the fifth evaluation criterion may include or may be at least one of an L3 score for the target sequence, a L4_OR(5 ') score for the target sequence, a L4_OR(3 ^') score for the target sequence, a cumulative position score (ACP) for the antisense oligonucleotide, a PE_SE score for the target sequence, and a length of the antisense oligonucleotide. ^'

[0073] According to various embodiments, the antisense oligonucleotide selector may further include: a sixth evaluation values determiner configured to determine for each antisense oligonucleotide of the fifth subset a sixth evaluation value according to a sixth evaluation criterion; and a sixth subset determiner configured to determine a sixth subset of the fifth subset of antisense oligonucleotides based on the sixth evaluation values.

[0074] According to various embodiments, the sixth evaluation criterion may be related to binding of the antisense oligonucleotide with the exon.

[0075] According to various embodiments, the sixth subset determiner may include: a sixth ordering circuit configured to order the antisense oligonucleotides of the fifth subset according to the sixth evaluation values; and a sixth selecting circuit configured to select as the sixth subset a pre-determined number or a pre-determined percentage of antisense oligonucleotides of the fifth subset based on the ordering of the antisense oligonucleotides of the fifth subset.

[0076] According to various embodiments, the sixth evaluation criterion may include or may be at least one of an L3 score for the target sequence, a L4_OR(5 ') score for the target sequence, a L4_OR(3 ') score for the target sequence, a cumulative position score (ACP) for the antisense oligonucleotide, a P_ESE score for the target sequence, and a length of the antisense oligonucleotide.

[0077] According to various embodiments, the first selection criterion may include or may be an L3 score for the target sequence, the second selection criterion may include or may be a L4_OR(5 '). score for the target sequence, the third selection criterion may include or may be a L4_OR(3 ') score for the target sequence^ the fourth selection criterion may include or may be a cumulative position score (ACP) for the antisense oligonucleotide, the fifth selection criterion may include or may be a P_ESE score for the target sequence, and the sixth selection criterion may include or may be a length of the antisense oligonucleotides.

[0078] According to various embodiments, the first selection criterion may include or may be an L3 score for the target sequence, the second selection criterion may include or may be a L4_OR(5 ') score for the target sequence, the third selection criterion may include or may be a L4_OR(3 ') score for the target sequence, the fourth selection criterion may include or may be a cumulative position score (ACP) for the antisense oligonucleotide, the fifth selection criterion may include or may be a length of the antisense oligonucleotide, and the sixth selection criterion may include or may be a P_ESE score for the target sequence.

[0079] According to various embodiments, the first selection criterion may include or may be an L3 score for the target sequence, the second selection criterion may include or may be a L4_OR(3 ') score for the target sequence, the third selection criterion may include or may be a L4_OR(5 ') score for the target sequence, the fourth selection criterion may include or may be a cumulative position score (ACP) for the antisense oligonucleotide, the fifth selection criterion may include or may be a P_ESE score for the target sequence, and the sixth selection criterion may include or may be a length of the antisense oligonucleotide.

[0080] According to various embodiments, the first selection criterion may include or may be an L3 score for the target sequence, the second selection criterion may include or may be a L4_OR(3 ^') score for the target sequence, the third selection criterion may include or may be a L4_OR(5') score for the target sequence, the fourth selection criterion may include or may be a cumulative position score (ACP) for the antisense oligonucleotide, the fifth selection criterion may include or may be a length of the antisense oligonucleotide, and the sixth selection criterion may include or may be a P_ESE score for the target sequence.

[0081] According to various embodiments, the first selection criterion may include or may be a cumulative position score (ACP) for the antisense oligonucleotide, the second selection criterion may include or may be an L3 score for the target sequence, the third selection criterion may include or may be a L4_OR(5 ^') score for the target sequence, the fourth selection criterion may include or may be a L4_OR(3 ^') score for the target sequence, the fifth selection criterion may include or may be a PE_SE score for the target sequence, and the sixth selection criterion may include or may be a length of the antisense oligonucleotide.

[0082] According to various embodiments, the first selection criterion may include or may be a cumulative position score (ACP) for the antisense oligonucleotide, the second selection criterion may include or may be an L3 score for the target sequence, the third selection criterion may include or may be a L4_OR(3 ^') score for the target sequence, the fourth selection criterion may include or may be a L4_OR(5 ^') score for the target sequence, the fifth selection criterion may include or may be a PE_SE score for the target sequence, and the sixth selection criterion may include or may be a length of the antisense oligonucleotide.

[0083] According to various embodiments, the first selection criterion may include or may be a cumulative position score (ACP) for the antisense oligonucleotide, the second selection criterion may include or may be an L3 score for the target sequence, the third selection criterion may include or may be a L4_OR(5 ^') score for the target sequence, the fourth selection criterion may include or may be a L4_OR(3 ^') score for the target sequence, the fifth selection criterion may include or may be a length of the antisense oligonucleotide, and the sixth selection criterion may include or may be a PESE score for the target sequence.

[0084] According to various embodiments, the first selection criterion may include or may be a cumulative position score (ACP) for the antisense oligonucleotide, the second selection criterion may include or may be an L3 score for the target sequence, the third selection criterion may include or may be : a L4_OR(3 ^') score for the target sequence, the fourth selection criterion may include or may be a L4_OR(5 ^') score for the target sequence, the fifth selection criterion may include of may be a length of the antisense oligonucleotide, the sixth selection criterion may include or may be a PESE score for the target sequence.

[0085] According to various embodiments, the first selection criterion may include or may be a L4_OR(5 ') score for the target sequence, the second selection criterion may include or may be a L4_OR(3 ') score for the target sequence, the third selection criterion may include or may be an L3 score for the target sequence, the fourth selection criterion may include or may be a cumulative position score (ACP) for the antisense oligonucleotide, the fifth selection criterion may include or may be a PESE score for the target sequence, the sixth selection criterion may include or may be a length of the antisense oligonucleotide.

[0086] According to various embodiments, the first selection criterion may include or may be a L4_OR(3 ') score for the target sequence, the second selection criterion may include or may be a L4_OR(5 ') score for the target sequence, the third selection criterion may include or may be ah L3 score for the target sequence, the fourth selection criterion may include or may be a cumulative position score (ACP) for the antisense oligonucleotide, the fifth selection criterion may include or may be a P_ESE score for the target sequence, and the sixth selection criterion may include or may be a length of the antisense oligonucleotide.

[0087] According to various embodiments, the first selection criterion may include or may be a L4_DR(5 ') score for the target sequence, the second selection criterion may include or may be a L4_OR(3 ') score for the target sequence, the third selection criterion may include or may be an L3 score for the target sequence, the fourth selection criterion may include or may be a cumulative position score (ACP) for the antisense oligonucleotide, the fifth selection criterion may include or may be a length of the antisense oligonucleotide, and the sixth selection criterion may include or may be a PE_SE score for the target sequence.

[0088] According to various embodiments, the first selection criterion may include or may be a L4_OR(3 ') score for the target sequence, the second selection criterion may include or may be a L4_OR(5 ') score for the target sequence, the third selection criterion may include or may be an L3 score for the target sequence, the fourth selection criterion may include or may be a cumulative position score (ACP) for the antisense oligonucleotide, the fifth selection criterion may include or may be a length of the antisense oligonucleotide, and the sixth selection criterion may include or may be a P_ESE score for the target sequence.

[0089] In a fourth aspect, an antisense oligonucleotide selector for selecting from a plurality of antisense oligonucleotides targeting a target sequence on one of a plurality of exons on a pre-mRNA may be provided. The antisense oligonucleotide selector may include: an exon selection circuit configured to select an exon on the pre-mRNA; a antisense oligonucleotide generation circuit configured to generate a plurality of antisense oligonucleotides for target sequences on the exon pre-mRNA, wherein each of the antisense oligonucleotides has a length of between 15 to 35 nucleotides; a score determination circuit configured to determining a L3 spore for a target sequence, a L4_OR(5 ') score for a target sequence, a L4_OR(3 ') score for a target sequence, a cumulative position score (ACP score) for an antisense oligonucleotide, a P_ESE score for a target sequence; an ordering circuit configured to order the antisense oligonucleotides at first according to the lowest L3 score in a first group; a first selection circuit configured to select a specified number of antisense oligonucleotides starting with the antisense oligonucleotide with the lowest L3 score from the first group and to order them secondly according to the lowest L4_OR(5 ') score in a second group; a second selection circuit configured to select a specified number of antisense oligonucleotides starting with the antisense oligonucleotide with the lowest L4_OR(5 ') from the second group and to order them thirdly according to the lowest L4_OR(3 ') score in a third group; a third selection circuit configured to select a specified number of antisense oligonucleotides starting with the antisense oligonucleotide with the lowest L4_OR(3 ') from the third group and to order them fourthly according to the lowest ACP score in a fourth group; a fourth selection circuit configured to select a specified number of antisense oligonucleotides starting with the antisense oligonucleotide with the lowest ACP score from the fourth group and to order them fifthly according to the highest PESE score in a fifth group; a fifth selection circuit configured to select a specified number of antisense oligonucleotides starting with the antisense oligonucleotide with the highest PESE score from the fifth group and to order them ^'. sixthly according to the length starting with the longest antisense oligonucleotides in a sixth group; and a sixth selection circuit configured to select at least one antisense oligonucleotides starting with the longest antisense oligonucleotide from the sixth group.

[0090] According to various embodiments, the specified number of antisense oligonucleotides for each group may be independently selected from the first 95% of each group or from the first 80 % of each group, or from the first 60% of each group or from the first 40% of each group, or from the first 20% of each group, or from the first 10% of each group, or from the first 5% of each group.

[0091] According to various embodiments, the specified number of antisense oligonucleotides may be selected from the first group may be 100 antisense oligonucleotides and wherein the selected specified number of antisense oligonucleotides from every subsequent group may be X minus 20% or 10% or 5% based on the number of selected antisense oligonucleotides in the previous group, wherein X may be the number of antisense oligonucleotides selected from the previous group. [0092] According to various embodiments, the specified number of antisense oligonucleotides selected from the first group may be selected from the group consisting of 90 antisense oligonucleotides, 80 antisense oligonucleotides, 70 antisense oligonucleotides, 60 antisense oligonucleotides and 50 antisense oligonucleotides.

[0093] According to various embodiments, the percentage deducted from every number of selected antisense oligonucleotides in the previous group may be selected from the group consisting of 15%, 10%, 9%, 8%, 1%, 6%, 5%, 4% and 3%.

[0094] According to various embodiments, the score determination circuit may be further configured to determine a L3 score for each target sequence, a L4_OR(5 ') score for each target sequence, a L4_OR(3 ^') score for each target sequence, a cumulative position score (ACP score) for each antisense oligonucleotide, a PESE score for each target sequence.

[0095] According to various embodiments, the score determination circuit may be further configured to determine the L3 score for each target sequence corresponding to an antisense oligonucleotide in the first group.

[0096] According to various embodiments, the score determination circuit may be further configured to determine the L4_OR(5') score for each target sequence corresponding to an antisense oligonucleotide in the second group.

[0097] According to various embodiments, the score determination circuit may be further configured to determine the L4_OR(3') score for each target sequence corresponding to an antisense oligonucleotide in the third group.

[0098] According to various embodiments, the score determination circuit may be further configured to determine the ACP score for each antisense oligonucleotide in the fourth group.

[0099] According to various embodiments, the score determination circuit may be further configured to determine the PESE score for each target sequence corresponding to an antisense oligonucleotide in the fifth group.

[00100] It is to be noted that the specified numbers for selection and/or the percentages for selection may also be used in the first and/or in the third aspect.

[00101] In a fifth aspect, there is provided an antisense oligonucleotide having a L3 score of about < 0.1 ; a L4JDR(5 ') score of about < 0.15; a L4_OR(3 ') score of about < 0.12; a PESE score of about > 70 %; and the antisense oligonucleotide binds to a target exon pre-mRNA starting in the first one third of the target exon pre-mRNA, counting from 5^' to 3 '; wherein the antisense oligonucleotides H3A (5'-GUAGGUCACUGAAGAGGUUCU-3 ') (SEQ ID NO: 24),

H4A (5'-UGUUCAGGGCAUGAACUCUUGUGGAUCCUU-3') (SEQ ID NO: 25), h29AON2 (5'-GGUUAUCCUCUGAAUGUCGC-3') (SEQ ID NO: 26),

h29AON6 (5 ' -UCUGUGCC AAU AUGCG AAUC-3 ') (SEQ ID NO: 27),

H32A (5'-CUUGUAGACGCUGCUCAAAAUUGGCUGGUU-3 ') (SEQ ID NO: 28),

H36A(1) (5'-UGUGAUGUGGUCCACAUUCUGGUCAAAAGU-3') (SEQ ID NO: 29), h40AONi: (5'-GAGCCUUUUUUCUUCUUUG-3') (SEQ ID NO: 30),

h49AONl (5'-CUUCCACAUCCGGUUGUUU-3 ') (SEQ ID NO: 31),

h55AON3 (5'-UGCAGUAAUCUAUGAGUUUC-3') (SEQ ID NO: 32),

h59AONl (5'-CAAUUUUUCCCACUCAGUAUU-3 ') (SEQ ID NO: 33),

h60AONl (5 '-GU UCUCU UIJCAG AGGCGC-3 ') (SEQ ID NO: 34),

h63AONl (5'-GGUCCCAGCAAGUUGUUUG-3') (SEQ ID NO: 35),

h71 AONr (5'-GCCAGAAGUUGAUCAG GU-3 ') (SEQ ID NO: 36),

h73AONl (5'-GAUCCAUUGCUGUUUUCC-3') (SEQ ID NO: 37),

h74AONl (5'-CUGGCUCAGGGGGGAGU-3') (SEQ ID NO: 38),

H75A (5'-GGACAGGCCUUUAUGUUCGUGCUGC-3 ') (SEQ ID NO: 39),

h78AONl (5'-GCUUUCCAGGGGUAUUUC-3') (SEQ ID NO: 40),

h78AON2 (5'-CAUUGGCUUUCCAGGGG-3') (SEQ ID NO: 41) are excluded.

[00102] In a sixth aspect, there is provided a method of treating Duchenne Muscular

Dystrophy (DMD) in a patient. The method includes administering a pharmaceutically effective amount of an antisense oligonucleotide as described above.

BRIEF DESCRIPTION OF THE DRAWINGS

[00103] The invention will be better understood with reference to the detailed description when considered in conjunction with the non-limiting examples and the accompanying drawings, in which:

[00104] Figure 1 shows the data of 23 AONs in which the respective nucleotide sequences of the AONs, the efficiency of the AONs and the target exons and sites of the dystrophin pre- mRNA that the respective AONs bind to during transcription are provided. AON efficiency is presented as a percentage of the density of transcripts with specific exon skipping (skipped band) to the sum of the skipped; arid unskipped (or "native transcript" as defined below) bands, are classified as "++" (>25%), "+" (<25%) and "-" (0%). [00105] Figure 2 shows the Reverse Transcription-PCR (RT-PCR) analysis of dystrophin mRNA treated with 100 nM of AON2 (SEQ ID NO: 2). The "native transcript" indicates the native mRNA transcript that maintains the targeted exon 2. The "transcripts with exon 2 skipping'' indicates the mRNA transcript derived from exon 2 skipping after incubation with AON2.

[00106] : Figure 3 shows the RTrPCR analysis of dystrophin mRNA treated with 200 nM of AON43-1 (SEQ ID NO: 3) and AON43-2 (SEQ ID NO: 4) respectively. The dystrophin mRNA was treated with the respective AON43-1 and AON43-2 in three separate experiments, as shown as "1^st exp' , "2^nd exp" and "3^rd exp". The "native transcript" indicates the native mRNA transcript that maintains the targeted exon 43. The "transcripts" with exon 43 skipping" indicates the mRNA transcript derived from exon 43 skipping after incubation with either AON43-1 or AON43-2,

[00107] Figure 4A shows the RT-PCR analysis dystrophin mRNA treated with 200 nM of AON45-1 (SEQ ID NO: 5), AON45-2 (SEQ ID NO: 6) and Mixture of AON45-1 and AON45-2 (indicated as "Mix. AON45-1 & AON45-2". . The "native transcripts" indicates the native mRNA transcript that maintains the targeted exon 45. The "transcripts with exon 45 skipping" indicates the mRNA transcripts derived from exon 45 skipping after incubation with the respective AON45-1, AON45-2 or Mixture of AON45-1 and AON45-2.

[00108] Figure 4B shows the RT-PCR analysis of dystrophin mRNA treated with 200nM of AON45-1 ; AON45-2; AON45-3 (SEQ ID NO: 7); AON45-Leiden obtained from Aartsma Rus et al, Oligonucleotides, 2005, 15:284-297; and Mixture of AON45-1 and AON45-2 (indicated as "Mix. AON45-1 & AON45-2") respectively. AON45-Leiden is used as a control. The "native transcripts" indicates the native mRNA transcript that maintains the targeted exon 45. The "transcripts with exon 45 skipping" indicates the mRNA transcripts derived from exon 45 skipping after incubation with the respective AON45-1, AON45-2, AON45-3, AON45-Leiden, Mixture of AON45-1 and AON45-2 and Control (no AON).

[00109] Figure 5 shows the RT-PCR analysis of dystrophin mRNA treated with 200nM of ΑΌΝ46-1 (SEQ ID NO: 8) and AON46-2 (SEQ ID NO: 9) respectively. The dystrophin mRNA was treated : with the respective AON46-1 and AON46-2 in three separate experiments, as shown as "1^st exp", "2^nd exp" and "3^rd exp". The "native transcripts" indicates the native mRNA transcript that maintains the targeted exon 46. The "transcripts with exon 46 skipping" indicates the mRNA transcripts derived from exon 46 skipping after incubation with the respective AON46-1 and AON46-2.

[00110] Figure 6A shows the RT-PCR analysis of dystrophin mRNA treated with 200 nM of AON47-1 (SEQ ID NO: 10), AON47-2 (SEQ ID NO: 1 1) and AON47-3 (SEQ ID NO: 12) respectively. The dystrophin mRNA was treated with the respective AON47-1, AON47-2 and AON47-3 in three separate experiments, as shown as "1^st exp", "2^nd exp" and "3^rd exp". The "native transcripts" indicates the native mRNA transcript that maintains the targeted exon 47. The "transcripts with exon 47 skipping" indicates the mRNA transcripts derived from exon 47 skipping after incubation with the respective AON47-1, AON47-2 and AON47-3.

[001 1 1 ] Figure 6B shows the RT-PCR analysis of dystrophin mRNA treated with AON47- 1 at concentrations of lOOnM, 200nM and 400nM in two separate experiments indicated as "1^st experiment" and "2^nd experiment". The "native transcript" indicates the mRNA transcript that maintains the targeted exon 47. The "dystrophin transcript with exon 47 skipping" indicates the mRNA transcripts derived from exon 47 skipping after incubation with the different concentrations of AON47-1.

[001 12] Figure 7 shows the RT-PCR analysis of dystrophin mRNA treated with lOOnM and 200nM of AON50-1 (SEQ ID NO: 13). The "native transcripts" indicate the mRNA transcript that maintains target exon 50. The "transcripts with exon 50 skipping" indicates the mRNA transcript derived from exon 50 after incubation with different concentrations of AON50-1.

[00113] Figure 8 A shows the RT-PCR analysis of dystrophin mRNA treated with 100 nM of AON51-1 (SEQ ID NO: 14); AON51-2 (SEQ ID NO: 15); AON51-3 (SEQ ID NO: 16); AON51 -Perth ("AON51-P") obtained from Wilton et al (Wilton, S.D., 2007, Molecular Therapy, vol.15, pp.1288); and AON45-Leiden ("AON51-L") obtained from Aartsma Rus et al, Oligonucleotides, 2005, 15:284-297. The "native transcripts" indicates the mRNA transcripts that maintain the targeted 51 exon. The "transcripts with exon 51 skipping" indicates the mRNA transcripts derived from exon 51 skipping after induction with the respective AONs as indicated in this figure.

[00114] Figure 8B shows the RT-PCR analysis of dystrophin mRNA treated with the respective AONs mentioned in Figure 8 A, with the exception that the control (without AON) and PCR control (H₂0) were also analyzed by agarose gel electrophoresis. [001 15] Figure 8C shows the RT-PCR analysis of dystrophin mRNA derived from DMD patient primary muscle cells treated with the respective AONs mentioned in Figure 8A, with the exception that the control (without AON) was also analyzed by agarose gel electrophoresis The band that forms between the native transcripts and the transcripts with exon 51 skipping on the agarose gel indicates the heteroduplex form of native Transcripts & transcripts with exon 51 skipping.

[001 16] Figures 9A and 9B show the RT-PCR analysis of dystrophin mRNA derived from DMD patient primary muscle cells treated with lOOnM of AON53-2 (SEQ ID NO: 18); AON53-3 (SEQ ID NO: 19); AON-Leiden obtained from Aartsma Rus et al, Oligonucleotides, 2005, 15:284-297; and AON-Perth obtained from Wilton et al. "NT" indicates the control in which no AON was used. The "native transcripts" indicates the mRNA transcripts that maintain the targeted 53 exon. The "transcripts with; exon 53 skipping" indicates the mRNA transcripts derived from exori 53 skipping after induction with the respective AONs as indicated in this figure.

[00117] Figure 10 A shows the RT-PCR analysis of dystrophin mRNA treated with AON57-1 (SEQ ID NO: 21); AON57-2 (SEQ ID NO: 22); and AON57-3 (SEQ ID NO: 23). The "native transcripts" indicate the mRNA transcripts that maintain the targeted 57 exon. The "transcript with exon 57 skipping" indicates the mRNA transcripts derived from exon 57 after induction with the respective AONs as indicated in this figure.

[00118] Figure 10B shows the RT-PCR analysis of dystrophin mRNA treated with AON57-1 at different concentrations of lOOnM, 200nM and 400nM in two separate experiments.

[00119] Figure 11 provides a plot that illustrates the occurrences of engaged nucleotides at each step of transcriptional analysis within an AON target site. In this figure, the horizontal axis denotes sequential steps of transcriptional analysis while the vertical axis denotes numbered nucleotides within the AON target site. At each step of transcriptional analysis, nucleotides in the target site that are engaged are depicted as a black dot in the plot. The calculations of L4_OR(5') and L4_OR(3') are described herein. The box which the arrow is pointed at refers to a representative group of 3 successive nucleotides with positions 20, 21 and 22 respectively.

[00120] Figure 12A provides a plot that illustrates the occurrences of engaged nucleotides (black dots) at each step of transcriptional analysis. [00121] Figure 12B provides a histogram that illustrates the proportion of steps of transcription analysis at which a nucleotide in the AON target site is engaged in relation to the total number of steps of transcriptional analysis (i.e., NEP: Nucleotide Engaged Proportion score), derived from the plot of engaged nucleotides in Figure 12A. The vertical axis denotes the NEP score; the horizontal axis denotes the numbered nucleotides in the AON target site counting from 5' to 3\ The MAXNEP is defined as the highest NEP among the NEPs of all nucleotides in the AON target site.

[00122] Figure 13 provides a histogram that illustrates the NEP score of each nucleotide in an AON target site, except that sections in which all consecutive nucleotides having NEP scores lower than 0.05 are identified, as indicated by the solid lines below the chart. For each of these sections, the Ρ_ΝΕΡ_{<0 05} score is obtained by dividing the number of nucleotides in the section by the total number of nucleotides in the AON target site. The MAX_PNEP<O.₀₅ score for the AON target site is the highest PNEPO.O_S score within the site. The vertical axis denotes the NEP score; the horizontal axis denotes the numbered nucleotides in the AON target site counting from 5' to 3 '. The dotted line within the chart denotes the 0.05 NEP Score level. .

[00123] Figure 14 provides a plot that illustrates the occurrences of engaged nucleotides at each step of transcriptional analysis within an AON target site for AON2 (SEQ ID NO: 2). The vertical axis denotes nucleotide numbers 17 to 44 of the target exon pre-m NA sequence of dystrophin gene. The horizontal axis denotes sequential steps of transcriptional analysis. At each step of transcriptional analysis, nucleotides in the target site that are engaged are depicted as a black dot in the plot; The L3 score for AON2 is 0.0679 and the PESE score for AON2 is 89.3%.

[00124] Figure 15; provides a plot that illustrates the occurrences of engaged nucleotides at each step of transcriptional analysis within an AON target site for AON43-1 (SEQ ID NO: 3). The vertical axis denotes nucleotide numbers 1 to 29 of the target exon pre-mRNA sequence of dystrophin gene. The horizontal axis denotes sequential steps of transcriptional analysis. At each step of transcriptional analysis, nucleotides in the target site that are engaged are depicted as a black dot in the plot. The L3 score for ΑΌΝ43-1 is 0.0488 and the PE_SE score for AON43-l is 86.2%. :

[00125] Figure 16 provides a plot that illustrates the occurrences of engaged nucleotides at each step of transcriptional analysis within an AON target site for AON45-1 (SEQ ID NO: 4). The vertical axis denotes nucleotide numbers 40 to 70 of the target exon pre-mRNA sequence of dystrophin gene. The horizontal axis denotes sequential steps of transcriptional analysis. At each step of transcriptional analysis, nucleotides in the target site that are engaged are depicted as a black dot in the plot. The L3 score for AON45-1 is 0.0489 and the PESE score for AON45-1 is 93.5%.

[00126] Figure 17 provides a plot that illustrates the occurrences of engaged nucleotides at each step of transcriptional analysis within an AON target site for AON46-1 (SEQ ID NO: 7). The vertical axis denotes nucleotide numbers 1 to 28 of the target exon pre-mRNA sequence of dystrophin gene. The horizontal axis denotes sequential steps of transcriptional analysis. At each step of transcriptional analysis, nucleotides in the target site that are engaged are depicted as a black dot in the plot. The L3 score for AON46-1 is 0.0628 and the P_ESE score for AON46-l is 75%.

[00127] ; Figure 18 provides a plot that illustrates the! occurrences of engaged nucleotides at each step of transcriptional analysis within an AON target site for AON47-1 (SEQ ID NO: 9). The vertical axis denotes nucleotide numbers 39 to 68 of the target exon pre-mRNA sequence of dystrophin gene. The horizontal axis denotes sequential steps of transcriptional analysis. At each step of transcriptional analysis, nucleotides in the target site that are engaged are depicted as a black dot in the plot: The L3 score for AON47-1 is 0.0154 and the PE_SE score for AON47-1 is 96.2%.

[00128] Figure 19 provides a plot that illustrates the occurrences of engaged nucleotides at each step of transcriptional analysis within an AON target site for AON50-1 (SEQ ID NO:

12) . The vertical axis denotes nucleotide numbers 6 to 28 of the target exon pre-mRNA sequence of dystrophin gene. The horizontal axis denotes sequential steps of transcriptional analysis. At each step of transcriptional analysis, nucleotides in the target site that are engaged are depicted as a black dot in the plot. The L3 score for AON50-1 is 0.0964 and the PE_SE score for AON50-1 is 100%.

[00129] Figure 20 provides a plot that illustrates the occurrences of engaged nucleotides at each step of transcriptional analysis within an AON target site for AON51-1 (SEQ ID NO:

13) . The vertical axis denotes nucleotide numbers 5 to 34 of the target exon pre-mRNA sequence of dystrophin gene. The horizontal axis denotes sequential steps of transcriptional analysis. At each step of transcriptional analysis, nucleotides in the target site that are engaged are depicted as a black dot in the plot. The L3 score for AON51-1 is 0.0241 and the P_ESE score for AON51-1 is 100%. [00130] Figure 21 provides a plot that illustrates the occurrences of engaged nucleotides at each step of transcriptional analysis within an AON target site for AON51-2 (SEQ ID NO: 14). The vertical axis denotes nucleotide numbers 42 to 72 of the target exon pre-mRNA sequence of dystrophin gene. The horizontal axis denotes sequential steps of transcriptional analysis. At each step of transcriptional analysis, nucleotides in the target site that are engaged are depicted as a black dot in the plot. The L3 score for AON51-2 is 0.0324 and the PE_SE score for AON51 -2 is 87.1 %: :

[00131] Figure 22 provides a plot that illustrates the occurrences of engaged nucleotides at each step of transcriptional analysis within an AON target site for AON53-1 (SEQ ID NO:

16) . The vertical axis denotes nucleotide numbers 17 to 41 of the target exon pre-mRNA sequence of dystrophin gene. The horizontal axis denotes sequential steps of transcriptional analysis. At each step of transcriptional analysis, nucleotides in the target site that are engaged are depicted as a black dot in the plot. The L3 score for AON53-1 is 0.0299 and the PESE score for AON53-1 is 96%.

[00132] Figure 23 provides a plot that illustrates the occurrences of engaged nucleotides at each step of transcriptional analysis within an AON target site for AON53-2 (SEQ ID NO:

17) . The vertical axis denotes nucleotide numbers 26 to 52 of the target exon pre-mRNA sequence of dystrophin gene. The horizontal axis denotes sequential steps of transcriptional analysis. At each step of transcriptional analysis, nucleotides in the target site that are engaged are depicted as a black dot in the plot. The L3 score for AON53-2 is 0.0536 and the PE_SE score for AON51 -2 is 96.3%. : :^■ ·^' . ^·■

[00133] Figure 24 provides a plot that illustrates the occurrences of engaged nucleotides at each step of transcriptional analysis within an AON target site for AON53-3 (SEQ ID NO: 18). The vertical axis denotes nucleotide numbers 32 to 62 of the target exon pre-mRNA sequence of dystrophin gene. The horizontal axis denotes sequential steps of transcriptional analysis. At each step of transcriptional analysis, nucleotides in the target site that are engaged are depicted as a black dot in the plot. The L3 score; for AON53-3 is 0.0789 and the

PESE score for AON51 80.6%

[00134] ; Figure 25 provides a plot that illustrates the occurrences of engaged nucleotides at each step of transcriptional analysis within an AON target site for AON57-1 (SEQ ID NO:

20). The; vertical axis denotes nucleotide numbers 4 to 25 of the target exon pre-mRNA sequence of dystrophin gene. The horizontal axis denotes sequential steps of transcriptional analysis. At each step of transcriptional analysis, nucleotides in the target site that are engaged are depicted as a black dot in the plot. The L3 score for AON57-1 is 0_^0087 and the PE_SE score for AON57-1 is 72.7%.

[00135] Figure 26 A shows a key legend for interpreting the vertical axis of Figures 26B to 26J. The segments of different shades on the vertical axis represent the prediction of exonic splicing enhancers (ESE) predicted by ESE Finder and RESCUE ESE as described in Example 4. The bottom panel of Figure 26A provides the list of SR Proteins used to predict ESEs. .■

[00136] igures 26B to J each provides a plot that illustrates the co-transcriptional binding accessibility of an AON target site for the respective target exqns 2, 43, 45, 46, 47, 50, 51 , 53 and 57 of the pre-mRNA of dystrophin gene. The occurrences of engaged nucleotides within the AON target sites ^'are denoted as black spots, i.e. they are inaccessible for AON binding. Therefore, sequences with minimal black spots (lowest possible L3(AON), L4_OR(5') and L4_OR(3') scores) should be selected. Segments of the target exon sequence that are predicted to contain ESE motifs are indicated on the vertical axis - bar charts (ESE-finder) and short sequences (Rescue-ESE).

[00137] Figure 27 lists the efficiency of each AON as described herein. AON efficiency is presented as a percentage of the density of the bands representing the transcripts amplicons exon skipping (skipped band) to the sum of the densities of the skipped and unskipped bands (transcripts without exon skipping), and are classified as "++" (>25%), "+" (<25%) and "-" (0%). In the third column, "1", "2" and "3" indicates that the first nucleotide of the AON target site is located at the first, second and last third of the exon 5'. ACP (last column) refers to Average Cumulative Position. Out of the 23 AONs described in this figure, 19 AONs were efficacious in inducing skipping of their specific target exon among which 14 are graded as "4-+".

[00138] ; Figure 28 shows the RT-PCR analysis of dystrophin mRNA derived from fibroblast , cells treated with 100 nM of AON47-1, AON47-2 and AON47-3 as shown in Figure 28A; 100 nM of AON57-1 , AON57-2 and AON57-3 as shown in Figure 28B; 100 nM of AON 51-1 , AON51-2 and AON51-3 as shown in Figures 28Ci and Cu; and 100 nM of AON53-1, AON53-2, AON53-3 and AON53-4 as shown in Figure 28Di and Dii. AON efficiency was assessed by comparing amplicons of dystrophin transcripts harboring exon skipping (lower arrow) with amplicons of native dystrophin transcript (upper arrow). AONs were able to induce exon skipping efficiently in human primary fibroblast cells for exons 47 (Figures 28A and B), 51 (Figure 28Ci) and 53 (Figure 28Di). Heteroduplex formation between amplicons carrying exon skipping with native transcripts that appear as an additional band with molecular weight of slightly lower than those of native transcript amplicons.

[00139] Figure 29 shows the immunocytochemistry of dystrophin protein in DMD patient skeletal muscle cells. AON induced restoration of dystrophin expression in DMD patient skeletal muscle cells. Double immunocytochemistry were performed to stain desmin that mark myogenic cells (a) and dystrophin (b). Restoration of dystrophin expression were observed in DMD patient's skeletal muscle cells treated with AONs targeting exons 51 (left panel) and 53 (middle panal). DMD patient's muscle cells treated with lipofectamine only did not show any dystrophin expression (right panel).

[00140]^■ Figure 30 shows the hit rates of AONs targeting dystrophin gene. Hit rates of highly efficient (++), efficient (+) and non-effective (-) AONs obtained in studies by Aartsma-Rus et al. (2005), Oligonucleotides 15: 284-297 ("Ref. 5"); Aartsma-Rus et al. (2009), Molecular Therapy 17:548-553 ("Ref. 2"); Harding et al. (2007), Molecular Therapy 15:157-166 ("Ref. 4"); Popplewell et al (2009) Molecular Therapy 17:554-561 ("Ref. 3"); and Wilton et al. (2007), Molecular Therapy 15: 1288-1296 ("Ref. 6"). Sum of columns 2 and 3 gives the hit rate for effective AONs. At each data source, the top row includes all AONs reported while the bottom row includes only AONs that target the same exons (2, 43, 45, 46, 47, 50, 51 , 53 and 57).

[00141] Figure 31 shows the boxplots of Average Cumulative Position (ACP) score and AON-target Gibbs energy of 14 efficient (++) and 9 non-efficient (+ and -) AONs. -S test were performed for each score and the ^-values are given on the respective boxplots. Statistical analysis reveals that efficient AONs has the lowest ACP scores (p-value is nearly zero (see Figure 31 A), i.e. they target sites nearest to exon 5'.

[00142] ;Figure 32 shows the comparison of L3 (AON) and P_ESE scores in each third of the target exons. The L3(AON) and PESE scores at each third of the 9 target exons are compared. The ineffective AONs target the last third of exons 45, 47, 51, 53 and 57. From the plots however, the last-third of. exons 45, 47 and 57 has much lower L3(AON) score (i.e. more accessible for AON binding) than their first-third. On the other hand, the last-third of exons 45, 53 and 57 has higher PESE scores than their first-third. [00143] Figure 33 shows the boxplots of 6 variables namely, AON melting temperature

(nearest neighbor method), 20-methyl-AON melting temperature (nearest neighbor method), percentage of GC content, Gibbs energy of AON -AON, binding energy of AON- AON and binding energy of AON-target of 14 efficient (++) and 9 non-efficient (+ and -) AONs. K-S test were performed for each score and the ^-values are given on the respective boxplots. With the exception of Gibbs energy of AON-target (Figure 3 IB), no statistical significance was detected. Specifically, efficient AONs have statistically lowest Gibbs energy of AON- target. As this variable is associated with GC content :and binding energy of AON-target, it is intriguing that no statistical significance was detected for the two latter variables. Moreover, the />-value obtained albeit significant is 345 times larger; than the p-value obtained using the ACP score. For these reasons, it is likely that low Gibbs energy of AON-target is an effect rather than the cause of AON efficiency. As such, Gibbs energy of AON-target is not included as a design variable.

[00144] Figure 34 shows a flow diagram 3400 illustrating a method of selecting at least one antisense oligonucleotide from a plurality of antisense oligonucleotides targeting a target sequence on one of a plurality of exons on a pre-mRNA.

[00145] Figure 35 shows an antisense oligonucleotide selector 3500 for selecting from a plurality of antisense oligonucleotides targeting a target sequence on one of a plurality of exons on a pre-mRNA may be provided.

[00146] Figure 36 shows a flow diagram 3600 illustrating a method for selecting at least one antisense oligonucleotide from a plurality of antisense oligonucleotides targeting a target sequence on an exon on a pre-mRNA may be provided.

[00147] Figure 37 shows an antisense oligonucleotide selector 3700 for selecting at least one antisense oligonucleotide from a plurality of antisense oligonucleotides targeting a target sequence on an exon on a pre-mRNA.

[00148] Figure 38 shows an antisense oligonucleotide selector 3800 for selecting at least one antisehse oligonucleotide from a plurality of antisense oligonucleotides targeting a target sequence on an exon on a pre-mRNA.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

[00149] ^'. Skipping of a specific exon in a gene to remove nonsense mutations and/or restore its reading frame is a viable therapeutic strategy for genetic diseases such as, but not limited to Duchenne muscular dystrophy (DMD), thalassemia, myotonic dystrophy (ClC-1 gene), hypercholesterolemia (ΑρόΒ gene), frontotempOral dementia with parkinsonism, ocular albinism and cancer. To induce exon skipping, AONs are designed to bind to appropriate sequences within the target exon pre-mRNA during; transcription. Current method of AON design is semi-empirical involving trial-and-error and preliminary experiments. For rational AON design, it was found that a set of design variables and their relative importance (hierarchy) is a pre-requisite. Therefore, in the present invention a set of hierarchical design variables (co-transcriptional binding accessibility of target site, average cumulative position, and presence of exonic splicing enhancers and optimum target length) is proposed and validated. The obtained hit rates for designing efficient AONs are significantly higher than prior studies, which range from 18% to 38%.

[00150] In a first aspect, a method for selecting at least one antisense oligonucleotide from a plurality of antisense oligonucleotides targeting a target sequence on an exon on a pre- mRNA may be provided. The method may include: determining for each antisense oligonucleotide of the plurality of antisense oligonucleotides a first evaluation value according to a first evaluation criterion; determining a first subset of the plurality of antisense oligonucleotides based on the first evaluation values; determining for each antisense oligonucleotide of the first subset a second evaluation value according to a second evaluation criterion; and determining a second subset of the first subset of antisense oligonucleotides based on the second evaluation values.

[00151] In certain embodiments, a method for selecting at least one antisense oligonucleotide from a plurality of antisense oligonucleotides targeting a target sequence on an exon on a pre-mRNA may be provided in Figure 36. In 3602, for each antisense oligonucleotide of the plurality of antisense oligonucleotides a first evaluation value according to a first evaluation criterion may be determined. In 3604, a first subset of the plurality of antisense oligonucleotides based on the first evaluation values may be determined. In 3606, for each antisense oligonucleotide of the first subset a second evaluation value according to; a second evaluation criterion may be determined. In 3608, a second subset of the first subset of antisense oligonucleotides based on the second evaluation values may be determined.

[00152] According to various embodiments, at least one of the first evaluation criterion and the second evaluation criterion may be related to binding of the antisense oligonucleotide with the exon. [00153] According to various embodiments, determining the first subset may include: ordering the antisense oligonucleotides of the plurality of antisense oligonucleotides according to the first evaluation values; and selecting as the first subset a pre-determined number or a pre-determined percentage of antisense oligonucleotides of the plurality of antisense oligonucleotides based on the ordering of the plurality of antisense oligonucleotides.

[00154] According to various embodiments, determining the second subset may include: ordering the antisense oligonucleotides of the first subset according to the second evaluation values; arid selecting as the second subset a pre-determined number or a pre-determined percentage of antisense oligonucleotides of the first subset based on the ordering of the antisense oligonucleotides of the first subset.

[00155] According to various embodiments, at least one of the first evaluation criterion and the second evaluation criterion may : include or may be at least one of an L3 score for the target sequence, a L4_OR(5 ^') score for the target sequence, a L4_OR(3 ') score for the target sequence, a cumulative position score (ACP) for the antisense oligonucleotide, a P_ESE score for the target sequence, and a length of the antisense oligonucleotide.

[00156] According to various embodiments, the method may further include: determining for each antisense oligonucleotide of the second subset a third evaluation value according to a third evaluation criterion; and determining a third subset of the second subset of antisense oligonucleotides based on the third evaluation values.

[00157] According to various embodiments, the third evaluation criterion may be related to binding of the antisense oligonucleotide with the exon.

[00158] According to various embodiments, determining the third subset may include: ordering the antisense oligonucleotides of the second subset according to the third evaluation values; and selecting as the third subset a pre-deteimined number or a pre-determined percentage of antisense oligonucleotides of the second subset based on the ordering of the antisense oligonucleotides of the second subset.

[00159] According to various embodiments, the third evaluation criterion may include or may be at least one of an L3 score for the target sequence, a L4_OR(5 ') score for the target sequence, a L4_OR(3 ^') score for the target sequence, a cumulative position score (ACP) for the antisense oligonucleotide, a P_ESE score for the target sequence, and a length of the antisense oligonucleotide. [00160] According to various embodiments, the method may further include: determining for each antisense oligonucleotide of the third subset a fourth evaluation value according to a fourth evaluation criterion; and determining a fourth subset of the third subset of antisense oligonucleotides based on the fourth evaluation values.

[00161 ] According to various embodiments, the fourth evaluation criterion may be related to binding of the antisense oligonucleotide with the exon.

[00162] According to various embodiments, determining the fourth subset may include: ordering the antisense oligonucleotides of the third subset according to the fourth evaluation values; and selecting as the fourth subset a pre-determined number or a pre-determined percentage of antisense oligonucleotides of the third subset based on the ordering of the antisense oligonucleotides of the third subset.

[00163] According to various embodiments, the fourth evaluation criterion may include or may be at least one of an L3 score for the target sequence, a L4_OR(5 ') score for the target sequence, a L4_OR(3 ^') score for the target sequence, a cumulative position score (ACP) for the antisense oligonucleotide, a PESE score for the target sequence, and a length of the antisense oligonucleotide.

[00164] According to various embodiments, the method may further include: determining for each antisense oligonucleotide of the fourth subset a fifth evaluation value according to a fifth evaluation criterion; and determining a fifth subset of the fourth subset of antisense oligonucleotides based on the fifth evaluation values.

[00165] According to various embodiments, the fifth evaluation criterion may be related to binding of the antisense oligonucleotide with the exon.

[00166] According to various embodiments, determining the fifth subset may include: ordering the antisense oligonucleotides of the fourth subset according to the fifth evaluation values; and selecting as the fifth subset a pre-determined number or a pre-determined percentage of antisense oligonucleotides of the fourth subset based on the ordering of the antisense oligonucleotides of the fourth subset.

[00167] According to various embodiments, the fifth evaluation criterion may include or may be at least one of an L3 score for the target sequence, a L4_OR(5 ') score for the target sequence, a L4_OR(3 ') score for the target sequence, a cumulative position score (ACP) for the antisense oligonucleotide, a PESE score for the target sequence, and a length of the antisense oligonucleotide. [00168] ; According to various embodiments, the method may further include: determining for each antisense oligonucleotide of the fifth subset a sixth evaluation value according to a sixth evaluation criterion; and determining a sixth subset of the fifth subset of antisense oligonucleotides based on the sixth evaluation values.

[00169] ; According to various embodiments, the sixth evaluation criterion may be related to binding of the antisense oligonucleotide with the exon.

[00170] According to various embodiments, determining the sixth subset may include: ordering the antisense oligonucleotides of the fifth subset according to the sixth evaluation values; and selecting as the sixth subset a pre-determined number or a pre-determined percentage of antisense oligonucleotides of the fifth subset based on the ordering of the antisense oligonucleotides of the fifth subset.

[00171 ] According to various embodiments, the sixth evaluation criterion may include or may be at least one of an L3 score for the target sequence, a L4_OR(5 ') score for the target sequence, a L4_OR(3 ') score for the target sequence, a cumulative position score (ACP) for the antisense oligonucleotide, a P_ESE score for the target sequence, and a length of the antisense oligonucleotide.

[00172] According to various embodiments, the first selection criterion may include or may be an L3 score for the target sequence, the second selection criterion may include or may be a L4_OR(5 ^') score for the target sequence, the third selection criterion may include or may be a L4_OR(3 ^') score for the target sequence, the fourth selection criterion may include or may be a cumulative position score (ACP) for the antisense oligonucleotide, the fifth selection criterion may include or may be a PESE score for the target sequence, and the sixth selection criterion may include or may be a length of the antisense oligonucleotide.

[00173] According to various embodiments, the first selection criterion may include or may be an L3 score for the target sequence, the second selection criterion may include or may be a L4 OR(5 ^') score for the target; sequence, the third selection criterion may include or may be a L4_OR(3 ^') score for the target sequence, the fourth selection criterion may include or may be a cumulative position score (ACP) for the antisense oligonucleotide, the fifth selection criterion may include or may be a length of the antisense oligonucleotide, and the sixth selection criterion may include or may be PESE score for the target sequence.

[00174] According to various embodiments, the first selection criterion may include or may be an L3 score for the target sequence, the second selection criterion may include or may be a L4_OR(3 ^') score for the target sequence, the third selection criterion may include or may be a L4_OR(5 ^') score for the target sequence, the fourth selection criterion may include or may be a cumulative position score (ACP) for the antisense oligonucleotide, the fifth selection criterion may include or may be a PESE score for the target sequence, and the sixth selection criterion may include or may be a length of the antisense oligonucleotide.

[00175] According to various embodiments, the first selection criterion may include or may be an L3 score for the target sequence, the second selection criterion may include or may be a L4_OR(3 ^') score for the target sequence, the third selection criterion may include or may be a L4_OR(5 ^') score for the target sequence, the fourth selection criterion may include or may be a cumulative position score (ACP) for the antisense oligonucleotide, the fifth selection criterion may include or may be a length of the antisense oligonucleotide, and the sixth selection criterion may include or may be a PESE score for the target sequence.

[00176] According to various embodiments, the first selection criterion may include or may be a cumulative position score (ACP) for the antisense oligonucleotide, the second selection criterion may include or may be an L3 score for the target sequence, the third selection criterion may include or may be a L4_OR(5 ') score for the target sequence, the fourth selection criterion may include or may be a L4_OR(3 ') score for the target sequence, the fifth selection criterion may include or may be a PESE score for the target sequence, and the sixth selection criterion may include or may be a length of the antisense oligonucleotide.

[00177] According to various embodiments, the first selection criterion may include or may be a cumulative position score (ACP) for the antisense oligonucleotide, the second selection criterion may include or may be an X3 score for the target sequence, the third selection criterion may include or may be a L4_OR(3 ^') score for the target sequence, the fourth selection; criterion may include or may be a L4_OR(5 ') score for the target sequence, the fifth selection criterion may include or may be a PESE score for the target sequence, and the sixth selection criterion may include or may be a length of the antisense oligonucleotide. :

[00178] According to various embodiments, the first selection criterion may include or may be a cumulative position score (ACP) for the antisense oligonucleotide, the second selection criterion may include or may be an L3 score for the target sequence, the third selection criterion may include or may be a L4_OR(5 ') score for the target sequence, the fourth selection criterion may include or may be a L4_OR(3 ') score for the target sequence, the fifth selection criterion may include or may be a length of the antisense oligonucleotide, and the sixth selection criterion may include or may be a P_ESE score for the target sequence.

[00179] According to various embodiments, the first selection criterion may include or may be a cumulative position score (ACP); for the antisense oligonucleotide, the second selection criterion may include or may be an L3 score for the target sequence, the third selection criterion may include or may be a L4_OR(3 ^') score for the target sequence, the fourth selection criterion may include or may be a L4 OR(5 ') score for the target sequence, the fifth selection criterion may include or may be a length of the antisense oligonucleotide, the sixth selection criterion may include or may be a PESE score for the target sequence.

[00180] According to various embodiments, the first selection criterion may include or may be a L4_OR(5 ^') score for the target sequence, the second selection criterion may include or may be a L4_OR(3 ') score for the target sequence, the third selection criterion may include or may be an L3 score for the target sequence, the fourth selection criterion may include or may be a cumulative position score (ACP) for the antisense oligonucleotide, the fifth selection criterion may include or may be a PESE score for the target sequence, the sixth selection criterion may include or may be a length of the antisense oligonucleotide.

[00181 ] According to various embodiments, the first selection criterion may include or may be a L4_OR(3 ') score for the target sequence, the second selection criterion may include or may be a L4_OR(5 ^') score for the target sequence, the third selection criterion may include or may be an L3 score for the target sequence, the fourth selection criterion may include or may be a cumulative position score (ACP) for the antisense oligonucleotide, the fifth selection criterion may include or may be a PESE score for the target sequence, and the sixth selection criterion may include or may be a length of the antisense oligonucleotide.

[00182] ! According to various embodiments, the first selection criterion may include or may be a L4_OR(5 ^') score for ^'the target sequence, the second selection criterion may include or may be a L4_OR(3 ') score for the target sequence, the third selection criterion may include or may be an L3 score for the target sequence, the fourth selection criterion may include or may be a cumulative position score (ACP) for the antisense oligonucleotide, the fifth selection criterion may include or may be a length of the antisense oligonucleotide, and the sixth selection criterion may include or maybe a PE_SE score for the target sequence.

[00183] According to various embodiments, the first selection criterion may include or may be a L4_OR(3 ') score for the target sequence, the second selection criterion may include or may be a L4_OR(5 ^'■) score for the target sequence, the third selection criterion may include or may be an L3 score for the target sequence, the fourth selection criterion may include or may be a cumulative position score (ACP) for the antisense oligonucleotide, the fifth selection criterion may include or may be a length of the antisense oligonucleotide, and the sixth selection criterion may include or may be a P_ESE score for the target sequence.

[00184] Furthermore, in a second aspect the present invention refers to a method of selecting at least one antisense oligonucleotide from a plurality of antisense oligonucleotides (AONs) targeting a target sequence on one of a plurality of exons on a pre-mRNA, wherein the method comprises:

■ selecting an exon on the pre-mRNA;

generating a plurality of antisense oligonucleotides for target sequences on the exon pre- mRNA, wherein each of the antisense oligonucleotides has a length of between 15 to 35 nucleotides;

determining a L3 score for a target sequence, a L4_OR(5 ^') score for a target sequence, a L4_OR(3 ') score for a target sequence, a cumulative position score (ACP score) for an antisense oligonucleotide, a P_Es_E score for a target sequence;

■ selecting a specified , number of antisense oligonucleotides starting with the antisense oligonucleotide with the lowest L4_OR(5') from the second group and ordering them thirdly according to the lowest L4_OR(3 ') score in a third group;

selecting a specified number of antisense oligonucleotides starting with the antisense oligonucleotide with the highest P_ESE score from the fifth group and ordering them sixthly according to the length starting with the longest antisense oligonucleotides in a sixth group; :

^■ selecting at least one antisense oligonucleotides starting with the longest antisense oligonucleotide from the sixth group.

[00185] ; This method has the advantage of speeding up AON design process and obtaining higher hit rates while expending less resource. This is valuable, for example, for genetic diseases, such as DMD therapy as different AONs are required to. address myriad patients' mutations, and for diseases genes amenable to exon skipping:

[00186] In a third aspect, an antisense oligonucleotide selector for selecting at least one antisense oligonucleotide from a plurality of antisense oligonucleotides targeting a target sequence on an exon on a pre-mRNA may be provided. The antisense oligonucleotide selector may include: a first evaluation values determiner configured to determine for each antisense oligonucleotide of the plurality of antisense oligonucleotides a first evaluation value according to a first evaluation criterion; a first subset determiner configured to determine a first subset of the plurality of antisense oligonucleotides based on the first evaluation values; a second evaluation values determiner configured to determine for each antisense oligonucleotide of the first subset a second evaluation value according to a second evaluation criterion; and a second subset determiner configured to determine a second subset of the first subset of antisense oligonucleotides based on the second evaluation values.

[00187] In certain embodiments, an antisense oligonucleotide selector 3700 for selecting at least one antisense oligonucleotide from a plurality of antisense oligonucleotides targeting a target sequence oh an exon on a pre-mRNA can be described in Figure 37. The antisense oligonucleotide selector 3700 may include: a first evaluation values determiner 3702 configured to determine for each^' antisense oligonucleotide of the plurality of antisense oligonucleotides a first evaluation value according to a first evaluation criterion; a first subset determiner 3704 configured to determine a first subset of the plurality of antisense oligonucleotides based on the first evaluation values; a second evaluation values determiner 3706 configured to determine for each antisense oligonucleotide of the first subset a second evaluation value according to a second evaluation criterion; and a second subset determiner 3708 configured to determine a second subset of the first subset of antisense oligonucleotides based on the second evaluation values. The first evaluation values determiner 3702, the first subset determiner 3704, the second values determiner 3706, and the second subset determiner 3708 may be coupled electrically or electronically, by a coupling 3710, for example by a cable or a bus.

[00188] According to various embodiments, at least one of the first evaluation criterion and the second evaluation criterion may be related to binding of the antisense oligonucleotide with the exon.

[00189] In certain embodiments, an antisense oligonucleotide selector 3800 for selecting at least one antisense oligonucleotide from a plurality of antisense oligonucleotides targeting a target sequence on an exon on a pre-mRNA can be described in Figure 38. The antisense oligonucleotide selector 3800 may, similar to the antisense oligonucleotide selector 3700 shown in Figure 37, include: a first evaluation values determiner 3702 configured to determine for each antisense oligonucleotide of the plurality of antisense oligonucleotides a first evaluation value according to a first evaluation criterion; a first subset determiner 3704 configured to determine a first subset of the plurality of antisense oligonucleotides based on the first evaluation values; a second evaluation values determiner 3706 configured to determine for each antisense oligonucleotide of the first subset a second evaluation value according to a second evaluation criterion; and a second subset determiner 3708 configured to determine a second subset of the first subset of antisense oligonucleotides based on the second evaluation values. According to various embodiments, the antisense oligonucleotide selector 3800 may furthermore include a third evaluation values determiner 3810, like will be described in more detail below. \ According to various embodiments, the antisense oligonucleotide selector 3800 may furthermore include a third subset determiner 3812, like will be described in more detail below. According to various embodiments, the antisense oligonucleotide selector 3800 may furthermore include a fourth evaluation values determiner 3818, like will be described in more detail below. According to various embodiments, the antisense pligonucleotide selector 38ΌΟ may furthermore include a fourth subset determiner 3820, like will be described in more detail below. According to various embodiments, the antisense oligonucleotide selector 3800 may furthermore include a fifth evaluation values determiner 3826, like will be described in more detail below. According to various embodiments, the antisense oligonucleotide selector 3800 may furthermore include a fifth subset determiner 3828, like will be described in more detail below. According to various embodiments, the antisense oligonucleotide selector 3800 may furthermore include a sixth evaluation values determiner 3834, like will be described in more detail below. According to various embodiments, the antisense oligonucleotide selector 3800 may furthermore include a sixth subset determiner 3836, like will be described in more detail below. The first evaluation values determiner 3702, the first subset determiner 3704, the second values determiner 3706, and the second subset determiner 3708, the third values determiner 3810, the third subset determiner 3812, the fourth values determiner 3818, the fourth subset determiner 3820, the fifth values determiner 3826, the fifth subset determiner 3828, the sixth values determiner 3834, and; the sixth subset determiner 3836, may be coupled; electrically or electronically, by a coupling 3842, for example by a cable or a bus.

[00190] According to various embodiments, the first subset determiner 3704 may include: a first ordering circuit 3802 configured to order the antisense oligonucleotides of the plurality of antisense oligonucleotides according to the first evaluation values; and a first selecting circuit 3804 configured to select as the first subset a pre-determined number or a predetermined percentage of antisense oligonucleotides of the plurality of antisense oligonucleotides based on the ordering of the plurality of antisense oligonucleotides.

[00191 ] According to various embodiments, the second subset determiner 3708 may include: a second ordering circuit 3806 configured to order the antisense oligonucleotides of the first subset according to the second evaluation values; and a second selecting circuit 3808 configured to select as the second subset a pre-determined number or a pre-determined percentage of antisense oligonucleotides of the first subset based on the ordering of the antisense oligonucleotides of the first subset.

[00192] According to various embodiments, at least one of the first evaluation criterion and the second evaluation criterion may include or may be at least one of an L3 score for the target sequence, a L4_OR(5 ^') score for the target sequence, a L4_OR(3 ^') score for the target sequence, a cumulative position score (ACP) for the antisense oligonucleotide, a P_ESE score for the target sequence, and a length of the antisense oligonucleotide.

[00193] ; According to various embodiments, the third evaluation values determiner 3810 may be configured to determine for each antisense oligonucleotide of the second subset a third evaluation value according to a third evaluation criterion. According to various embodiments, the third subset determiner 3812 may be configured to determine a third subset of the second subset of antisense oligonucleotides based on the third evaluation values.

[00194] According to various embodiments, the third evaluation criterion may be related to binding of the antisense oligonucleotide with the exon. [00195] According to various embodiments, the third subset determiner 3812 may include: a third ordering circuit 3814 configured to order the antisense oligonucleotides of the second subset according to the third evaluation values; and a third selecting circuit 3816 configured to select as the third subset a pre-determined number or a pre-determined percentage of antisense oligonucleotides of the second subset based on the ordering of the antisense oligonucleotides of the second subset.

[00196] According to various embodiments, the third evaluation criterion may include or may be at least one of an L3 score for the target sequence, a L4_OR(5 ') score for the target sequence, a L4_OR(3 ') score for the target sequence, a cumulative position score (ACP) for the antisense oligonucleotide, a PESE score for the target sequence, and a length of the antisense oligonucleotide.

[00197] According to various embodiments, the fourth evaluation values determiner 3818 may be configured to determine for each antisense oligonucleotide of the third subset a fourth evaluation value according to a fourth evaluation criterion. According to various embodiments, the fourth subset determiner 3820 may be configured to determine a fourth subset of the third subset of antisense oligonucleotides based on the fourth evaluation values.

[00198] According to various embodiments, the fourth evaluation criterion may be related to binding of the antisense oligonucleotide with the exon.

[00199] According to various embodiments, the fourth subset determiner 3820 may include: a fourth ordering circuit 3822 configured to order the antisense oligonucleotides of the third subset according to the fourth evaluation values; and a fourth selecting circuit 3824 configured to select as the fourth subset a pre-determined number or a pre-determined percentage of antisense oligonucleotides of the third subset based on the ordering of the antisense oligonucleotides of the third subset.

[00200] According to various embodiments, the fourth evaluation criterion may include or may be at least one of an L3 score for the target sequence, a L4_OR(5 ') score for the target sequence, a L4_OR(3 ') score for the target sequence, a cumulative position score (ACP) for the antisense oligonucleotide, a P_ESE score for the target sequence, and a length of the antisense oligonucleotide.

[00201] According to various embodiments, the fifth evaluation values determiner 3826 may be configured to determine for each antisense oligonucleotide of the fourth subset a fifth evaluation value according to a fifth evaluation criterion. According to various embodiments, the fifth subset determiner 3828 may be configured to determine a fifth subset of the fourth subset of antisense oligonucleotides based on the fifth evaluation values.

[00202] According to various embodiments, the fifth evaluation criterion may be related to binding of the antisense oligonucleotide with the exon.

[00203] According to various embodiments, the fifth subset determiner 3828 may include: a fifth ordering circuit 3830 configured to order the antisense oligonucleotides of the fourth subset according to the fifth evaluation values; and a fifth selecting circuit 3832 configured to select as the fifth subset a predetermined number or a pre-determined percentage of antisense oligonucleotides of the fourth subset based on the ordering of the antisense oligonucleotides of the fourth subset.

[00204] According to various embodiments, the fifth evaluation criterion may include or may be at least one of an L3 score for the target sequence, a L4_OR(5 ') score for the target sequence, a L4_OR(3 ") score for the target sequence, a cumulative position score (ACP) for the antisense oligonucleotide, a PE_SE score for the target sequence, and a length of the antisense oligonucleotide.

[00205] According to various embodiments, the sixth evaluation values determiner 3834 may be configured to determine for each antisense oligonucleotide of the fifth subset a sixth evaluation value according to a sixth evaluation criterion. According to various embodiments, the sixth subset determiner 3836 may be configured to determine a sixth subset of the fifth subset of antisense oligonucleotides based on the sixth evaluation values.

[00206] According to various embodiments, the sixth evaluation criterion may be related to binding of the antisense oligonucleotide with the exon.

[00207] According to various embodiments, the sixth subset determiner 3836 may include: a sixth ordering circuit 3838 configured to order the antisense oligonucleotides of the fifth subset according to the sixth evaluation values; and a sixth selecting circuit 3840 configured to select! as the sixth subset a pre-determined number or a pre-determined percentage of antisense oligonucleotides of the fifth subset based on the ordering of the antisense oligonucleotides of the fifth subset.

[00208] According to various embodiments, the sixth evaluation criterion may include or may be at least one of an L3 score; for the target sequence, a L4_OR(5 ^') score for the target sequence, a L4_OR(3 ') score for the target sequence; a cumulative position score (ACP) for the antisense oligonucleotide, a PE_SE score for the target sequence, and a length of the antisense oligonucleotide.

[00209] : According to various embodiments, the first selection criterion may include or may be an L3 score for the target sequence, the second selection criterion may include or may be a L4_OR(5 ') score for the target sequence, the third selection criterion may include or may be a L4_OR(3 ') score for the target sequence, the fourth selection criterion may include or may be a cumulative position score (ACP) for the antisense oligonucleotide, the fifth selection criterion may include or may be a PESE score for the target sequence, and the sixth selection criterion may include or may be a length of the antisense oligonucleotide.

[00210] According to various embodiments, the first selection criterion may include or may be an L3 score for the target sequence, the second selection criterion may include or may be a L4_OR(5 ^') score for the target sequence, the third selection criterion may include or may be a L4_OR(3 ') score for the target sequence, the fourth selection criterion may include or may be a cumulative position score (ACP) for the antisense oligonucleotide, the fifth selection criterion may include or may be a length of the antisense oligonucleotide, and the sixth selection criterion may include or may be a PESE score for the target sequence .

[00211] According to various embodiments, the first selection criterion may include or may be an L3 score for the target sequence, the second selection criterion may include or may be a L4_OR(3 ') score for the target sequence, the third selection criterion may include or may be a L4_OR(5 ^') score for the target sequence, the fourth selection criterion may include or may be a cumulative position score (ACP) for the antisense oligonucleotide, the fifth selection criterion may include or may be a PESE score for the target sequence, and the sixth , selection criterion may include; or may be a length of the antisense oligonucleotide,

[00212] ^' According to various embodiments, the first selection criterion may include or may be an L3 score for the target sequence, the second selection criterion may include or may be a L4_OR(3 ^') score for the target sequence, the third selection criterion may include or may be a L4_OR(5 ^') score for the target sequence, the fourth selection criterion may include or may be a cumulative position score (ACP) for the antisense oligonucleotide, the fifth selection criterion may include or may be a length of the antisense oligonucleotide, and the sixth selection criterion may include or may be a PESE score for the; target sequence.

[00213] ; According to various embodiments, the first selection criterion may include or may be a cumulative position score (ACP) for the antisense; oligonucleotide, the second selection criterion may include or may be an L3 score for the target sequence, the third selection criterion may include or may be a L4_OR(5 ^') score for the target sequence, the fourth selection criterion may include or may be a L4_OR(3 ') score for the target sequence, the fifth selection criterion may include or may be a P_ESE score for the target sequence, and the sixth selection criterion may include or may be a length of the antisense oligonucleotide.

[00214] According to various embodiments, the first selection criterion may include or may be a cumulative position score (ACP) for the antisense oligonucleotide, the second selection criterion may include or may be an L3 score for the target sequence, the third selection criterion may include or may be a L4_OR(3 ^') score for the target sequence, the fourth selection criterion may include or may be a L4_OR(5^') score for the target sequence, the fifth selection criterion may include or may be a P_ESE score for the target sequence, and the sixth selection criterion may include or may be a length of the antisense oligonucleotide.

[00215] According to various embodiments, the first selection criterion may include or may be a cumulative position score (ACP) for the antisense oligonucleotide, the second selection criterion may include or may be an L3 score for the target sequence, the third selection criterion may include or may be a L4_OR(5 ^') score for the target sequence, the fourth selection criterion may include or may be a L4_OR(3 ') score for the target sequence, the fifth selection criterion may include or may be a length of the antisense oligonucleotide, and the sixth selection criterion may include or may be a PE_SE score for the target sequence.

[00216] According to various embodiments, the first selection criterion may include or may be a cumulative position score (ACP) for the antisense oligonucleotide, the second selection criterion may include or may be an L3 score for the target sequence, the third selection criterion may include or may be a L4_OR(3 ') score for the target sequence, the fourth selection criterion may include or may be a L4_OR(5') score for the target sequence, the fifth selection criterion may include or may be a length of the antisense oligonucleotide, the sixth selection criterion may include or may be a PESE score for the target sequence.

[00217] ; According to various embodiments, the first selection criterion may include or may be a L4_OR(5 ') score for the target sequence, the second selection criterion may include or may be a L4_OR(3 ') score for the target sequence, the third selection criterion may include or may be an L3 score for the target sequence, the fourth selection criterion may include or may be a cumulative position score (ACP) for the antisense oligonucleotide, the fifth selection cntenon may include or niay be a score for the target sequence, the sixth selection criterion may include or may be a length of the antisense oligonucleotide.

[00218] According to various embodiments, the first selection criterion may include or may be a L4_OR(3 ^') score for the target sequence, the second selection criterion may include or may be ! a L4_OR(5 ^') score for the target sequence, the third selection criterion may include or may be an L3 score for the target sequence, the fourth selection criterion may include or may be a cumulative position score (ACP) for the antisense oligonucleotide, the fifth selection; criterion may include or may be a P_ESE score for the target sequence, and the sixth selection criterion may include or may be a length of the antisense oligonucleotide.

[00219] According to various embodiments, the first selection criterion may include or may be a L4_OR(5 ^') score for the target sequence, the second selection criterion may include or may be a L4_OR(3 ^') score for the target sequence, the third selection criterion may include or may be an L3 score for the target sequence, the fourth selection criterion may include or may be a cumulative position score (ACP) for the antisense oligonucleotide, the fifth selection criterion may include or may be a length of the antisense oligonucleotide, and the sixth selection criterion may include or may be a PE_SE score for the target sequence.

[00220] According to various embodiments, the first selection criterion may include or may be a L4_OR(3 ') score for the target sequence, the second selection criterion may include or may be a L4_OR(5 ^') score for the target sequence, the third selection criterion may include or may be an L3 score for the target sequence, the fourth selection criterion may include or may be a cumulative position score (ACP) for the antisense oligonucleotide, the fifth selection criterion may include or may be a length of the antisense oligonucleotide, and the sixth selection criterion may include or may be a PES score for the target sequence.

[00221] In a fourth aspect, an antisense oligonucleotide: selector for selecting from a plurality of antisense oligonucleotides targeting a target sequence on one of a plurality of exons on a pre-mRNA may be provided. The antisense oligonucleotide selector may include: an exon selection circuit configured to select an exon on the pre-mRNA; a antisense oligonucleotide generation circuit configured to generate a plurality of antisense oligonucleotides for target sequences on the exon pre-mRNA, wherein each of the antisense oligonucleotides has a length of between 15 to 35 nucleotides; a score determination circuit configured to determining a L3 score for a target sequence, a L4_OR(5 ') score for a target sequence, a L4_OR(3 ^') score for a target sequence, a cumulative position score (ACP score) for an antisense oligonucleotide, a P_ESE score for a target sequence; an ordering circuit configured to order the antisense oligonucleotides at first according to the lowest L3 score in a first group; a first selection circuit configured to select a specified number of antisense oligonucleotides starting with the antisense oligonucleotide with the lowest L3 score from the first group and to order them secondly according to the lowest L4 OR(5 ') score in a second group; a second selection circuit; configured to select a specified number of antisense oligonucleotides starting with the antisense oligonucleotide with the. lowest L4_OR(5') from the second group and to order them thirdly according to the lowest L4_OR(3 ') score in a third group; a third selection circuit configured to select a specified number of antisense oligonucleotides starting with the antisense oligonucleotide with the lowest L4_OR(3') from the third group and to order them fourthly according to the lowest AC? score in a fourth group; a fourth selection circuit configured to select a specified number of antisense oligonucleotides starting with the antisense oligonucleotide with the lowest ACP score from the fourth group and to order them fifthly according to the highest P_ESE score in a fifth group; a fifth selection circuit configured to select a specified number of antisense oligonucleotides starting with the antisense oligonucleotide with the highest P_ESE score from the fifth group and to order them sixthly according to the length starting with the longest antisense oligonucleotides in a sixth group; and a sixth selection circuit configured to select at least one antisense oligonucleotides starting with the longest antisense oligonucleotide from the sixth group. - .

[00222] In certain embodiments,! an antisense oligonucleotide selector 3500 for selecting from a plurality of antisense oligonucleotides targeting a target sequence on one of a plurality of exons on a pre-mRNA can be described in Figure 35. The antisense oligonucleotide selector 3500 may include: an exon selection circuit 3502 configured to select an exon on the pre-mRNA; a antisense oligonucleotide generation circuit 3504 configured to generate a plurality of antisense oligonucleotides for target sequences on the exon pre-mRNA, wherein each of the antisense oligonucleotides has a length of between 15 to 35 nucleotides; a score determination circuit 3506 configured to determining a L3 score for a target sequence, a L4_OR(5') score for a target sequence, a L4_OR(3 ') score for a target sequence, a cumulative position score (ACP score) for an antisense oligonucleotide, a P_ESE score for a target sequence; an ordering circuit 3508 configured to order the antisense oligonucleotides at first according to the lowest L3 score in a first group; a first selection circuit 3510 configured to select a specified number of antisense oligonucleotides starting with the antisense oligonucleotide with the lowest L3 score from the first group and to order them secondly according to the lowest L4_OR(5 ') score in a second group; a second selection circuit 3512 configured to select a specified number of antisense oligonucleotides starting with the antisense oligonucleotide with the lowest L4_OR(5') from the second group and to order them thirdly according to the lowest L4_OR(3 ^') score in a third group; a third selection circuit 3514 configured to select a specified number of antisense oligonucleotides starting with the antisense oligonucleotide with the lowest L4_OR(3 ') from the third group and to order them fourthly according to the; lowest ACP score in a fourth group; a fourth selection circuit 3516 configured to select a specified number of antisense oligonucleotides starting with the antisense oligonucleotide with the lowest ACP score from the fourth group and to order them fifthly according to the highest PESE score! in a fifth group; a fifth selection circuit 3518 configured to select a specified number of antisense oligonucleotides starting with the antisense oligonucleotide with the highest PESE score from the fifth group and to order them sixthly according to the length starting with the longest antisense oligonucleotides in a sixth group; and a sixth selection circuit 3520 configured to select at least one antisense oligonucleotides starting with the longest antisense oligonucleotide from the sixth group. The exon selection circuit 3502, the antisense oligonucleotide generation circuit 3504, the score determination circuit 3506, the ordering circuit 3508, the first selection circuit 3510, the second selection circuit 3512, the third selection circuit 3514, the fourth selection circuit 3516, the fifth selection circuit 3518, and the sixth selection circuit 3520 may be coupled electrically or electronically, by a coupling 3522, for example by a cable or a bus - ^~

[00223]: According to various embodiments, a method of selecting at least one antisense oligonucleotide from a plurality of antisense oligonucleotides targeting a target sequence on one of a plurality of exons on a pre-mRNA may be provided. The method may include: selecting an exon on the pre-mRNA; generating a plurality of antisense oligonucleotides for target sequences on the exon pre-mRNA, wherein each of the antisense oligonucleotides has a length of between 15 to 35 nucleotides; determining a L3 score for a target sequence, a L4_OR(5') score for a target sequence, a L4_OR(3 ') score for a target sequence, a cumulative position score !(ACP) for an. antisense oligonucleotide, a PESE score for a target sequence; ordering the antisense oligonucleotides at first according to a first parameter in a first group! wherein the first parameter is selected from a group consisting of the L3 score for the target sequence, the L4_OR(5 ') score for the target sequence, the L4_OR(3 ') score for the target sequence, the cumulative position score (ACP) for the antisense oligonucleotide, the PE_SE score for the target sequence and the length of the antisense oligonucleotide; selecting a specified number of antisense oligonucleotides from the first group and ordering them subsequently in a second group according to any one of the above parameters except the parameter already used to order the antisense oligonucleotides in the first group; and repeating the steps of ordering and selecting the antisense oligonucleotides until the antisense oligonucleotides have been ordered and selected according to each of the parameters.

[00224] According to various embodiments, the antisense oligonucleotides may be ordered at first according to a first parameter in a first group wherein the first parameter is selected from a group consisting of the L3 score for the target sequence, the L4_OR(5') score for the target sequence, the L4_OR(3 ^') score for the target sequence, and the cumulative position score (ACP) for the antisense oligonucleotide.

[00225] According to various embodiments, a specified number of antisense oligonucleotides may be selected from the first group and ordering them secondly in a second group according to a second parameter.

[00226] According to various embodiments, the second parameter may be selected from the group consisting of the L3 score for the target sequence, the L4_OR(5 ') score for the target sequence, and the L4_OR(3 ^') score for the target sequence; wherein the second parameter is different from the first parameter.

[00227] According to various embodiments, a specified number of antisense oligonucleotides may be selected from the second group and they may be ordered thirdly in a third group according to a third parameter; wherein the third parameter is selected from the group consisting of the L3 score for the target sequence, the L4_OR(5 ') score for the target sequence, and the L4_OR(3 ') score for the target sequence; wherein the third parameter is different from the first and second parameter.

[00228] According to various embodiments, a specified number of antisense oligonucleotides may be selected from the third group and they may be ordered fourthly in a fourth group according to a fourth parameter.

[00229] ; According to various embodiments, the fourth parameter may be selected from the group consisting of the cumulative position score (ACP) for the antisense oligonucleotide, the L4_OR(5 ^') score for the target sequence, and the L4_OR(3 ^') score for the target sequence. [00230] According to various embodiments, the fourth parameter may be different from the first, second and third parameter.

[0023 1 ] According to various embodiments, a specified number of antisense oligonucleotides may be selected from the fourth group and they may be ordered Fifthly in a fifth group according to a fifth parameter.

[00232] According to various embodiments, the fifth parameter may be selected from the group consisting of the PESE score and the length of the antisense oligonucleotide; wherein the fifth parameter may be different from the first, second, third and fourth parameter.

[00233] According to various embodiments, a specified number of antisense oligonucleotides may be selected from the fifth group and they may be ordered sixthly in a sixth group according to a sixth parameter.

[00234]! According to various embodiments, the sixth parameter may be selected from the group consisting of the PESE score and the length of the antisense oligonucleotide; wherein the sixth parameter may be different from the first, second; third, fourth and fifth parameter.

[00235] According to various embodiments, the L3 score for the target sequence, the L4_OR(5 ^') score for the target sequence, the L4_OR(3 ^') score for the target sequence, the cumulative position score (ACP) for the antisense oligonucleotide are ordered according to the lowest score within the respective group, and the PESE score may be ordered according to the highest score in the respective group, and the length of the antisense oligonucleotide may be ordered according to the longest antisense oligonucleotide in the respective group:

[00236] According to various embodiments, at least one antisense oligonucleotide may be selected from the sixth group starting with the highest PESE or the longest antisense oligonucleotide.

[00237] According to various embodiments, the antisense oligonucleotides may be ordered at first according to the lowest L3 score in a first group.

[00238] According to various embodiments, a specified number of antisense oligonucleotides may be selected starting with the antisense oligonucleotide with the lowest L3 score from the first group and ordering them secondly according to the lowest L4_OR(5 ^') score in a second group.

[00239] According to various embodiments, specified number of antisense oligonucleotides may be selected starting with the antisense oligonucleotide with the lowest L4_OR(5 ') from the second group ordering them thirdly according to the lowest L4_OR(3 ^') score in a third group. ;

[00240] According to ; various - embodiments, a specified number of antisense oligonucleotides may be selected starting with the antisense oligonucleotide with the lowest L4_OR(3 ') from the third group and ordering them fourthly according to the lowest ACP score in a fourth group.

[00241] According to various embodiments, a specified number of antisense oligonucleotides may be selected starting with the antisense oligonucleotide with the lowest ACP score from the fourth group and ordering them fifthly according to the highest P_ESE score in a fifth group.

[00242] According ; to various embodiments, a specified number of antisense oligonucleotides may be selected starting with the antisense oligonucleotide with the highest PESE score from the fifth group and ordering them sixthly according to the length starting with the longest antisense oligonucleotides in a sixth group.

[00243] According to various embodiments, at least one antisense oligonucleotides may be selected starting with the longest antisense oligonucleotide from the sixth group.

[00244] According to various embodiments, from the group in which the antisense oligonucleotides are ordered according to the length of the antisense oligonucleotides the longest antisense oligonucleotides among the antisense oligonucleotides having a length of 20 to 35 nucleotides may be selected and in case no antisense nucleotides are included in the group of antisense oligonucleotides, having a length of 20 to 35 nucleotides, the longest antisense oligonucleotides having a length of 15 to 19 nucleotides may be selected.

[00245] According to various embodiments, the specified number of antisense oligonucleotides for each group may be independently selected from the first 95% of each group or from the first 80 % of each group, or from the first 60% of each group or from the first 40% of each group, or from the first 20% of each group, or from the first 10% of each group, or from the first 5% of each group.

[00246] According to various embodiments, the specified number of antisense oligonucleotides selected from the first group may be 100 antisense oligonucleotides and the selected specified number of antisense oligonucleotides- from every subsequent group may be X minus 20% or 10% or 5% based on the number of selected antisense oligonucleotides in the previous group, wherein X may be the number of antisense oligonucleotides selected from the previous group.

[00247] According to various embodiments, the specified number of antisense oligonucleotides selected from the first group may be selected from the group consisting of 90 antisense oligonucleotides, 80 antisense oligonucleotides, 70 antisense oligonucleotides, 60 antisense oligonucleotides and 50 antisense oligonucleotides.

[00248] According to various embodiments, the percentage deducted from every number of selected antisense oligonucleotides i the previous group may be selected from the group consisting of 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4% and 3%. :

[00249] According to various embodiments, the method may further include determining a L3 score for each target sequence, a L4_OR(5 ^') score for each target sequence, a L4_OR(3 ') score for each target sequence, a cumulative position score (ACP score) for each antisense oligonucleotide, a PESE score for each target sequence.

[00250] According to various embodiments, the L3 score may be determined for each target sequence corresponding to an antisense oligonucleotide in the first group.

[00251] According to various embodiments, the L4_OR(5') score may be determined for each target sequence corresponding to an antisense oligonucleotide in the second group.

[00252] According to various embodiments, the L4_OR(3') score may be determined for each target sequence corresponding to an antisense oligonucleotide in the third group.

[00253] According to various embodiments, the ACP score may be determined for each antisense oligonucleotide in the fourth group.

[00254] According to various embodiments, the PESE score may be determined for each target sequence corresponding to an antisense oligonucleotide in the fifth group.

[00255] In certain embodiments, a method of selecting at least one antisense oligonucleotide from a plurality of antisense oligonucleotides targeting a target sequence on one of a plurality of exons on a pre-mRNA can be described in Figure 34. In 3402, an exon on the pre-mRNA may be selected.! In 3404, a plurality of antisense oligonucleotides for target sequences on the exon pre-mRNA may be generated, wherein each of the antisense oligonucleotides has a length of between 15 to 35 nucleotides, In 3406, a L3 score for a target sequence,; a L4_OR(5 ^') score for a target sequence, a L4_OR(3 ^') score for a target sequence, a cumulative position score (ACP score) for an antisense oligonucleotide, a PESE score for a target sequence may be determined, In 3408, the antisense oligonucleotides may be ordered at first according to the lowest L3 score in a first group. In 3410, a specified number of antisense oligonucleotides; may be selected starting with the antisense oligonucleotide with the lowest L3 score from the first group and they may be ordered secondly according to the lowest L4_OR(5 ') score in a second group. In 3412, a specified number of antisense oligonucleotides may be selected starting with the antisense oligonucleotide with the lowest L4_OR(5') from the second group and they may be ordered thirdly according to the lowest L4_OR(3 ') score in a third group. In 3414, a specified number of antisense oligonucleotides may be sjelected starting with the antisense oligonucleotide with the lowest L4_OR(3 ') from the third group and they may be ordered fourthly according to the lowest ACP score in a fourth group. In 3416, a specified number of antisense oligonucleotides may be selected starting with the antisense oligonucleotide with the lowest ACP score from the fourth group and they may be ordered fifthly according to the highest PESE score in a fifth group. In 3418, a specified number of antisense oligonucleotides may be selected starting with the antisense oligonucleotide with the highest PE_SE score from the fifth group and they may be ordered sixthly according to the length starting with the longest antisense oligonucleotides in a sixth group. In 3420, at least one antisense oligonucleotides may be selected starting with the longest antisense oligonucleotide from the sixth group

[00256] According to various embodiments, the antisense oligonucleotide selector may comprise a memory which is for example used in the processing carried out by the antisense oligonucleotide selector. A memory used in the embodiments may be a volatile memory, for example a DRAM (Dynamic Random Access Memory) or a non- volatile memory, for example a PROM (Programmable Read Only Memory), an EPROM (Erasable PROM), EEPROM (Electrically Erasable PROM), or a flash memory, e.g., a floating gate memory, a charge trapping memory, an MRAM (Magnetoresistive Random Access Memory) or a PCRAM (Phase Change Random Access Memory) ._

[00257] In an embodiment, a "circuit" may be understood as any kind of a logic implementing entity, which may be special purpose circuitry or a processor executing software stored in a memory, firmware, or any combination thereof. Thus, in an embodiment, a "circuit" may be a hard-wired logic circuit or a programmable logic circuit such as a programmable processor, e.g. a microprocessor (e.g. a Complex Instruction Set Computer (CISC) processor or a Reduced Instruction Set Computer (RISC) processor). A "circuit" may also be a processor executing software, e.g. any kind of computer program, e.g. a computer program using a virtual machine code such as e.g. Java. Any other kind of implementation of the respective functions which will be described in more detail below may also be understood as a "circuit" in accordance with an alternative embodiment

[00258] Co-transcriptional binding accessibility of target site (L3, L4_OR(5') and L4_OR(3') scores). Since exon recognition and splicing is co-transcriptional, AONs need to compete with splicing factors for binding to target exon during transcription. Throughout this process however, the target exon secondary structure is dynamic as newly transcribed nucleotides create new combinations of complementary Watson-Crick base-pairings within the pre-mRNA. As a result, the target exon accessibility for AON binding is dynamic. This is accounted; for by the design variable, co-transcriptional binding accessibility of target site, which was shown (Wee, K.B., et al., 2008, Oligonucleotides, PLoS One, vol.3, no.3, pp. el 844) to be a key factor in influencing AON efficiency in more than one AON datasets published by different labs. Despite its relevance, it has not been used in AON design thus far.

[00259] Average Cumulative Position (ACP) A trend between AON efficiency and starting position of the AON target site was discerned - first target nucleotide of efficient, inefficient and non-effective AONs lies in the first-, middle- and last-third of the target exon (from 5' to 3'), respectively. The Average Cumulative Position score (ACP) is defined to quantify the average position of each target nucleotide from exon 5'. The results indicate that AONs with target sites hearer to the 5 ' end could have better efficiency. In fact, the correlation of AON efficiency with position of target site from exon 5' has been reported through retrospective analysis by several labs. This consistent correlation demonstrates its significant influence on AON efficiency. A plausible explanation is that as dystrophin pre-mRNA is co- transcriptionally spliced, binding competition between AONs and splicing factors begins once the exon 5' is transcribed. As a result, AONs should target sites as soon as the exon begins transcribing, which is at the 5 ' side of the exon.

[00260]! Presence of ESE motifs Due to the presence of long introns in the dystrophin gene, ESE-deperident exori recognition is particularly important. ESEs are short motifs within an exon that splicing factors bind to. An AON induces exon skipping by binding to these motifs and thereby prevent the spliceosome from identifying the target as an exori, which will be removed along with the introns subsequently. Therefore, it is anticipated that the presence of ESEs within an AON target site will influence exon skipping efficiency. [00261] AON target length. Using the two design variables, many short AON target sites (< 10 bases) with high co-transcriptional -binding accessibilities and ESE motifs are possible. A target site of this sequence length may not be sufficiently unique to avoid unspecific binding to other genes and furthermore, it cannot anneal firmly to the target under physiological temperature due to its associated low annealing temperature. In fact, longer AON length correlates with AON efficiency. On the other hand, too long a target site has the issue of binding to partial complementary sequence. Therefore, the length of an AON target must be within an optimal range of 20 to 35 nucleotides.

[00262] An important finding of the present invention was that the above method for selecting at least one AON from a plurality of AONs depends on a hierarchical order according to which the above design parameters are considered when selecting AONs from the manifold possible AONs which can be used to target specific target sequences on the pre- mRNA of an exori of a gene. This means that at first AONs are ordered in a group or list according to their lowest L3 values. Instead of now selecting AONs from the group thus formed which lie below a certain threshold value for L3, a fixed number or fixed percentage of AONs is selected from the group or list starting with the AONs with the lowest values for L3 rather than selecting AONs with a value of L3 lying below a specific threshold value. These AONs are then ordered in a new list now according to their lowest L4_OR(5 ^:) values. Also from this new list a fixed number of AONs or percentage of AONs is selected rather than AONs having the lowest values for L4_OR(5 ^'). This process is continued with all other design parameters as well, i.e. L4_OFL(3 '), ACP etc.

[00263] Notably, as no specific threshold values or ranges are required when selecting AONs from the lists or groups of AONs the method confers generality and thus allows to be used to design AONs for all kinds of genes whether they are associated with a genetic disease or not. Diseases known to be amenable to modulation of pre-mRNA splicing by AONs inducing, for example by inducing exon skipping include, but are not limited to Duchenne muscular dystrophy or DMD, thalassemia, spinal riiuscular atrophy (SMA), Hutchinson- Gilford progeria syndrome, frontotemporal dementia with parkinsonism, ocular albinism and cancer (eg. bcl-x, HER2 gene), cystic fibrosis, ataxia telangiectasia, propionic & methylmalonic academia, myotonic dystrophy (ClC-1 gene), inflammatory diseases (TNFR2 gene) and hypercholestrolnemia (ApoB gene). As mentioned, the method can also be used to design AONs in all eukaryotic system for the purposes of researching on different gene functions. AONs can be used not only to induce exon skipping but in general can be used to induce mRNA degradation, inhibit protein translation, or modulate pre-mRNA splicing.

[00264] The: method of selecting the at least one AON from a plurality of AONs according to the present invention is generally based on the basic principal of determining "engaged" nucleotides and the information derivable thereof. In this context, without wishing to be bound by theory, single stranded RNA sequences, such as pre-mRNA, have a natural tendency to form secondary structures, in which nucleotides in the RNA sequence may bind to or "pair" with another nucleotide to form double stranded sequences/ Each nucleotide in the pre-mRNA sequence will have different capability or tendency to pair with another nucleotide to form a secondary structure. It is therefore expected that this capability or tendency of a nucleotide to pair changes continuously as the pre-mRNA sequence is formed or emerges during transcription. To analyze the changing secondary structure formation during transcription, the following model of transcriptional analysis can be used.

[00265] This transcriptional analysis model involves the use of a "window of analysis". Such a window of analysis can be in the range of about 800 to about 2000 nucleotides; about 1000 to about 2000 nucleotides; about 1200 to about 2000 nucleotides; about 1500 to about 2000 nucleotides in the pre-mRNA . sequence. In certain embodiments, the size of the "window of analysis" can consist of about 1500 nucleotides in the pre-mRNA sequence. This window is moved forward in a series of steps in the direction of transcription, moving forward by one nucleotide at each step. At every step, the window of selected nucleotides, such as the 1500 nucleotides comprised in an exemplary window of analysis, can be analyzed for secondary structure formation using the m-fold prediction software. In this way, at each step of transcriptional analysis, it is possible to obtain a set of predicted secondary structures, which will indicate whether a particular nucleotide is paired or unpaired with another nucleotide in secondary structure formation. When the nucleotide is paired, it will be unavailable for binding to an AON. Therefore, for each "window of analysis", a nucleotide within the target exon is determined to be "engaged" if it was predicted to be completely inaccessible or paired in all the predicted structures.

[00266] For each nucleotide in an AON target site or target exon, with respect to all the steps or "windows of analysis", a nucleotide engaged score may be derived as follows:

Total number of steps of transcriptional analysis at which the nucleotide is engaged / Total number of steps of transcriptional analysis.

[00267] Once the engaged nucleotides are determined further parameters can be calculated which facilitate predicting the design of AONs which are suitable for binding and modulating the mRNA structure, such as by influencing exon splicing (e.g. exon skipping), degrade mRNA or inhibit protein translation.! Determination and calculation of different parameters are explained in the following.

[00268] Definition of L3

[00269] To characterize the AON target site, an AON target site engaged score (L3) may be derived as follows:

Sum of nucleotide engaged scores for all nucleotides within the AON target site / Total number of nucleotides in AON target site

[00270] Definition of L4 OR(5'

[00271] The derivation of the L4_OR(5') score involves analysis of the first three nucleotides at the 5' end of the AON target site (or "5' nucleotides" for short). At each step of transcriptional analysis, it can be determined if one or more of these three 5' nucleotides is engaged or not engaged. Froni this concept, the L4_OR(5') score is defined as follows:

The number of steps of transcriptional analysis at which one or more of the three 5' nucleotides of the AON target site sequence engaged / The total number of steps of transcriptional analysis employed for the AON target site.

[00272] Definition of L4 OR(3')

[00273] The L4_OR(3') score involves analysis of the last three nucleotides at the 3' end of the AON target site (or "3' nucleotides" for short); Similarly, , at each step of transcriptional analysis, it can be determined if one or more the three 3' nucleotides is engaged or not engaged. The L4_OR(3') score is defined as follows: The number of steps of ^' transcriptional; analysis; at which one or more of the three 3 ' nucleotides of the AON target site is engaged I The total number of steps of transcriptional analysis employed for the AON target site.

[00274] L3(AON), L4_OR(5') and L4_OR(3') scores are used to quantify the co- transcriptional binding accessibility for AON design according to the present invention. Therefore, lower scores indicate high co-transcriptional binding accessibility, and vice-versa. As mentioned above, co-transcriptional binding accessibility of target site was shown to be a key facto in influencing AON efficiency and has not been; used in AON design thus far. Even though it is shown in Wee, K B., et al., 2008, Oligonucleotides, PLoS One, vol.3, no.3, pp.el 844 that AON; efficiency is more sensitive to L4_OR(5') than L4_OR(3'), AON efficiency is influenced by the co-transcriptional binding accessibility of an entire target site more than at the 5' or 3 ' ends. Therefore, L3(AON) is ranked the most important among the

[00279] : In some/embodiments, the; scores of each level of analysis described above can be correlated with the efficiency and efficacy of the AONs as described herein. The term "efficacy" used in relation to an AON refers to the ability of an AON to induce specific exon skipping in a given target exon prerrhRNA for example. The "efficiency" as described herein refers to the extent or degree at which an AON has efficacy In this context, the AONs selected using the method of the present invention can not only bind to the target exon pre- mRNA, but also have an increased probability of inducing exon skipping of a pre-mRNA.

[00280] As a non-limiting illustrative example, an AON as described herein which induces specific exon skipping in for example one out of ten pre-mRNAs has efficacy, but may not be necessarily efficient. For example, ; the efficiency of an AON predicted using the method of the present invention can afterwards be analyzed and confirmed in cell culture tests in which cells are incubated with a given concentration or a range of concentrations of at least one AON. Depending on the gene analyzed different cell types can be used. For example, in one embodiment cells that can be used include but are not limited to human fibroblasts, or primary muscle cells or human myotubes. After in vitro transfection, exon skipping efficiency can then be estimated by densitometry analysis of gel images in which the densitometry of the bands representing the transcripts with specific exon skipping (skipped band) and without exon skipping (Unskipped band) are determined, (see Examples 1 to 3). In some embodiments, the AON efficiency can be presented as a percentage of the density of the skipped band to the sum of the densities of the skipped and unskipped bands. As an example, AON efficiency can be classified as "++" if the percentage of the density of the skipped band to the sum of the densities of skipped and unskipped bands is more than 30% ± 5% or ± 4% or ± 3% or ± 2% or^■±; 1%. AON efficiency can be classified as "+" if the percentage of the ; density of the skipped band to the sum of densities of skipped and unskipped bands is less than 20% ± 5% or ± 4% or ± 3% or ± 2% or ±1%. AON efficiency can be classified as "-" if the percentage of the density of the skipped band to the sum of densities of skipped arid unskipped; bands is 0%. As another non-limiting example, equal densities of the skipped and unskipped; band can be regarded as representing 50% efficiency.

[00281] The "hit rate" can be defined as the number of AONs confirmed to fit a specific category of AON efficiency/total number of AONs evaluated or validated. Thus, the hit rate for "++" refers to the number of AONs confirmed to be "++"/ total number of AONs evaluated or validated. The hit rate for "+" refers to the number of AONs confirmed to be "+"/ total number Of AONs evaluated or validated. The hit rate for "-" refers to the number of AONs confirmed to be "-"/ total number of AONs evaluated or validated.

[00282] The term "nucleotide" as used herein includes native (naturally occurring) nucleotides, which include a nitrogenous base selected from the group consisting of adenine (A), thymidine (T), cytosine (C), guanine (G) and uracil (U), a sugar selected from the group of ribose, arabifiose, xylose, and pyranose, and deoxyribose (the combination of the base and sugar generally referred to. as a "nucleoside"), and one to three phosphate groups, and which can form phosphodiester intemucleosidyl linkages. The "nucleotide" can also refer to nucleotide analogs. Such analogs can have a sugar analog, a base analog and/or an intemucleosidyl linkage analog. Additionally, analogs exhibiting non-standard base pairing are also included (see for example U.S. Pat. No. 5,432,272). Such nucleotide analogs include nucleotides that are chemically modified in the natural base ("base analogs"), chemically modified in the natural sugar ("sugar analogs"), and/or chemically modified in the natural phosphodiester or any other intemucleosidyl linkage ("intemucleosidyl linkage analogs"). In certain embodiments, the aromatic ring or rings contain at least one nitrogen atom. In certain embodiments, the nucleotide base is : capable of forming Watson-Crick and/or Hoogsteen hydrogen bonds with an appropriately complementary nucleotide base. Exemplary nucleotide bases and analogs thereof include, but are not limited to, naturally occurring nucleotide bases, e.g., adenine, guanine, cytosine, uracil, and thymine, and analogs of the naturally occurring nucleotide bases, e.g., 7-deazaadenine, 7-deazaguanine, 7-deaza-8-azaguanine, 7-deaza-8- azaadenine, N652-isopentenyladenine, N2-dimethyl guanine (dmG), 7-methyl guanine (7mG), inosine, nebularine, 2-amin0purine, 2-amino-6-chloropurine, 2,6-diaminopurine, hypoxanthine, pseudouridine, pseudocytosine, pseudoisocytosine, 5-propynylcytosine, isocytosine, isoguanine, 7-deazaguanine, 2thiopyrimidine, 6-thioguanine, 4-thiothymine, 4- thiouracil, 0.sup.6-miethylguanine, \ N.sup.6-methyladenine, 0.sup.4-methylthymine, 5,6- dihydrothymine, 5 -6-dihydrouracil,: pyrazolo[3,4-D]pyrimidines (see for example U.S. Pat. Nos. 6,143,877 and: 6,127,121), ethenoadenine, indoles such as nitroindole and 4- methylindole, and pyrroles such as nitropyrrole.

[00283] The term "antisense oligonucleotide" or "AON" as described herein refers to a molecule that is complementary to a "sense" nucleic acid encoding a gene expression product, for example, complementary to the coding strand of a double stranded cDNA molecule: or complementary to an RNA sequence, e.g., a pre-mRNA (precursor mRNA) or mRNA. Accordingly an AON can form hydrogen bonds with a sense target nucleic acid sequence. In some embodiments, the AON as described herein can bind or hybridize to a complementary nucleotide sequence of one of a plurality of exons of any part of a target pre- mRNA during transcription. In other embodiments, the AON described herein binds to nascent pre-mRNA during transcription.. In some embodiments, the AONs can be a DNA or a RNA molecule. In this context, an "oligonucleotide" refers to a polymer of repeating units generically known as nucleotides or nucleosides. An unmodified (naturally occurring) nucleotide has three components: (1) a nitrogenous base linked by one of its nitrogen atoms to (2) a 5-carbon cyclic sugar and (3) a phosphate, esterified to carbon 5 of the sugar. When incorporated into an oligonucleotide chain, the phosphate of a first nucleotide is also esterified to carbon 3 of the sugar of a second, adjacent nucleotide. The "backbone" of an unmodified oligonucleotide consists of (2) and (3), that is, sugars linked together by phosphodiester linkages between the C5 (5') position of the sugar of a first nucleotide and the C3 (3') position of a second, adjacent nucleotide. A "nucleoside" is the combination of (1) a nucleobase and (2) a sugar in the - absence of a phosphate moiety. The backbone of an oligonucleotide positions a series of bases in a specific order; the written representation of this series of bases, which is conventionally written in 5 ' to 3 ' order, is known as a nucleotide sequence.

[00284] Oligonucleotides can be obtained from existing nucleic acid sources, including genomic or cDNA, or produced by synthetic methods. In some embodiments, each nucleoside unit can include a heterocyclic base and a pentofuranosyl, 2'-deoxypentfuranosyl, trehalose, arabinose, 2 '-deoxy-2 '-substituted arabinose, 2'-0-substituted arabinose or hexose sugar group. The nucleoside residues can be coupled to each other by any of the numerous known internucleoside linkages. Such internucleoside linkages include, without limitation, phosphodiester, ^'. phosphorothioate, phosphorodithioate, alkylphosphonate, alkylphosphonothioate, phosphotri ester, phosphoramidate, siloxane, carbonate, carboalkoxy, acetamidate, carbamate, morpholino, borano, thioether, bridged phosphoramidate, bridged methylene phosphonate, bridged phosphorothioate, and sulfone internucleoside linkages. The term "oligonucleotide" also encompasses polynucleosides having one or more stereospecific internucleoside linkage (e.g., (Rp)- or (Sp)-phosphorothioate, alkylphosphonate, or phosphotriester linkages). As used herein, the term "oligonucleotide" is expressly intended to include polynucleosides and dinucleosides having any such ihtemucleoside linkage, whether or not the linkage comprises a phosphate group. In certain embodiments, these internucleoside linkages may be phosphodi ester, phosphorothioate, or phosphorodithioate linkages, or combinations thereof. ;

[00285] . ^' As used herein, the term "complementary" or "complement" is intended to mean the relationship of nucleotides/bases on two different strands of DNA or RNA, where the bases are paired (guanine with cytosine, adenine with thymine (DNA) or uracil (RNA)). Therefore, an AON of the present invention or selected according to the method of the present invention can form hydrogen bond(s) with another nucleotide sequence, for example a target exon pre-mRNA, by either; conventional Watson-Crick base pairing or other non- traditional types of pairing such as Hoogsteen or reversed Hoogsteen hydrogen bonding between complementary nucleosides or nucleotides. In this context, it is understood in the art that a nucleic acid molecule need not be 100% complementary to a target nucleic acid sequence to be specifically or selectively hybridizable. That is, two or more nucleic acid molecules may be less than fully complementary. Complementarity is indicated by a percentage of contiguous residues in a nucleic acid molecule that can form hydrogen bonds with a second nucleic acid molecule.; For example, if a first nucleic acid molecule has 10 nucleotides and a second nucleic acid molecule has 10 nucleotides, then base pairing of 5, 6, 7, 8, 9, or 10 nucleotides between the first and second nucleic acid molecules represents 50%, 60%, 70%, 80%, 90%, and 100% complementarity, respectively, not to mention a few.

[00286] In certain embodiments, an AON that "binds" to a target site or target exon pre- mRNA refers to an AON that specifically hybridizes or duplexes to the respective target site or target exon pre-mRNA via complementary base pairing during transcription, thereby inducing exon skipping. The "target site" or "AON target site" refers to a nucleotide sequence which contains at least a region or sequence that is the reverse complement of an AON as described herein. Accordingly, the "target exon" used in relation to a pre-mRNA refers to the particular region or sequence that is present in the pre-mRNA or the messenger mRNA. In some embodiments, an AON as described herein that targets a target sequence on one of a plurality of exons on a pre-mRNA may not induce exon skipping.

[00287] The "target exon pre-mRNA" or "target sequence on one of a plurality of exons on a pre-mRNA" as described herein can be derived from any kinds of genes, such as genes associated with diseases amenable to modulation of pre-mRNA splicing, eg., exon skipping. Such diseases include diseases caused by mutations of the ubiquitin-activating enzyme El

(UBE1) gene, mutations of the lamin A (LMNA gene) or mutations of the microtubule- associated protein tail (MAPT) gene, not to mention a few. Examples of such disease can include but are not limited to Duchenne muscular dystrophy or DMD, thalassemia, spinal muscular atrophy (SMA), Hutchinson-Gilford progeria syndrome, frontotemporal dementia with parkinsonism, ocular albinism, cancer (eg. bcl-x, HER2 gene), cystic fibrosis, ataxia telangiectasia, propionic & methylmalonic acidemia, myotonic dystrophy (ClC-1 gene), inflammatory diseases (TNFR2 gene) and hypercholesterolemia (ApoB gene), In some embodiments, the exon of dystrophin pre-mRNA (Accession No. NM 004006); can be selected from the group consisting exons 1 to 79, In other embodiments, the exon of dystrophin pre-mRNA can also be selected from the group consisting of exons 1 to 50 and 52 to 79. In further embodiments, the exon can be selected from the group consisting of exons 2, 43, 45, 46, 47, 50, 51, 53 and 57.

[00288] As used herein, the terms "precursor messenger mRNA" or "pre-mRNA" refer to an immature single strand of messenger ribonucleic acid (mRNA) that contains one or more intervening sequence(s) (introns). Pre-mRNA is transcribed by an RNA polymerase from a DNA template in the cell nucleus and is comprised of alternating sequences of introns and coding regions (exons). Without wishing to be bound by theory, pre-mRNA undergoes splicing to remove the introns to form mRNA or messenger RNA. In the process of transcription, pre-mRNA is formed by the consecutive addition of one nucleotide after another by the RNA polymerase II, starting with the 5' end of pre-mRNA. Therefore, pre- mRNA is not generated instantaneously in its entirety, but built up by nucleotide by nucleotide. Hence, at any moment during the continuous process of transcription, a part of the incomplete pre-mRNA which has just been formed (or "extruded" from the RNA polymerase II) by the addition of nucleotides, parts further away towards the 5' end which were formed some time earlier. Therefore, the "nascent pre-mRNA" refers to the pre-mRNA strand that is being extruded out from the RNA polymerase II during co-transcriptional pre-mRNA processing. This nascent pre-mRNA" is part of the pre-mRNA under construction. As mentioned above, once a pre-mRNA has been completely processed by the splicing out of introns and joining of exons, it is referred to as "messenger RNA" or "mRNA", which is an RNA that is comprised exclusively of exons. Eukaryotic pre-mRNAs exist only transiently before being fully processed into mRNA. When a pre-mRNA has been properly processed to an mRNA sequence, it is exported out of the nucleus and eventually translated into a protein by ribospmes in the cytoplasm.

[00289] The term "generating" in "generating a plurality: of antisense oligonucleotides" means primarily that the AONs are generated with the use of a computer system and not in physical form even though the latter one is not excluded. In other words the term ''generating" means that a list of AQNs is provided that can theoretically be binding due to its complementarity to a specific target sequence on the exon mRNA. From this theoretical list of possible AONs specific AONs are selected using the method of the present invention.

[00290] -The plurality of AONs used according to the method of the present invention can be of any specified number, depending on the specific sequence that the AON is designed to bind. For instance, the average length of a dystrophin exon of 140 bases would generate 1276 possible AON target sequences with lengths of 20 to 30 nucleotides.

[00291] In certain embodiments, the specified number of AONs for each group as described according to the method of the present invention can be independently selected from the first 95% of each group or from the first 80 % of each group, or from the first 60% of each group or from the first 40% of each group, or from the first 20% of each group, or from the first 10% of each group, or from the first 5% of each group.

[00292] In other embodiments, the specified number of AONs selected from the first group as described according to the method of the present invention can be 100 antisense oligonucleotides and the selected specified number of antisense oligonucleotides from every subsequent group is X minus 20% or 10% or 5% based on the number of selected antisense oligonucleotides in the previous group, wherein X is the number of antisense oligonucleotides selected from the previous group. In this context, the specified number of AONs selected frbm the first group can be one selected from the group consisting of 90 antisense oligonucleotides, 80 antisense oligonucleotides, 70 antisense oligonucleotides, 60 antisense oligonucleotides and 50 antisense oligonucleotides: The percentage deducted from every number of selected antisense oligonucleotides in the previous group may be selected from the group consisting of 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4% and 3%.

[00293] In some embodiments, the top 10% of AONs can be selected from the first group as described according to the method of the present invention, followed by the top 80% from the second group, followed by the top 80% from the third group, followed by the top 30% from the fourth group, followed by the top 50% from the fifth group, followed by the top 10 AONs from the sixth group (if available). In certain embodiments, the first group can be AONs sorted by ascending L3 scores, the second group can be AONs sorted by ascending L4_OR(5') scores, the third group can be AONs sorted by ascending L4_OR(3') scores, the fourth group can be sorted by ascending ACP values, the fifth group can be sorted by descending PESE and the sixth group can be AONs sorted by descending target length.

[00294] ; In some embodiments, the AONs of the present invention or selected according to the method of the present invention can include a backbone. The "backbone" of an unmodified AON generally refers to a 5-carbon cyclic sugar and a phosphate, esterified to carbon 5 of the sugar, that is, sugars linked together by phosphodi ester linkages between the C5(5') position of the sugar of a first nucleotide and the C3 (3') position of a second, adjacent nucleotide. In some embodiments, the backbone can include one that retains a phosphorous atom and/or one that does not have a phosphorous atom. In other embodiments, the backbone can be one of ribonucleic acid, deoxyribonucleic acid, DNA phosphorothioate, RNA phosphorothioate, 2'-0-hydrocarbyl ribonucleic acid, 2'-0-hydrocarbyl DNA, 2'-0- hydrocarbyl RNA phosphorothioate, 2'-0-hydrocarbyl DNA phosphorothioate, 2'-F- phosphprothioate, 2'-F-phbsphodiester, 2'-methoxyethyl phosphorothioate, 2-methoxyethyl phosphodiester, deoxy methylene(methylimino) (deoxy MMI), 2'-0-hydrocarby MMI, deoxy-methylphos-phonate, 2'-0-hydrocarbyl methylphosphonate, morpholino, 4'-thio DNA, 4'-thio RNA, peptide nucleic acid, 3'-amidate, deoxy 3'-amidate, 2'-0- hydrocarbyl 3'- amidate, locked nucleic acid, cyclohexane nucleic acid, tricycle-DNA, 2'fluoro-arabino nucleic acid, N3'-P5' phosphoroamidate, carbamate linked, phosphotriester linked, a nylon backbone modification and mixtures of the aforementioned backbones.

[00295] In some embodiments, the AONs of the present invention or selected according to the method of the present invention can; also include molecules selected from one of phosphorodiamidate morpholino oligomer (PMO), peptide-conjugated phosphorodiamidate morpholino oligomer (PPMO), and non-peptide dendrimeric octaguanidine moiety-tagged morpholino oligomer and combinations thereof.

[00296] ; In some embodiments, the AONs of the present invention or selected according to the method of the present invention can include at least one chemically modified nucleotide base. The synthesis of oligonucleotides for example AONs as described herein comprising a modified or non-natural base are within the knowledge of a person of average skill in the art and can be found in for example, US Patent No. 5,457,191, drawn to modified nucleobases based on the 3-dezazpurine ring system and methods of synthesis thereof; and US Patent No. 5,459,255, drawn to modified nucleobases based on N-2 substituted purines. Examples of chemically modified; nucleotide bases can also include but are not limited to 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2- aminopropyladenine, 5-propynyluracil and 5-propynylcytosine, not to mention a few.

[00297] ; Another : modification of the AONs of the present invention or selected according to the method of the present invention can involve chemically linking to the AON one or more moieties or conjugates that enhance the activity, cellular distribution or cellular uptake of the oligonucleotide. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety, cholic acid, a thioether, e.g., hexyl-5-tritylthiol, a thiocholesterol, an aliphatic chain, e.g., dodecandiol or Undecyl residues, a phospholipid, e.g., di-hexadecyl-rac- glycerol or triethylammonium l,2-di-0-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or a polyethylene glycol chain, or adamantane acetic acid, a palmityl moiety, or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety.

[00298]: The present invention also relates to an antisense oligonucleotide (AON) having a L3 score of about < 0: 1 ; a L4_OR(5 ') score of about < 0.15; a L4_OR(3 ') score of about < 0.12; a P_ESE score of about > 70 %; and the antisense oligonucleotide binds to a target exon pre-mRNA starting in the first one third of the target exon pre-mRNA, counting from 5 ' to 3 '; wherein the antisense oligonucleotides

H3A (5'-GUAGGUCACUGAAGAGGUUCU-3') (SEQ ID NO: 24),

h29AON6 (5'-UCUGUGCCAAUAUGCGAAUC-3') (SEQ ID NO: 27),

H32A (5 '-CUUGUAGACGCUGCUCAAAAUUGGCUGGUU-3 ') (SEQ ID NO: 28), H36A(1) (5'-UGUGAUGUGGUCCACAUUCUGGUCAAAAGU-3 ') (SEQ ID NO: 29), h40AONl (5'-GAGCCUUUUUUCUUCUUUG-3') (SEQ ID NO: 30),

h49AONl (5'-CUUCCACAUCCGGUUGUUU-3') (SEQ ID NO: 31),

h55AON3 (5'-UGCAGUAAUCUAUGAGUUUC-3') (SEQ ID NO: 32),

h59AONl (5'-CAAUUUUUCCCACUCAGUAUU-3') (SEQ ID NO: 33),

h60AONl (5'-GUUCUCUUUCAGAGGCGC-3') (SEQ ID NO: 34),

h63 AON1 (5 '-GGUCCCAGCAAGUUGUUUG-3 ') (SEQ ID NO: 35),

h71 AONl (5'-GCCAGAAGUUGAUCAGAGU-3') (SEQ ID NO: 36), h73 AGN1 (5'-GAUCCAUUGCUGUUUUCC-3 ') (SEQ ID NO: 37),

h74AONl (5'-CUGGCUCAGGGGGGAGU-3') (SEQ ID NO: 38),

H75A (5'-GGACAGGCCUUUAUGUUCGUGCUGC-3') (SEQ ID NO: 39),

h78AONl (5'-GCUUUCCAGGGGUAUUUC-3') (SEQ ID NO: 40),

h78AON2 (5'-CAUUGGCUUUCCAGGGG-3') (SEQ ID NO: 41) are excluded.

[00299] The feature that the antisense oligonucleotide binds to a target exon pre-mRNA starting in the first one third of the target exon pre-mRNA means that the AON should bind to a target site which starts within the first one third of the target exon, counting from 5' to 3'

(i.e. the first 5' nucleotide of the target sequence is one of the nucleotides comprising the first one third o f the exon, within which the target site resides).

[00300] ; To clarify this definition, the following examples are provided. The number of nucleotides that makes up an exon varies from one exon: to another. If the number of nucleotides in an exon divided by three results in a whole number, then each one third portion of the exon consists of a similar whole number of nucleotides. Thus, for an exon with 30 nucleotides, the first one third of the exon will be the first 10 nucleotides, counting from the 5' end. In this case, the first nucleotide of the AON target site should be one of these first 10 nucleotides. However, for an exon with 29 nucleotides, each one third portion of the exon will consist of 9.66" nucleotides, which is not a whole number of nucleotides. In this case, the first nucleotide of the AON target site should be one of the first 9 nucleotides. It should not be the 10^th nucleotide, as only 0.66" of the nucleotide, but not the whole nucleotide, is within the first one third of the exon.

[00301] In certain embodiments, the AON of the present invention can have a length of > 21 nucleotides, provided that the antisense oligonucleotides H4A (5'-UGU UCA GGG CAU GAA CUC UUG UGG AUC CUU-3') (SEQ ID NO:. 25), H32A (5'-CUU GUA GAC GCU GCU CAA AAU UGG CUG GUU-3') (SEQ ID NO: 28), H36A (5'-UGU GAU GUG GUC CAC AUU CUG GUC AAA AGU-3') (SEQ ID NO: 29) and H75A (5'-GGA CAG GCC UUU AUG UUC GUG CUG C-3') (SEQ ID NO: 39) are excluded.

[00302] In some embodiments, the AON of the present invention has a MAXNEP score of about < 0.95; and a MAX_P_NEP<o.o5 score of about > 0.32,; provided that H75A (5'-GGA CAG GCC UUU AUG UUC GUG CUG C-3') (SEQ ID NO: 39) is excluded.

[00303] Definition of MAXNgg : ; [00304] As evident from in the definition of "engaged" nucleotide given above, at each step of transcription analysis, a nucleotide in an AON target site will either be engaged or not engaged. It follows that for each nucleotide in an AON target site, it can be determined that the proportion of steps of transcription analysis when the nucleotide is engaged in relation to the total number of steps , of transcriptional analysis applied for the site. This is named the Nucleotide Engaged Proportion or NEP score:

Number of steps of transcriptional analysis at which a nucleotide in the AON target site is engaged / ' Total number of steps of transcriptional analysis employed for the AON target site. The MAXNEP is defined as the highest NEP among the NEPs of all nucleotides in the AON target site.

[00305] The derivation of NEP score from the Plot of Engaged Nucleotides is illustrated in Figure 12.

[003061 ; Definition of MAX PNFP_<( rvs Γ

[00307] This score is derived from the NEP score, A histogram can be drawn to illustrate the NEP score of each nucleotide in an AON target site, as illustrated by Figure 12. From such a histogram, sections can be identified in which all consecutive nucleotides have NEP scores which are lower than 0.05. For each section with 2 or more consecutive nucleotides having NEP scores < 0.05, a PNEP_<O_.O_S score may be obtained as follows:

Number of nucleotides in the section of the AON; target site wherein all consecutive nucleotides have NEP scores less than 0.057 Total number of nucleotides in the AON target site

[00308] In certain embodiments, the PESE score is between about 72.7 % to about 100%. In other embodiments , the L3 score is between about 0.0087 to about 0.0964.

[00309] In some embodiments, the AON of the present invention can have a length of between about 22 to 31 nucleotides. In other embodiments, the AON of the present invention can be any of the following:

AON 2 (5 '-UAC CCA UUU UGU GAA UGU UUU CUU UtJG A-3 ') (SEQ ID NO: 2), AON 43-1 (5 -GAG CUU UGU UGU AGA CUA UCU UUU AUA UU-3') (SEQ ID NO: 3), N 45-1 (5'-UUA UUU CUU CCC CAG UUG CAU UCA AUG UUC U-3') (SEQ ID : 5),

N 46 1 (5'-UGA CAA GAU AUU CUU UUG UUC UUC UAG C-3') (SEQ ID NO: 8), N 47 1 (5'-ACG GGU CCU CCA GUU UCA UUU AAU UGU UUG-3') (SEQ ID NO:),

N 50 1 (5 '-ACU CAG AGC UCA GAU CUU CUA AC-3') (SEQ ID NO: 13),

N 51 1 (5'-GGU UGU GUC ACC AGA GUA ACA GUC UGA GUA-3') (SEQ ID NO:),

N 51 2 (5'-UUU CUA GUU UGG AGA UGG CAG UUU CCU UAG U-3') (SEQ ID : 15),

N 53-1 (5'-GUG UUC UUG UAC UUC AUC CCA CUG A-3') (SEQ ID NO: 17), N 53-2 (5/-CGG UUC UGA AGG UGU UCU UGU ACU UCA-3') (SEQ ID NO: 18), N 53-3 (5 '-CUG UUG CCU CCG GUU CUG AAG GUG UUC UUG U-3') (SEQ ID : 19), and

N 57-1 (5'-GUC AGA ACU GGC UUC CAA AUG G-3'j (SEQ ID NO: 21).

310] In other embodiments, the AONs as described herein can be any of the following: N 2-1 (5 '-UGU UUU CUU UUG AAC AUC UUC UCU UU-3 ^') (SEQ ID NO: 1) N 2-2 (5 '-UAC CCA UUU UGU GAA UGU UUU CUU UUG A-3') (SEQ ID NO: 2), N 43-1 (5'-GAG CUU UGU UGU AGA CUA UCU UUU AUA UU-3') (SEQ ID NO: 3), N 45-1 (5'-UUA UUU CUU CCC CAG UUG CAU UCA AUG UUC U-3') (SEQ ID : 5),

N 46-1 (5'-UGA CAA GAU AUU CUU UUG UUC UUC UAG C-3') (SEQ ID NO: 8), N 46-2 (5'-UCU UUU CCA GGU UCA AGU GGC AUA CU-3') (SEQ ID NO: 9)

47-1 (5' -ACG GGU CCU CCA GUU UCA UUU AAU UGU UUG-3') (SEQ ID NO:),

N 50- 1 (5'-ACU CAG AGC UCA GAU CUU CUA AC-3') (SEQ ID NO: 13),

N 51 - 1 (5'-GGU UGU GUC ACC AGA GUA ACA GUC UGA GUA-3') (SEQ ID NO:),

N 51-2 (5'-UUU CUA GUU UGG AGA UGG CAG UUU CCU UAG U-3') (SEQ ID : 15),

N 53 1 (5'-GUG UUC UUG UAC UUC AUC CCA CUG A-3') (SEQ ID NO: 17), N 53-2 (5'-CGG UUC UGA AGG UGU UCU UGU ACU UCA-3') (SEQ ID NO: 18), AON 53-3 (5 '-CUG UUG CCU CCG GUU CUG AAG GUG UUC UUG U-3') (SEQ ID NO: 19), and

AON 57-1 (5'-GUC AGA ACU GGC UUC CAA AUG G-3') (SEQ ID NO: 21).

[0031 1] In specific embodiments, a total of 12 AONs of the present invention fulfilled all the above 8 primary and secondary criteria when tested for skipping of various dystrophin exons. All of these 12 AONs showed hig efficiency (++ or >25%) in induction of the iritended exon skipping. In contrast, none of the target sequences, of the AONs with low efficiency (+ or <25% efficiency) of which were not efficacious (-), fulfilled all the six primary criteria.

Table 1: summarizes the characteristics of the AONs referred to herein according to an embodiment of the present invention: . ^' . ,

List of "Criteria":

1. L3(AON) < 0.1

2. L4_OR(5') < 0 15

3. L4_OR(3') < 0 12

4. AON targets first third of exori (from 5' to 3')

5. PE_SE 0.7

6. AON length > 21 nt

7. MAX_NEP < 0.95

8: MAX PNEP<₀.₀₅ > 0.32

[00312] The following Table 2 summarizes the characteristics of known AONs from

Wilton SD, Fall AM, Harding PL, McClorey G, Coleman C et al. (2007) Antisense

Qligonucleotide-induced Exon Skipping Across the Human Dystrophin Gene Transcript. Mbl Ther 15: 1288-1296; and: Aartsma-Rus A, De Winter CL, Janson AA, Kaman WE, van

providing a subject with a sufficient amount of the complex or pharmaceutical composition or medicament thereof so as to alleviate or eliminate a disease state and/or the symptoms of a disease state, and a weakened and/or unhealthy state.

[00314] The term "pharmaceutically effective amount" as used herein means that amount of an AON as described above or a pharmaceutical composition or medicament comprising the AON compound which is effective for producing some desired therapeutic effect in at least a sub^population of cells in the patient at a reasonable benefit/risk ratio applicable to any medical treatment. ;

[00315] In some embodiments, the AON of the present invention or selected according to the method of the present invention can be administered together with a carrier. The "carrier" can include any pharmaceutically acceptable carrier as long as the carrier can is compatible with other ingredients of the formulation and not injurious to the patient. Accordingly, pharmaceutical compositions for use in accordance with the present invention may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.

[00316] In some embodiments, the carrier as used herein can include a nanoparticle, such as a polymeric nanoparticle. Polymeric nanoparticles ^"are within the knowledge of the person of averaged skill in the art and can for example be formed from chitosan and hyaluronic acid; or chitosan and alginic acid; as described in PCT Publication WO 2009/035438 drawn to polymeric nanoparticles by ion-ion interactions. The carrier can also.be a liposome, such as a pH-sensitive liposome or an antibody conjugated liposome. Liposomes are artificial membrane vesicles which are useful delivery vehicles in vitro and in vivo. These formulations may have net cationic, anionic or neutral charge characteristics and are useful characteristics with in vitro, in vivo and ex vivo delivery methods. Therefore, in some embodiments, a liposome carrier can be a combination of phospholipids or other lipids. The physical characteristics of such liposomes can for example depend on the pH, ionic strength, and the presence of divalent cations. The carrier as used herein can also be a viral vector, or a cationic lipid, or a polymer, or a UsnRNA, such as U7 snRNA, or Ul RNA or U6 RNA, or a cell penetrating peptide.

[00317] In other embodiments, the carrier as used herein can also include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives,: such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragaeanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such -as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and; soybean oil; (10) glycols, such as propylene glycol; (11) pblyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters; such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and/or polyanhydrides; and (22) other non-toxic compatible substances employed in pharmaceutical formulations.

[00318] The AONs as described above or pharmaceutical composition or medicament thereof can be administered in a number of ways depending upon whether local or systemic administration is desired and Upon the area to be treated. In some embodiments, the AONs as described above can be administered to the patient orally, or rectally, or transmucosally, or intestinally, or intramuscularly, or siibcutaneously, or intramedullary, or intrathecally, or direct intraventricularly, or intravenously, or intravitreally, or intraperitoneally, or intranasally, or intraocularly.

[00319] The AONs of the invention or selected according to the method of the invention can also encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other compound, which, upon administration to an animal, including a human, is capable of providing the biologically active metabolite or residue thereof. Accordingly, also described herein is drawn to prodrugs and pharmaceutically acceptable salts of such pro-drugs, and other bioequivalents. The term "pharmaceutically acceptable salt" refers to physiologically and pharmaceutically acceptable salt(s) of the AONs as described above; i.e. salts that retain the desired biological activity of - the parent compound and do not impart undesired toxicological effects thereto. Examples of such pharmaceutically acceptable salts include but arc not limited to (a) salts formed with cations such as sodium, potassium, ammonium, magnesium, calcium, polyamines such as spermine and spermidine, etc; (b) acid addition salts formed with inorganic acids, for example hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid and the like; (c) salts formed with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfohic acid, polygalacturonic acid, and the like; and (d) salts formed from elemental anions such as chorine, bromine, and iodine.

[00320] The invention illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising", "including", "containing", etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed; Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

[00321] The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[00322] Other embodiments are within the following claims and non-limiting examples. In addition, where features or aspects; of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

EXAMPLES

Example 1: Transfection of AONs into Human Fibroblast Cells

[00323] Human fibroblast cells are cultured in growth medium containing DMEM with 10%FBS on six-well cell culture dish (diameter of 30mm). When the cells reach confluence, the cells are prepared for the transfection by replacing the growth medium with Opti-MEM containing 5% horse serum. The transfection mixture is prepared by mixing the pre-diluted AON and pre-diluted Lipofectamine2000 (Life Technologies, Inc.) (both in opti-MEM) followed by 30 minutes incubation at room temperature. After 30 minutes incubation, the transfection mixture is added to the prepared fibroblast cells followed by incubatioii in 37°C with 5%C0₂. After 5 hours of incubation, the transfection mixture containing medium is replaced with fresh growth medium and incubation to be continued until the cells to be harvested for mRN A analysis.

Example 2: Transfection of AONs into primary cultures of DMD patients' myogenic cells

[00324] Primary cell cultures derived from DMD patients' skeletal muscle (provided by Dr. Muntoni, UK) were seeded in 6-well plates coated with collagen (Biomed), and grown in supplemented muscle cell growth medium. Cultures were switched to supplemented muscle cell differentiation medium when myoblasts fused to form visible myotubes (elongated cells containing multiple nuclei and myofibrils). Both media were purchased from Promocell (Heidelberg, Germany). Transfection of AON was then performed as described in Example 1. Example 3: Assessment of the AON Efficiency in Inducing Targeted Exon Skipping

(mRNA analysis)

[00325] Reverse Transcription-PCR - Total RNA is extracted from fibroblast 24 hours after AON treatment using Trizoi (Invitrogen, Carlsbad, Canada). Single step RT-PCR is performed on ~400ng total RNA using a single step RT-PCR analysis kit, Access RT-PCR system (Promega, Madison, USA), according to the manufacturer's instructions for 20 cycles, followed by nested PGR for 22 cycles. Sequences of dystrophin exon-specific primers used for single step RT-PCR and nested PC R are listed in Table 3. Exon skipping efficiency is estimated by densitometry analysis of the gel images^" comparing the density of amplicons from dystrophin mRNA with exon; skipping to the sum of native dystrophin mRNA plus dystrophin mRNA with exdri skipping as previously described in Graham IR et al, J. Gene

Med., 2004, 6: 1 149-58. The PCR products! are then verified by sequencing to confirm the skipping of the specific exon targeted.

Table 3 : List of primers used for RT-PCR analyzing the efficacy and efficiency of the AONs in inducing specific exon skipping

Primer set for Nested PCR cDMD-54R 56 5 '-G AAGTTTC AGGGCC AAGTC A-3 '

To assess AON57

cDMD-54F 58 5'-GCCAGTGGCAGACAAATGTA-3'

; Primer set for Single step RT-PCR CDMD-60R 59 5'-GCAATTTCTCCTCGAAGTGC-3'

CDMD-55F 60 5 '-C AGG ATGCT ACCCGT AAGG A-3 '

Primer set for Nested PCR cDMD^59R 61 5 '-GCTTTCGT AG AAGCCG AGTG-3 '

*F = forward

R = reverse

Example 4: AON Design

[00326]. 23 AONs targeting exons 2, 43, 45, 46, 47, 50, 51, 53 and 57 of dystrophin gene are. designed based on the hierarchical; set of design variables^ in accordance with the method of the present invention. The target exons were chosen based on their therapeutic potential on DMD patients' mutations (Aartsma-Rus et al. (2006). Entries in the Leiden Duchenne muscular dystrophy mutation database: an Overview of mutation types and paradoxical cases that confirm the reading-frame rule. Muscle Nerve. 34: 135-144). The co-transcriptional binding accessibility of each target exon is obtained as described herein. Next, target exon sequences were predicted for ESE motifs using ESE-finder (Cartegni L, Wang J, Zhu Z, Zhang MQ, Krainer AR (2003) ESEfinder: A web resource to identify exonic splicing enhancers. Nucleic Acids Res 31 : 3568-3571) and Rescue-ESE (Fairbrother WG, Yeh RF, Sharp PA, Burge CB (2002) Predictive identification of exonic splicing enhancers in human genes. Science 297: 1007-1013). The predicted ESE motifs were then merged with the co- transcriptional binding accessibility plot. AON target sequences were subsequently selected by first having the highest co-transcriptional binding accessibility (lowest possible L3(AON), L4_OR(5') and L4_OR(3') scores), followed by the highest possible P_ESE. The range of target length selected is between 20 to 35 nucleotides. Multiple AONs were designed for each target exon if more than one optimal target sequences are available (Figure

26). ;

Example 5: Assessment of AONs targeting exons 51 and 53 efficiency in restoring

■ dystrophin expression in DMD muscle cells

[00327] Muscle cells of DMD patients do not express dystrophin, which is normally present i the sarcolemma. To analyze the effect of AON therapy, the expression of dystrophin was detected by immunpchemistry staining of AON treated and untreated muscle cells. Primary antibody anti C -terminal dystrophin was used to assess the success of the restoration of the dystrophin expression. As DMD patients carry mutations that lead to fnRNA reading frame shift with a stop codon/truncation of the protein, the antibody against C-terminal (3') of dystrophin should not be able to detect any dystrophin in their muscle tissues. The success of the AON in restoring dystrophin expression should be marked by the detection of the dystrophin in the patients's muscle cells. The efficiency of the AON was reflected by the number of dystrophin positive cells compared to dystrophin negative myogenic cells.

[00328] On the third day after transfection the muscle cells on the cover slips were fixed with methanol for 5 minutes followed by acetone treatment for 1 minute. Double staining immunocytochemistry method was used to detect dystrophin and desmin. Mouse monoclonal anti-dystrophin antibody (No vocastra; Laboratories Ltd.) and rabbit polyclonal anti-desmin antibody (Sigma) were used as the respective primary antibodies. Following the primary antibody, Alexa fluor 594 goat anti mouse lgG (Invitfogen): and Alexa fiuor 488 goat anti rabbit lgG (Invitrogen) were applied to satin dystrophin and desmin, respectively.

[00329] Specific exon skipping induced by AONs targeting exons 51 and 53 respectively were demonstrated in DMD patient's ! muscle cells. Immunocytochemistry staining showing the restoration of dystrophin expression in DMD patient's muscle cells induced by AON targeting exons 51 and 53 are shown in Figure 29.

Example 5: Comparison of hit rates in designing efficient AONs with prior studies

[00330] 14 AONs were found to be efficient among 19 effective AONs; at least one efficient AON is obtained per target exon. This translates to hit rates of 61% and 83% for designing efficient and effective AONs respectively. In comparison, corresponding hit rates of between 18 to 38% and between 56 to 79% were obtained in previous studies as shown in Figure 30. When only AONs targeting the same exons as our study is compared, the hit rates obtained in previous studies are lower at between 21 to 27% and between 44 to 73%, respectively (Figure 30). The hit rates obtained in the present invention is thus a significant improvement over previous efforts, especially for the design of efficient AONs according to the present invention.

[00331] ; The higher hit rates obtained in the present invention than all prior studies supports the plausibility of designing efficient AONs by a rational approach that eliminates the need for trial-and-error and preliminary experimentations (for instance, hybridization array analysis of each target exon). Therefore, the present method confers another advantage of expending less resource while speeding up the AON design process. Given that different

AONs are needed to address specific DMD patients' mutations, the ability to design efficient

AONs with a high hit rate requiring minimal resources in a short time is valuable.

Claims

1. An antisense oligonucleotide having a L3 score of about < 0.1 ; a L4_OR(5 ) score of about < 0.15; a L4_OR(3 ^') score of about < 0.12; a P_ESE score of about > 70 %; and the antisense oligonucleotide binds to a target exon pre-mRNA starting in the first one third of the target, exon pre-mRNA, counting from 5 ^' to 3 ^'; wherein the antisense oligonucleotides

H3A (5'-GUAGGUCACUGAAGAGGUUCU-3 ') (SEQ ID NO: 24),

H4A (5'-UGUyCAGGGCAUGAACUCUUGUGGAUCCUU-3') (SEQ ID NO: 25), h2?AON2 (5'-GGUUAUCCUCUGAAUGUCGC-3 ') (SEQ ID NO: 26),

h29AON6 (5'-UCUGUGCCAAUAUGCGAAUC-3 ') (SEQ ID NO: 27),

H32A (5' CUUGUAGACGCUGCUCAAAAUUGGCUGGUU-3 ') (SEQ ID NO:

28) ,

H36A(1) (5'-UGUGAUGUGGUCCACAUUCUGGUCAAAAGU-3 ') (SEQ ID NO:

29) ,

h40AONl (5'-GAGCCUUUUUUCUUCUUUG-3 ') (SEQ ID NO: 30),

h49AONl (5'-CUUCCACAUCCGGUUGUUU-3 ') (SEQ ID NO: 31),

h55AON3 (5'-UGCAGUAAUCUAUGAGUUUC-3') (SEQ ID NO: 32),

h59AONl (5 '-C AAUUU ϋ UCCCACUC AGU AUl J-3 ') (SEQ ID NO: 33),

h60AONl (5'-GUUCUCUUUCAGAGGCGC-3 ') (SEQ ID NO: 34),

h63AONl (5'-GGUCCCAGCAAGUUGUUUG-3 ') (SEQ ID NO: 35),

h71AONl (5'-GCCAGAAGUUGAUCAGAGU-3 ') (SEQ ID NO: 36),

h73AONl (5'-GAUCCAUUGCUGUUUUCC-3') (SEQ ID NO: 37),

h74AONl (5'-CUGGCUCAGGGGGGAGU-3 ') (SEQ ID NO: 38),

H75A (5'-GGACAGGCCUUUAUGUUCGUGCUGC-3 ') (SEQ ID NO: 39), h78AONl (5'-GCUUUCCAGGGGUAUUUC-3 ') (SEQ ID NO: 40),

h78AON2 (5'-CAUUGGClJUUCCAGGGG-3 ') (SEQ ID NO: 41) are excluded.

2. The antisense oligonucleotide of claim 1 , wherein the antisense oligonucleotide has a length of > 21 nucleotides; wherein the antisense oligonucleotides H4A (5'-UGU UCA GGG CAU GAA CUC UUG UGG AUC CUU-3 ') (SEQ ID NO: 25), H32A (5'-CUU GUA GAC GCU GCU CAA AAU UGG CUG GUU-3') (SEQ ID NO: 28), H36A (5 '-UGU GAU GUG GUC CAC AUU .CUG GUC AAA AGU-3 ') (SEQ ID NO: 29) and H75A (5 '-GGA CAG GCC UUU AUG UUC GUG CUG C-3 ') (SEQ ID NO: 39) are excluded.

3. The antisense oligonucleotide of claim 2, wherein the antisense oligonucleotide has a MAXNEP score of about < 0.95; and a MAX_P_NEp<o.o5 score of about > 0.32; wherein H75A (5 '-GGA CAG GCC UUU AUG UUC GUG CUG C-3 ') (SEQ ID NO: 39) is excluded.

4. The antisense oligonucleotide of any one of claims 1 to 3, wherein the PESE score is between about 72.7 % to about 100%.

5: The antisense oligonucleotide of any one of the preceding claims, wherein the L3 score is between about 0.0087 to about 0.0964.

6. The antisense oligonucleotide of any one of the preceding claims, wherein the antisense oligonucleotide has a; length of between about 22 to 31 nucleotides.

7. The antisense oligonucleotide of any one of the preceding claims, wherein the antisense oligonucleotide comprises a backbone which is selected from the group consisting of ribonucleic acid, deoxyribonucleic acid, DNA phosphorothioate, RNA phosphorothioate, 2'-0-hydrocarbyl ribonucleic acid, 2^'-0-hydrocarbyl DNA, 2'-0- hydrocarbyl RNA phosphorothioate, 2'-0-hydrocarbyl DNA phosphorothioate, 2 -F- phosphorothioate, 2 -F-phosphodiester, 2'-methoxyethyl phosphorothioate, 2- methoxyethyl phosphodi ester, deoxy methylene(methylimino) (deoxy MMI), 2'-0- hydrocarby MMI, deoxy-methylphos-phonate, 2'-0-hydrocarbyl methylphosphonate, mprpholino, 4'-thio DNA, 4 ^!-thio RNA, peptide nucleic acid, 3'-amidate, deoxy 3'- amidate, 2'-0- hydrocarbyl 3 - amidate, locked nucleic acid, cyclohexane nucleic acid, tricycle-DNAi 2'fluoro-arabino nucleic acid, N3 '-P5 ' phosphoroamidate, carbamate linked, phosphotriester linked, a nylon backbone modification and mixtures of the aforementioned backbones.

The; antisense oligonucleotide of any one of claims 1 to 7, wherein the antisense oligonucleotide comprises ^' molecules which are selected from the group consisting of phosphorodiamidate morpholino oligomer (PMO), peptide-conjugated phosphorodiamidate morpholino oligomer (PPMO), and non-peptide dendrimeric octaguanidine moiety-tagged morpholino oligomer.

The antisense oligonucleotide of any one of the preceding claims, wherein the target exon pre-mRNA is at least one of the exons of dystrophin pre-mRNA.

10 The antisense oligonucleotide of claim 9, wherein the exon Of dystrophin pre-mRNA is selected from the group consisting of exons 1 to 79.

1 1. The antisense oligonucleotide of claim 10, wherein the exon is selected from the group consisting of exons 1 to 50 and 52 to 79.

12. The antisense oligonucleotide of claim 10, wherein the exon is selected from the group consisting of exons 2, 43, 45, 46, 47, 50, 51 , 53 and 57.

13. The antisense oligonucleotide of any one of claims 1 to 12, wherein the antisense oligonucleotide is a DNA or RNA molecule.

14. The antisense oligonucleotide of any one of claims 1 to 13, wherein the antisense oligonucleotide comprises at least one chemically modified nucleotide base.

15 The antisense oligonucleotide Of any one of the ; preceding claims, wherein the antisense oligonucleotide is _; -selected from the group consisting of

ON 2 (5 '-UAC CCA UUU UGU GAA UGU UUU CUU UUG A-3 ') (SEQ ID NO: 2), ON 43-1 (5 '-GAG CUU UGU UGU AGA CUA UCU UUU AUA UU-3') (SEQ ID NO: 3), ON 45-1 (5'-UUA UUU CUU CCC CAG UUG CAU UCA AUG UUC U-3') (SEQ ID O: 5),

ON 46-1 (5'-UGA CAA GAU AUU CUU UUG UUC UUC UAG C-3 ') (SEQ ID NO: 8), AON 47-1 (5'-ACG GGU CCU CCA GUU UCA UUU AAU UGU UUG-3') (SEQ ID NO: 10),

AON 50-1 (5 '-ACU CAG AGC UCA GAU CUU CUA AC-3') (SEQ ID NO: 13),

AON 51-1 (5'-GGU UGU GUC ACC AGA GUA ACA GUC UGA GUA-3') (SEQ ID NO: 14),

AON 51 2 (5'-UUU CUA GUU UGG AGA UGG CAG UUU CCU UAG U-3') (SEQ ID NO: 15),

AON 53- (5'-GUG UUC UUG UAC UUC AUC CCA CUG A-3') (SEQ ID NO: 17), ΑΟΝ 53- (5'-CGG UUC UGA AGG UGU UCU UGU ACU UCA-3') (SEQ ID NO: 18), AON 53 (5 '-CUG UUG CCU CCG GUU CUG AAG GUG UUC UUG U-3') (SEQ ID NO: 19),

AON 57- 1 (5'-GUC AGA ACU GGC UUC CAA AUG G-3') (SEQ ID NO: 21).

16. A method of treating Duchenne Muscular Dystrophy (DMD) in a patient comprising administering a pharmaceutically effective amount of an antisense oligonucleotide of any one of the preceding claims to a patient.

17. The method of claim 16, wherein the antisense oligonucleotide is administered together with a carrier.

18. The method of claim 17, wherein the carrier is selected from the group consisting of a nanoparticle, such as a polymeric nanoparticle; a liposome, such as pH-sensitive liposome, an antibody conjugated liposome; a viral vector, a cationic lipid, a polymer, a UsnRNA. such as U7 snRNA and a cell penetrating peptide.

19. The method of any one of claims 16 to 18, wherein; the antisense oligonucleotide is administered orally, or rectal, or transmucosal, or intestinal, or intramuscular, or subcutaneous, or intramedullary, or intrathecal, or direct intraventricular, or intravenous, or intravitreal, or intraperitoneal, or intranasal, or intraocular.

20. A method for selecting at least one antisense oligonucleotide from, a plurality of antisense oligonucleotides targeting a target sequence on an exon on a pre-mRNA, wherein the method comprises:

determining for each antisense oligonucleotide of the plurality of antisense oligonucleotides a first evaluation value according to a first evaluation criterion: determining a first subset of the plurality of antisense oligonucleotides based on the first evaluation values;

determining for each antisense oligonucleotide of the first subset a second evaluation -value according to a second evaluation criterion; and

determining a second subset of the first subset of antisense oligonucleotides based on the second evaluation values.

21. The method of claim 20,

wherein at least one of the first evaluation criterion and the second evaluation criterion is related to binding of the antisense oligonucleotide with the exon.

22. The method of claim 20 or 21 ,

wherein determining the first subset comprises:

ordering the antisense oligonucleotides of the plurality of antisense oligonucleotides according to the first evaluation values; and

selecting as the first subset a pre-determined number or a pre-determined percentage of antisense oligonucleotides of the plurality of antisense oligonucleotides based on the ordering of the plurality of antisense oligonucleotides.

23. The method of any one of claims 20 to 22, :

wherein determining the second subset comprises:

ordering the antisense oligonucleotides of the first subset according to the second evaluation values; and

selecting as the second subset a pre-determined number or a pre-determined percentage of antisense oligonucleotides of the first subset based on the ordering of the antisense oligonucleotides of the first subset.

The method of any one of claims 20 to 23,

wherein at least one of the first evaluation criterion and the second evaluation criterion comprises at least one of an L3 score for the target sequence, a L4_OR(5 ') score for the target sequence, a L4_OR(3 ^') score for the target sequence, a cumulative position score (ACP) for the antisense oligonucleotide, a PESE score for the target sequence, and a length of the antisense oligonucleotide.

25. The method of any one of claims 20 to 24, further comprising:

determining for each antisense oligonucleotide of the second subset a third evaluation value according to a third evaluation criterion; and

determining a third subset of the second subset of antisense oligonucleotides based on the third evaluation values.

The method of claim 25,

wherein the third evaluation criterion is related to binding of the antisense oligonucleotide with the exon.

27. The method of claims 25 or 26,

wherein determining the third subset comprises:

ordering the antisense oligonucleotides of the second subset according to the third evaluation values; and

selecting as the third subset a pre-determined number or a pre-determined percentage of antisense oligonucleotides of the second subset based on the ordering of the antisense oligonucleotides of the second subset.

28. The method of any one of claims 25 to 27, .

wherein the third evaluation criterion comprises at least one of an L3 score for the target sequence, a L4_OR(5 ') score for the target sequence, a L4_OR(3 ') score for the target sequence, a cumulative position score (ACP) for the antisense oligonucleotide, a PESE score for the target sequence, and a length of the antisense oligonucleotide.

29. The method of any one of claims 25 to 28, further comprising: determining for each antisense oligonucleotide of the third subset a fourth evaluation value according to a fourth evaluation criterion; and

determining a fourth subset of the third subset of antisense oligonucleotides based on the fourth evaluation values.

30. The method of claim 29,

wherein the fourth evaluation criterion is related to binding of the antisense oligonucleotide with

31. The method of claims 29 or 30,

wherein determining the fourth subset comprises:

ordering the antisense oligonucleotides of the third subset according to the fourth evaluation values; and

selecting as the fourth subset a pre-determined number or a pre-determined percentage of antisense oligonucleotides of the third subset based on the ordering of the antisense oligonucleotides of the third subset.

32. The method of any one of claims 29 to 31 ,

wherein the fourth evaluation criterion comprises at least one of an L3 score for the target sequence, a L4_OR(5 ') score for the target sequence, a L4_OR(3 ') score for the target sequence, a cumulative position score (ACP) for the antisense oligonucleotide, a PE_SE score for the target sequence, and a length of the antisense oligonucleotide.

The method of any one of claims 29 to 32, further comprising:

determining for each antisense oligonucleotide of the fourth subset a fifth evaluation value according to a fifth evaluation criterion; and

determining a fifth subset of the fourth subset of antisense oligonucleotides based on the fifth evaluation values.

34. The method of claim 33,

wherein the fifth evaluation criterion is related to binding of the antisense oligonucleotide with the exon.

35. The method of claims 33 or 34,

wherein determining the fifth subset comprises:

ordering the antisense oligonucleotides of the fourth subset according to the fifth evaluation values; and

selecting as the fifth subset a pre-determined number or a pre-determined percentage of antisense oligonucleotides of the fourth subset based on the ordering of the antisense oligonucleotides of the fourth subset.

36. The method of any one of claims 33 to 35,

wherein the fifth evaluation criterion comprises at least one of an L3 score for the target sequence, a L4_OR(5 ^') score for the target sequence, a L4_OR(3 ') score for the target sequence, a cumulative position score (ACP) for the antisense oligonucleotide, a PE_SE score for the target sequence, and a length of the antisense oligonucleotide.

37. The method of any one of claims 33 to 36, further comprising:

determining for each antisense oligonucleotide of the fifth subset a sixth evaluation value according to a sixth evaluation criterion; and

determining a sixth subset of the fifth subset of antisense oligonucleotides based on the sixth evaluation values.

38. The method of claim 37,

wherein the sixth evaluation criterion is related to binding of the antisense oligonucleotide with the exori.

The method of claims 37 or 38,

wherein determining the sixth subset comprises:

ordering the antisense oligonucleotides of the fifth subset according to the sixth evaluation values; and

selecting as the sixth subset a pre-determined number or a pre-determined percentage of antisense oligonucleotides of the fifth subset based on the ordering of the antisense oligonucleotides of the fifth subset.

The method of any one of claims 37 to 39,

wherein the sixth evaluation criterion comprises at least one of an L3 score for the target sequence, a L4_OR(5 ^') score for the target sequence, a L4_OR(3 ') score for the target sequence, a cumulative position score (ACP) for the antisense oligonucleotide, a PESE score for the target sequence, and a length of the antisense oligonucleotide.

The method of claim 22 and claim 23 and claim 27^' and claim 31 and claim 35 and claim 3 ,

wherein the first selection criterion comprises an L3 score for the target sequence, the second selection criterion comprises a L4_OR(5 ') score for the target sequence, the third selection criterion comprises a L4_OR(3 ') score for the target sequence, the fourth selection criterion comprises a cumulative position score (ACP) for the antisense oligonucleotide, the fifth selection criterion comprises a P_ESE score for the target sequence, arid the sixth selection criterion comprises a length of the antisense oligonucleotide, or

wherein the first selection criterion comprises an L3 score for the target sequence, the second selection criterion comprises a L4_OR(5 ') score for the target sequence, the third selection criterion comprises a L4_OR(3 ') score for the target sequence, the fourth selection criterion comprises a cumulative position score (ACP) for the antisense oligonucleotide, the fifth selection criterion comprises a length of the antisense oligonucleotide, and the sixth selection criterion comprises a PESE score for the target sequence, or

wherein the first selection criterion comprises an L3 score for the target sequence, the second selection criterion comprises a L4_OR(3 ') score for the target sequence, the third selection criterion comprises a L4_OR(5 ') score for the target sequence, the fourth selection criterion comprises a cumulative position score (ACP) for the antisense oligonucleotide, the fifth selection criterion comprises a PESE score for the target sequence, and the sixth selection criterion comprises a length of the antisense oligonucleotide, or

wherein the first selection criterion comprises an L3 score for the target sequence, the second selection criterion comprises a L4_OR(3 ') score for the target sequence, the third selection criterion comprises a L4_OR(5 ^') score for the target sequence, the fourth selection criterion comprises a cumulative position score (ACP) for the antisense oligonucleotide, the fifth selection criterion comprises a length of the antisense oligonucleotide, and the sixth selection criterion comprises a PESE score for the target sequence, or

wherein the first selection criterion comprises a cumulative position score (ACP) for the antisense oligonucleotide, the second selection criterion comprises an L3 score for the target sequence, the third selection criterion comprises a L4_OR(5 ') score for the target sequence, the fourth selection criterion comprises a L4_OR(3 ^') score for the target sequence, the fifth selection criterion comprises a PESE score for the target sequence, and the sixth selection criterion comprises a length of the antisense oligonucleotide, or

wherein the first selection criterion comprises a cumulative position score (ACP) for the antisense oligonucleotide, the second selection criterion comprises an L3 score for the target sequence, the third selection criterion comprises a L4_OR(3 ') score for the target sequence, the fourth selection criterion comprises a L4_OR(5 ^') score for the target sequence, the fifth selection criterion comprises a PESE score for the target sequence, and the sixth selection criterion comprises a length of the antisense oligonucleotide, or

wherein the first selection criterion comprises a cumulative position score (ACP) for the antisense oligonucleotide, the second selection criterion comprises an L3 score for the target sequence, the third selection criterion comprises a L4_OR(5 ') score for the target sequence, the fourth selection criterion comprises a L4_OR(3 ^') score for the target sequence, the fifth selection criterion comprises a length of the antisense oligonucleotide, and the sixth selection criterion comprises a PESE score for the target sequence, or

wherein the first selection criterion comprises a cumulative position score (ACP) for the antisense oligonucleotide, the second selection criterion comprises an L3 score for the target sequence, the third selection criterion comprises a L4_OR(3 ^') score for the target sequence, the fourth selection criterion comprises a L4_OR(5 ^') score for the target sequence, the fifth selection criterion comprises a length of the antisense oligonucleotide, the sixth selection criterion comprises a PE_SE score for the target sequence, or

wherein the first selection criterion comprises a L4_OR(5 ^') score for the target sequence, the second selection criterion comprises a L4_OR(3 ^') score for the target sequence, the third selection criterion comprises an L3 score for the target sequence, the fourth selection criterion comprises a cumulative position score (ACP) for the antisense oligonucleotide, the fifth selection criterion comprises a P_ESE score for the target sequence, the sixth selection criterion comprises a length of the antisense oligonucleotide, or

wherein the first selection criterion comprises a L4_OR(3 ^') score for the target sequence, the second selection criterion comprises a L4_OR(5 ^') score for the target sequence, the third selection criterion comprises an L3 score for the target sequence, the; fourth selection criterion comprises a cumulative position score (ACP) for the antisense oligonucleotide, the fifth selection criterion comprises a P_ESE score for the target sequence, and the sixth selection criterion comprises a length of the antisense oligonucleotide, or

wherein the first selection criterion comprises a L4_OR(5 ^') score for the target sequence, the second selection criterion comprises a L4_OR(3 ^') score for the target sequence, the third selection criterion comprises an L3 score for the target sequence, the fourth selection criterion comprises a cumulative position score (ACP) for the antisense oligonucleotide, the fifth selection criterion comprises a length of the antisense oligonucleotide, and the sixth selection criterion comprises a PESE score for the target sequence, or

wherein the first selection criterion comprises a L4_OR(3 ^') score for the target sequence, the second selection criterion comprises a L4_OR(5 ^') score for the target sequence, the third selection criterion comprises an L3 score for the target sequence, the fourth selection criterion comprises a cumulative position score (ACP) for the antisense oligonucleotide, the fifth selection criterion comprises a length of the antisense oligonucleotide, and the sixth selection criterion comprises a P_Es_E score for the target sequence.

An antisense oligonucleotide selector for selecting at least one antisense oligonucleotide from a plurality of antisense oligonucleotides targeting a target sequence on an exon on a. pre-mRNA, the antisense oligonucleotide selector comprising:

a first evaluation values determiner configured to determine for each antisense oligonucleotide of the plurality of antisense oligonucleotides a first evaluation value according to a first evaluation criterion;

a first subset determiner configured to determine a first subset of the plurality of antisense oligonucleotides based on the first evaluation values;

a second evaluation values: determiner configured to determine for each antisense oligonucleotide of the first subset a second evaluation value according to a second evaluation criterion; and

a second subset determiner configured to determine a second subset of the first subset of antisense oligonucleotides based on the second evaluation values.

43. The antisense oligonucleotide selector of claim 42,

44. The antisense oligonucleotide selector of claim 42 or 43,

wherein the first subset determiner comprises:

a first ordering circuit configured to order the antisense oligonucleotides of the plurality of antisense oligonucleotides according to the first evaluation values; and a first selecting circuit configured to select as the first subset a pre-determined number or a pre-determined percentage of antisense oligonucleotides of the plurality of antisense oligonucleotides based on the ordering of the plurality of antisense oligonucleotides.

45. The antisense oligonucleotide selector of any one of claims 42 to 44,

wherein the second subset determiner comprises:

a second ordering circuit configured to order the antisense oligonucleotides of the first subset according to the second evaluation values; and a second selecting circuit configured to select as the second subset a pre-determined number or a pre-determined percentage of antisense oligonucleotides of the first subset based on the ordering of the antisense oligonucleotides of the first subset.

46. The antisense oligonucleotide selector of any one of claims 42 to 45,

wherein at least one of the first evaluation criterion and the second evaluation criterion comprises at least one of an L3 score for the target sequence, a L4_OR(5 ') score for the target sequence, a L4_OR(3 ') score for the target sequence, a cumulative position score (ACP) for the antisense oligonucleotide, a P_ESE score for the target sequence, and a length of the antisense oligonucleotide.

47. The antisense oligonucleotide selector of any one of claims 42 to 46, further comprising:

« a third evaluation values determiner configured to determine for each antisense oligonucleotide of the second subset a third evaluation value according to a third evaluation criterion; and

a third subset determiner configured to determine a third subset of the second subset of antisense oligonucleotides based on the third evaluation values.

The antisense oligonucleotide selector of claim 47,

wherein the third evaluation criterion is related to binding of the antisense oligonucleotide with the exori.

The antisense oligonucleotide selector of claims 47 or 48,

wherein the third subset determiner comprises:

a third ordering circuit configured to order the antisense oligonucleotides of the second subset according to the third evaluation values; and

a third selecting circuit configured to select as the third subset a pre-determined number or a pre-determined percentage of antisense oligonucleotides of the second subset based on the ordering of the antisense oligonucleotides of the second subset.

50. The antisense oligonucleotide selector of any one of claims 47 to 49, wherein the third evaluation criterion comprises at least one of an L3 score for the target sequence, a L4_OR(5 ^') score for the target sequence, a L4_OR(3 ') score for the target sequence, a cumulative position score (ACP) for the antisense oligonucleotide, a PE_SE score for the target sequence, and a length of the antisense oligonucleotide.

51. The antisense oligonucleotide selector of any one of claims 47 to 50, further comprising:

^■ a fourth evaluation values determiner configured to determine for each antisense oligonucleotide of the third subset a fourth evaluation value according to a fourth evaluation criterion; and

^■ a fourth subset determiner configured to determine a fourth subset of the third subset of antisense oligonucleotides based on the fourth evaluation values.

52. The antisense oligonucleotide selector of claim 51 ,

wherein the fourth evaluation criterion is related to binding of the antisense oligonucleotide with the exon. ^'

53. The antisense oligonucleotide selector of claims 51 or 52,

wherein the fourth subset determiner comprises:

a fourth ordering circuit configured to order the antisense oligonucleotides of the third subset according to the fourth evaluation values; and

a fourth selecting circuit configured to select as the fourth subset a pre-determined number or a pre-determined percentage of antisense oligonucleotides of the third subset based on the ordering of the antisense oligonucleotides of the third subset.

54. The antisense oligonucleotide selector of any one of claims 51 to 53,

55. The antisense oligonucleotide selector of any one of claims 51 to 54, further comprising:

a fifth evaluation values determiner configured to determine for each antisense oligonucleotide of the fourth subset a fifth evaluation value according to a fifth evaluation criterion; and

a fifth subset determiner configured to determine a fifth subset of the fourth subset of antisense oligonucleotides based on the fifth evaluation values.

56. The antisense oligonucleotide selector of claim 55,

57. The antisense oligonucleotide selector of claims 55 or 56,

wherein the fifth subset determiner comprises:

a fifth ordering circuit configured to order the antisense oligonucleotides of the fourth subset according to the fifth evaluation values; and

a ; fifth selecting circuit configured to select as the fifth subset a pre-determined number or a pre-determined percentage of antisense oligonucleotides of the fourth subset based on the ordering of the antisense oligonucleotides of the fourth subset.

58. The antisense oligonucleotide selector of any one of claims 55 to 57,

wherein the fifth evaluation criterion comprises at least one of an L3 score for the target sequence, a L4_OR(5 ) score for the target sequence, a L4_OR(3 ^') score for the target sequence, a cumulative position score (ACP) for the antisense oligonucleotide, a PE_SE score for the target sequence, and a length of the antisense oligonucleotide.

59. The antisense oligonucleotide selector of any one of claims 55 to 58, further comprising:

■ a sixth evaluation values determiner configured to determine for each antisense oligonucleotide of the fifth subset a sixth evaluation value according to a sixth evaluation criterion; and a sixth subset determiner configured to determine a sixth subset of the fifth subset of antisense oligonucleotides based on the sixth evaluation values.

The antisense oligonucleotide selector of claim 59,

wherein the sixth evaluation criterion is related to binding of the antisense oligonucleotide with the exon.

61. The antisense oligonucleotide selector of claims 59 or 60,

wherein the sixth subset determiner comprises:

a sixth ordering circuit configured to order the antisense oligonucleotides of the fifth subset according to the sixth evaluation values; and

a sixth selecting circuit configured to select as the sixth subset a pre-determined number or a pre-determined percentage of antisense oligonucleotides of the fifth subset based on the ordering of the antisense oligonucleotides of the fifth subset.

62. The antisense oligonucleotide selector of any one of claims 59 to 61 ,

wherein the sixth evaluation criterion comprises at least one of an L3 score for the target sequence, a L4_OR(5 ') score for the target sequence, a L4_OR(3 ') score for the target sequence, a cumulative position score (ACP) for the antisense oligonucleotide, a PE_SE score for the target sequence, and a length of the antisense oligonucleotide.

63. The antisense oligonucleotide selector of claim 44 and claim 45 and claim 49 and claim 53 and claim 57 and claim 61,

wherein the first selection criterion comprises an L3 score for the target sequence, the second selection criterion comprises a L4_OR(5 ') score for the target sequence, the third selection criterion comprises a L4_OR(3 ') score for the target sequence, the fourth selection criterion comprises a cumulative position score (ACP) for the antisense oligonucleotide, the fifth selection criterion comprises a P_ESE score for the target sequence, and the sixth selection criterion comprises a length of the antisense oligonucleotide, or ^'

wherein the first selection criterion comprises an L3 score for the target sequence, the second selection criterion comprises a L4_OR(5 ') score for the target sequence, the third selection criterion comprises a L4_OR(3 ') score for the target sequence, the fourth selection criterion comprises a cumulative position score (ACP) for the antisense oligonucleotide, the fifth selection criterion comprises a length of the antisense oligonucleotide, and the sixth selection criterion comprises a PE_SE score for the target sequence, or

wherein the first selection criterion comprises an L3 score for the target sequence, the second selection criterion comprises a L4_OR(3 ^') score for the target sequence, the third selection criterion comprises a L4_OR(5 ^') score for the target sequence, the fourth selection criterion comprises a cumulative position score (ACP) for the antisense oligonucleotide, the fifth selection criterion comprises a PESE score for the target sequence, and the sixth selection criterion comprises a length of the antisense oligonucleotide, or

wherein the first selection criterion comprises an L3 score for the target sequence, the second selection criterion comprises a L4_OR(3 ') score for the target sequence, the third selection criterion comprises a L4_OR(5 ^') score for the target sequence, the fourth selection criterion comprises a cumulative position score (ACP) for the antisense oligonucleotide, the fifth selection criterion comprises a length of the antisense oligonucleotide, and the sixth selection criterion comprises a PE_SE score for the target sequence, or

wherein the first selection criterion comprises a cumulative position score (ACP) for the antisense oligonucleotide, the second selection criterion comprises an L3 score for the target sequence, the third selection criterion comprises a L4_OR(5 ^') score for the target sequence, the fourth selection criterion comprises a L4_OR(3 ^') score for the target sequence, the fifth selection criterion comprises a PESE score for the target sequence, and the sixth selection criterion comprises a length of the antisense oligonucleotide, or

wherein the first selection criterion comprises a cumulative position score (ACP) for the antisense oligonucleotide, the second selection criterion comprises an L3 score for the target sequence, the third selection criterion comprises a L4_OR(3 ^') score for the target sequence, the fourth selection criterion comprises a L4_OR(5 ^') score for the target sequence, the fifth selection criterion comprises a PE_SE score for the target sequence, and the sixth selection criterion comprises a length of the antisense oligonucleotide, or

wherein the first selection criterion comprises a cumulative position score (ACP) for the antisense oligonucleotide, the second selection criterion comprises an L3 score for the: target sequence, the third: selection criterion comprises a L4_OR(5^') score for the target sequence, the fourth selection criterion comprises a L4_OR(3 ') score for the target sequence, the fifth selection criterion comprises a . length of the antisense oligonucleotide, and the sixth selection criterion comprises a P_ESE score for the target sequence, or

wherein the first selection criterion comprises a cumulative position score (ACP) for the; antisense oligonucleotide, the second selection criterion comprises an L3 score for the target sequence, the third selection criterion comprises a L4_OR(3 ') score for the target sequence, the fourth selection criterion comprises a L4_OR(5') score for the target sequence, the fifth selection criterion comprises a length of the antisense oligonucleotide, the sixth selection criterion comprises a P_ESE score for the target sequence, or

wherein the first selection criterion comprises a L4_OR(5') score for the target sequence, the second selection criterion comprises a L4_OR(3 ^') score for the target sequence, the third selection criterion comprises an L3 score for the target sequence, the fourth selection criterion comprises a cumulative position score (ACP) for the antisense oligonucleotide, the fifth selection criterion comprises a P_ESE score for the target sequence, the sixth selection criterion comprises a length of the antisense oligonucleotide, or

wherein the first selection criterion comprises a L4_OR(3 ^') score for the target sequence, the second selection criterion comprises a L4_OR(5 ') score for the target sequence, the third selection criterion comprises an L3 score for the target sequence, the fourth selection criterion comprises a cumulative position score (ACP) for the antisense oligonucleotide, the fifth selection criterion comprises a PESE score for the target sequence, and the sixth selection criterion comprises a length of the antisense oligonucleotide, or

wherein the first selection criterion comprises a L4_OR(5 ') score for the target sequence, the second selection criterion comprises a L4_OR(3 ) score for the target sequence, the third selection criterion comprises an L3 score for the target sequence, the fourth selection criterion comprises a cumulative position score (ACP) for the antisense oligonucleotide, the fifth selection criterion comprises a length of the antisense oligonucleotide, and the sixth selection criterion comprises a PE_SE score for the target sequence, or

wherein the first selection criterion comprises a L4_OR(3 ^') score for the target sequence, the second selection criterion comprises a L4_OR(5 ') score for the target sequence, the third selection criterion comprises an L3 score for the target sequence, the fourth selection criterion comprises a cumulative position score (ACP) for the antisense oligonucleotide, the fifth selection criterion comprises a length of the antisense oligonucleotide, and the sixth selection criterion comprises a PESE score for the target sequence.