EP4522745A1

EP4522745A1 - Antisense oligonucleotides targeting cfp-elk1 intergene region

Info

Publication number: EP4522745A1
Application number: EP23726061.7A
Authority: EP
Inventors: Katarzyna CHYZYNSKA; Lars Joenson; Bettina NORDBO; Jonas VIKESAA
Original assignee: F Hoffmann La Roche AG
Current assignee: F Hoffmann La Roche AG
Priority date: 2022-05-10
Filing date: 2023-05-10
Publication date: 2025-03-19
Also published as: JP2025516554A; CN119173632A; WO2023217890A9; WO2023217890A1

Abstract

The present invention relates to antisense oligonucleotides that are complementary to an ELK1-CFP pre-mRNA transcript and which lead to reduced levels of said transcript in cells. The present invention further relates to conjugates, salts and pharmaceutical compositions thereof; and methods for treatment of diseases associated with increased levels of ELK1-CFP pre-mRNA transcript, including Amyotrophic lateral sclerosis.

Description

ANTISENSE OLIGONUCLEOTIDES TARGETING CFP-ELK1 INTERGENE REGION

FIELD OF INVENTION

The present invention relates to antisense oligonucleotides that are complementary to a transcribed human CFP-ELK1 intergene region. These antisense oligonucleotides may lead to reduced levels of ELK1-CFP pre-mRNA transcript in cells. The present invention further relates to conjugates, salts and pharmaceutical compositions thereof; and methods for treatment of diseases associated with increased levels of ELK1-CFP pre-mRNA transcript, including Amyotrophic lateral sclerosis.

BACKGROUND

TAR DNA-binding protein 43 (TDP-43) is encoded by TARDBP, and has roles in transcriptional repression, pre-mRNA splicing and translational regulation. This also includes polyadenylation of RNA transcripts. Removing or decreasing the expression of TDP-43 can therefore lead to poly(A) tails being left off pre-mRNA transcripts.

TDP-43 depletion is indicated in a range of diseases, referred to as TDP-43 pathologies, and including for example diseases such as amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD), Progressive supranuclear palsy (PSP), Primary lateral sclerosis, Progressive muscular atrophy, Alzheimer’s disease, Parkinson’s disease, autism, Hippocampal sclerosis dementia, Down syndrome, Huntington’s disease, polyglutamine diseases, such as spinocerebellar ataxia 3, myopathies and Chronic Traumatic Encephalopathy.

ALS is also known as motor neurone disease or Lou Gehrig's disease. It is neurodegenerative and results in progressive loss of motor neurones in the brain and spine. Mutations in TARDBP are associated with ALS, as are mutations in the C9orf72, SOD1 and FUS genes (Edgar et al. 2021 , Neurobiol Aging, 108). Currently, there is no known cure for ALS, and it can affect those of any age. It can occur in families with a history of the disease, but may also arise sporadically in subjects with no familial cases. Typical treatment involves forms of assisted ventilation, which can extend a subject’s life but cannot cure the disease. Currently available medications, e.g., riluzole (US 5,527,81), are similarly only able to prolong life by short stretches rather than provide a cure. The present invention aims to devise new treatments for neurodegenerative disorders such as ALS.

The CFP gene codes for properdin, a plasma glycoprotein with a role in activation of the innate immune system’s complement system. Expression of CFP triggers a complement cascade, and it is typically only expressed in the liver. Properdin allows for stable C3 and C5-convertase attack complexes to form, through binding to human cell membranes. Formation of the attack complex can then lead to lysis of dying cells. Overexpression of CFP elsewhere in the body may be linked to neurodegenerative diseases. In particular, increasing complement activation in the central nervous system has been linked to neurodegenerative diseases, including Amyotrophic lateral sclerosis (ALS) (Kjaeldgaard et al. 2018, Mol. Immun. 102).

The present invention seeks to provide antisense oligonucleotides that ameliorate the effects of TDP-43 depletion by targeting CFP.

SUMMARY OF INVENTION

The present invention relates to antisense oligonucleotides that are complementary to a transcribed human CFP-ELK1 intergene region.

The ELK1 gene codes for ETS Like-1 protein, which functions as a transcription activator. It is adjacent to the complement factor properdin gene (CFP). The inventors have surprisingly determined that lack of TDP-43 causes the poly(A) tail of the ELK1 pre-mRNA transcript to be left off. This in turn results in transcription of ELK1 to continue on to the adjacent CFP gene, resulting in transcription of a single combined pre-mRNA ELK1-CFP transcript. This transcript includes transcribed mRNA from both genes as well as from the intergene region between them.

It would have been expected that the combined pre-mRNA ELK1-CFP transcript would undergo nonsense mediated (NMD) decay, due to the presence of exon-exon splice junctions more than 50 bases downstream of the stop codon. However, the inventors have surprisingly determined that this may not always occur, and may instead lead to increased expression of CFP.

A lack of, or reduced level of, TDP-43 may lead to increased CFP expression in the brain, as the properdin protein may be translated from the combined ELK1-CFP transcript. In this way a lack of, or reduced level of, TDP-43 has been linked to neurodegenerative diseases, including ALS. It is an aim of the present invention to provide antisense oligonucleotides which ameliorate the effects of TDP-43 depletion by targeting the ELK1-CFP intergene region.

The invention provides antisense oligonucleotides that are complementary to the ELK1-CFP intergene region. These antisense oligonucleotides may be capable of reducing the levels of ELK1-CFP pre-mRNA in a cell

The antisense oligonucleotides of the invention may reduce ELK1-CFP pre-mRNA levels by binding to ELK1-CFP pre-mRNA.

The antisense oligonucleotides of the invention are believed to bind to ELK1-CFP pre-mRNA post-transcription, leading to recruitment of RNaseHI , which results in cleavage of ELK1-CFP pre-mRNA. The cleaved ELK1-CFP pre-mRNA transcript is believed to be degraded. Hence there are reduced levels of ELK1-CFP pre-mRNA transcript in the cells.

The antisense oligonucleotides of the invention may therefore be used to remove or to reduce the level of ELK1-CFP pre-mRNA in cells.

In one aspect, the invention provides an antisense oligonucleotide 8 to 40 nucleotides in length that comprises a contiguous nucleotide sequence of at least 8 nucleotides in length and which is complementary to a transcribed human CFP-ELK1 intergene region.

In some embodiments, the antisense oligonucleotide complementary to a transcribed human CFP-ELK1 intergene region may comprise a contiguous nucleotide sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or fully complementary to the transcribed human ELK1-CFP intergene region.

In some embodiments, the transcribed human ELK1-CFP intergene region is within a human ELK1-CFP pre-mRNA transcript.

In some embodiments, the contiguous nucleotide sequence is 8 to 40 nucleotides in length.

In some embodiments, the antisense oligonucleotide is single stranded.

In some embodiments, the antisense oligonucleotide comprises one or more modified nucleosides. In some embodiments, the antisense oligonucleotide is capable of recruiting RNase H1.

In some embodiments, the antisense oligonucleotide is a gapmer.

In some embodiments, the antisense oligonucleotide may comprise at least one modified internucleoside linkage.

In some embodiments, one or more, or all, of the modified internucleoside linkages may comprise a phosphorothioate linkage.

In some embodiments, all the internucleoside linkages present within the antisense oligonucleotide may be phosphorothioate internucleoside linkages.

In some embodiments, the antisense oligonucleotide may be capable of reducing the level of the ELK1-CFP pre-mRNA transcript by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 100% in a cell, compared to a control.

In some embodiments, the control may be a cell that has not been exposed to the antisense oligonucleotide.

In some embodiments, the antisense oligonucleotide may be covalently attached to at least one conjugate moiety.

In some embodiments, the antisense oligonucleotide may be in the form of a pharmaceutically acceptable salt.

In some embodiments, the salt may be a sodium salt or a potassium salt.

In some embodiments, the antisense oligonucleotide may be encapsulated in a lipid-based delivery vehicle, covalently linked to or encapsulated in a dendrimer, or conjugated to an aptamer.

The invention provides for a pharmaceutical composition comprising the antisense oligonucleotide of the invention, and a pharmaceutically acceptable diluent, carrier, salt and/or adjuvant. In some embodiments, the pharmaceutical composition may comprise an aqueous diluent or solvent, such as phosphate buffered saline.

The invention provides for an in vivo or in vitro method for reducing the level of ELK1-CFP pre-mRNA transcript in a cell which is transcribing ELK1-CFP pre-mRNA, the method comprising exposing said cell to an effective amount of the antisense oligonucleotide of the invention, or the pharmaceutical composition of the invention.

In some embodiments, the cell may be either a human cell or a mammalian cell.

In some embodiments, the level of human ELK1-CFP pre-mRNA transcript may be decreased by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 100%, compared to a control.

In some embodiments, the control is a cell that has not been exposed to the antisense oligonucleotide.

The present invention also provides a method of treating or preventing a disease comprising administering a therapeutically or prophylactically effective amount of the antisense oligonucleotide of the invention, or the pharmaceutical composition of the invention, to a subject suffering from or susceptible to the disease.

The present invention also provides an antisense oligonucleotide of the invention, or the pharmaceutical composition of the invention, for use as a medicament for treatment or prevention of a disease in a subject.

The present invention also provides the use of the antisense oligonucleotide of the invention, or the pharmaceutical composition of the invention, for the preparation of a medicament for the treatment or prevention of a disease in a subject.

In some embodiments, the disease may be associated with increased levels of human ELK1- CFP pre-mRNA transcript.

In some embodiments, the disease may be Amyotrophic lateral sclerosis (ALS).

BRIEF DESCRIPTION OF THE FIGURES Figure 1 - Figure 1 displays Next-Gen Sequence read mapping from the ELK1 and CFP genes, with mapped reads from two samples of untreated cells (PBS control) and two samples from cells treated with compound A (SEQ ID 2) to deplete TDP-43 protein expression. The increased expression of mRNA reads that map just downstream of the ELK1 mRNA in cells treated with compound A (SEQ ID 2) is indicated by grey arrows. The black arrows indicate the increased expression (approx. 10-fold) of the downstream CFP gene.

Figure 2 - Figure 2 shows the expression level of the CFP gene relative to the positioning of the tested gapmer ASO (antisense oligonucleotide). The levels of CFP mRNA in fully untreated cells and in TDP-43 depleted cells before ASO use are superimposed as benchmarks.

Figure 3 - Figure 3 shows the expression level of the ELK1 gene relative to the positioning of the tested gapmer ASO. The levels of ELK1 mRNA in fully untreated cells and in TDP-43 depleted cells before ASO use are superimposed as benchmarks.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the determination that in TDP-43 depleted cells, transcription of the ELK1 gene continues on to the adjacent CFP gene, resulting in transcription of a single combined pre-mRNA ELK1-CFP transcript. This transcript includes transcribed mRNA from both genes as well as from the intergene region between them.

The inventors have identified that the level of ELF1-CFP pre-mRNA transcript can be reduced by targeting the ELF1-CFP pre-mRNA with antisense oligonucleotides. This can reduce CFP expression in the brain, which can be used to treat neurodegenerative disorders such as Amyotrophic lateral sclerosis (ALS).

Described herein are target sites present in the human ELF1-CFP pre-mRNA transcript, which can be targeted by antisense oligonucleotides. In some embodiments the antisense oligonucleotide may be capable of reducing the level of ELF1-CFP pre-mRNA transcript.

The inventors have surprisingly determined that targeting the intergene region of human ELF1- CFP pre-mRNA transcript can be particularly effective, for example in reducing CFP expression in the brain. Without wishing to be bound by theory, it is considered that the antisense oligonucleotides of the invention can reduce ELF1-CFP pre-mRNA transcript levels by binding to the ELF1-CFP pre-mRNA transcript. Binding of the antisense oligonucleotides of the invention to the intergene region of the ELF1-CFP pre-mRNA transcript is believed to lead to the recruitment of RNaseHI to the ELF1-CFP pre-mRNA transcript, resulting in cleavage of ELF1-CFP pre- mRNA transcript and subsequent degradation of the cleaved pre-mRNA. Hence there are reduced levels of ELF1-CFP pre-mRNA transcript and therefore reduced expression of CFP. The reduced expression of CFP may be in the brain, or elsewhere.

Reduced levels of ELF1-CFP pre-mRNA transcript and CFP expression are desirable to treat a range of disorders which are characterised by, or caused by, increased expression of CFP in the brain. These include Amyotrophic lateral sclerosis (ALS).

Antisense Oligonucleotide

The term “antisense oligonucleotide” as used herein is defined as an oligonucleotide capable of modulating levels of a target mRNA transcript by hybridising to a target nucleic acid, in particular to a contiguous sequence on a target nucleic acid. The term “oligonucleotide” as used herein is defined as it is generally understood by the skilled person as a molecule comprising two or more covalently linked nucleosides that are complementary to the nucleotides of an mRNA target. Such covalently bound nucleosides may also be referred to as nucleic acid molecules or oligomers.

Antisense oligonucleotides are not generally double stranded and are therefore not siRNAs or shRNAs.

Antisense oligonucleotides are commonly made in the laboratory by solid-phase chemical synthesis followed by purification and isolation. When referring to a sequence of the antisense oligonucleotide, reference is made to the sequence or order of nucleobase moieties, or modifications thereof, of the covalently linked nucleotides or nucleosides. The antisense oligonucleotides of the invention are man-made, and are chemically synthesised, and are typically purified or isolated. The antisense oligonucleotides of the invention may comprise one or more modified nucleosides such as 2’ sugar modified nucleosides. The antisense oligonucleotides of the invention may comprise one or more modified internucleoside linkages, such as one or more phosphorothioate internucleoside linkages. In some embodiments, the antisense oligonucleotides of the invention are single stranded antisense oligonucleotides. It is understood that single stranded antisense oligonucleotides of the present invention can form hairpins or intermolecular duplex structures (duplex between two molecules of the same antisense oligonucleotide), as long as the degree of intra or inter self-complementarity is less than approximately 50% across of the full length of the antisense oligonucleotide.

In some embodiments, the single stranded antisense oligonucleotides of the invention may not contain RNA nucleosides.

Advantageously, the antisense oligonucleotides of the invention comprise one or more modified nucleosides or nucleotides, such as 2’ sugar modified nucleosides. Furthermore, in some antisense oligonucleotides of the invention, it may be advantageous that the nucleosides which are not modified are DNA nucleosides.

In some embodiments, the antisense oligonucleotides of the invention are 8 to 40 nucleotides in length.

In some embodiments, the antisense oligonucleotides of the invention are 8 to 40 nucleotides in length and comprise a contiguous nucleotide sequence at least 40 nucleotides in length, such as 8 to 40 nucleotides in length.

In some embodiments, the antisense oligonucleotides of the invention are 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39 or 40 nucleotides in length.

In some embodiments the antisense oligonucleotides of the invention are at least 12 nucleotides in length.

In some embodiments the antisense oligonucleotides of the invention are at least 14 nucleotides in length.

In some embodiments the antisense oligonucleotides of the invention are at least 16 nucleotides in length.

In some embodiments the antisense oligonucleotides of the invention are at least 18 nucleotides in length. Preferably, the antisense oligonucleotides of the invention are 16 to 20 nucleotides in length.

More preferably, the antisense oligonucleotides of the invention are 18 to 20 nucleotides in length.

In some embodiments, the contiguous nucleotide sequence is 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39 or 40 nucleotides in length.

In some embodiments the antisense oligonucleotide comprises the contiguous nucleotide sequence.

In some embodiments the antisense oligonucleotide consists of the contiguous nucleotide sequence.

In some embodiments the antisense oligonucleotide is the contiguous nucleotide sequence.

Modified Antisense Oligonucleotide

The antisense oligonucleotide according to the invention may be a modified antisense oligonucleotide.

The term modified antisense oligonucleotide describes an antisense oligonucleotide comprising one or more sugar-modified nucleosides and/or modified internucleoside linkages. The term “chimeric antisense oligonucleotide” is a term that has been used in the literature to describe antisense oligonucleotides comprising sugar modified nucleosides and DNA nucleosides. In some embodiments, it may be advantageous for the antisense oligonucleotide according to the invention to be a chimeric antisense oligonucleotide.

In some embodiments, the antisense oligonucleotide according to the invention, or contiguous nucleotide sequence thereof, may include modified nucleobases, which function as the typical nucleobase in base pairing, for example 5-methyl cytosine may be used in place of methyl cytosine. Inosine may be used as a universal base.

It is understood that the contiguous nucleobase sequences (motif sequence) can be modified to, for example, increase nuclease resistance and/or binding affinity to the target nucleic acid. The pattern in which the modified nucleosides (such as high affinity modified nucleosides) are incorporated into the antisense oligonucleotide sequence is generally termed antisense oligonucleotide design.

In an embodiment, the antisense oligonucleotide according to the invention comprises at least 1 modified nucleoside, such as at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, or at least 19 modified nucleosides.

In some embodiments all of the nucleosides of the antisense oligonucleotide may be modified nucleosides.

Suitable modifications are described herein under the headings “modified nucleoside”, “high affinity modified nucleosides”, “sugar modifications”, “2’ sugar modifications” and Locked nucleic acids (LNA)”.

Gapmer

Gapmers are short DNA antisense oligonucleotides, with RNA-mimicking segments on either end of the central DNA region. A gapmer will bind to a pre-mRNA transcript containing a sequence complementary to that of the gapmer DNA, with the RNA-mimic segments ensuring a high binding affinity. High binding affinity ensures there are reduced off-target effects, while hybridisation between the gapmer and the pre-mRNA can prevent full transcription of the pre- mRNA by RNA polymerases.

Gapmers also induces cleavage of a pre-mRNA transcript through recruiting RNase H, which cleaves RNA-DNA hybrids. Gapmers can be engineered to have increased nuclease resistance and reduced immunogenicity and toxicity through modification, particularly by use of locked nucleic acids. Pre-mRNA that has been cleaved by RNase H is then degraded, preventing translation of the gapmer-targeted transcript. In this way, gapmers can be used as therapeutic agents to limit levels of pre-mRNA of genes where overexpression may cause or contribute to disease and negative patient outcomes. Gapmers can therefore be engineered and synthesised to target specific pre-mRNA transcripts, to treat or prevent diseases caused by overexpression of identified genes.

In some embodiments the antisense oligonucleotide of the invention is a gapmer. The term “gapmer” as used herein refers to an antisense oligonucleotide which comprises a region of RNase H recruiting antisense oligonucleotides (gap) which is flanked 5' and 3' by one or more affinity enhancing modified nucleosides (flanks). Various gapmer designs are described herein.

Headmers and tailmers are antisense oligonucleotides capable of recruiting RNase H where one of the flanks is missing, i.e. only one of the ends of the antisense oligonucleotide comprises affinity enhancing modified nucleosides. For headmers the 3' flank is missing (i.e. the 5' flank comprises affinity enhancing modified nucleosides) and for tailmers the 5' flank is missing (i.e. the 3' flank comprises affinity enhancing modified nucleosides).

Within the invention, the antisense oligonucleotide may be a headmer or a tailmer.

The term “LNA gapmer” is a gapmer antisense oligonucleotide wherein at least one of the affinity enhancing modified nucleosides is an LNA nucleoside. Within the invention the antisense oligonucleotide may be an LNA gapmer.

The term “mixed wing gapmer” refers to a LNA gapmer wherein the flank regions comprise at least one LNA nucleoside and at least one non-LNA modified nucleoside, such as at least one 2' substituted modified nucleoside, such as, for example, 2'- O-alkyl-RNA, 2'- O-methyl-RNA, 2'-alkoxy-RNA, 2'- O-methoxyethyl-RNA (MOE), 2'-amino-DNA, 2'-Fluoro-RNA, and 2'-F-ANA nucleoside(s). In some embodiments the mixed wing gapmer has one flank which comprises LNA nucleosides (e.g. 5' or 3') and the other flank (3' or 5' respectfully) comprises 2' substituted modified nucleoside(s). Within the invention the antisense oligonucleotide may be a mixed wing gapmer.

Gapmer Design

In a preferred embodiment the antisense oligonucleotide of the invention has a gapmer design or structure, also referred herein merely as "gapmer".

In a gapmer structure the antisense oligonucleotide comprises at least three distinct structural regions: a 5'-flank, a gap and a 3'-flank, F-G-F' in '5 -> 3' orientation. In this design, flanking regions F and F' (also termed wing regions) comprise a contiguous stretch of modified nucleosides, which are complementary to the intergene region of the ELF1-CFP pre-mRNA transcript target nucleic acid, while the gap region, G, comprises a contiguous stretch of nucleotides which are capable of recruiting a nuclease, preferably an endonuclease such as RNase, for example RNase H, when the antisense oligonucleotide is in duplex with the target nucleic acid. Nucleosides which are capable of recruiting a nuclease, in particular RNase H, can be selected from the group consisting of DNA, alpha-L-oxy-LNA, 2'-Flouro-ANA and UNA.

Regions F and F', flanking the 5' and 3' ends of region G, preferably comprise non-nuclease recruiting nucleosides (nucleosides with a 3' endo structure), more preferably one or more affinity enhancing modified nucleosides.

In some embodiments, the 3' flank comprises at least one LNA nucleoside, preferably at least 2 LNA nucleosides. In some embodiments, the 5' flank comprises at least one LNA nucleoside, preferably at least 2 LNA nucleosides. In some embodiments both the 5' and 3' flanking regions comprise a LNA nucleoside, preferably at least 2 LNA nucleosides. In some embodiments all the nucleosides in the flanking regions are LNA nucleosides.

In other embodiments, the flanking regions may comprise both LNA nucleosides and other nucleosides (mixed flanks), such as DNA nucleosides and/or non-LNA modified nucleosides, such as 2' substituted nucleosides. In this case, the gap is defined as a contiguous sequence of at least 5 RNase H recruiting nucleosides (nucleosides with a 2' endo structure, preferably DNA) flanked at the 5' and 3' end by an affinity enhancing modified nucleoside, preferably LNA, such as beta-D-oxy-LNA. Consequently, the nucleosides of the 5' flanking region and the 3' flanking region which are adjacent to the gap region are modified nucleosides, preferably non-nuclease recruiting nucleosides. In antisense oligonucleotides with mixed flanks where the flanks comprise DNA the 5' and 3' nucleosides are modified nucleosides.

Nucleotides and Nucleosides

Nucleotides and nucleosides are the building blocks of antisense oligonucleotides and polynucleotides and, for the purposes of the present invention, include both naturally occurring and non-naturally occurring nucleotides and nucleosides. In nature, nucleotides, such as DNA and RNA nucleotides comprise a ribose sugar moiety, a nucleobase moiety and one or more phosphate groups (which is absent in nucleosides). Nucleosides and nucleotides may also interchangeably be referred to as “units” or “monomers”.

Modified Nucleoside The term “modified nucleoside” or “nucleoside modification” as used herein refers to nucleosides modified as compared to the equivalent DNA or RNA nucleoside by the introduction of one or more modifications of the sugar moiety or the (nucleo)base moiety.

Advantageously, the antisense oligonucleotide according to the invention may comprise one or more modified nucleosides.

In some embodiments the contiguous nucleobase sequences (motif sequence) can be modified to, for example, increase nuclease resistance and/or binding affinity to the target nucleic acid. Advantageously, high affinity modified nucleosides are used.

Advantageously, one or more of the modified nucleosides of the antisense oligonucleotide according to the invention may comprise a modified sugar moiety. The term modified nucleoside may also be used herein interchangeably with the term “nucleoside analogue” or modified “units” or modified “monomers”. Nucleosides with an unmodified DNA or RNA sugar moiety are termed DNA or RNA nucleosides herein. Nucleosides with modifications in the base region of the DNA or RNA nucleoside are still generally termed DNA or RNA if they allow Watson Crick base pairing. Exemplary modified nucleosides which may be used in the antisense oligonucleotide according to the invention include LNA, 2’-O-MOE, 2’oMe and morpholino nucleoside analogues.

Locked Nucleic Acid Nucleosides (LNA Nucleoside)

A “LNA nucleoside” is a 2’- modified nucleoside which comprises a biradical linking the C2’ and C4’ of the ribose sugar ring of said nucleoside (also referred to as a “2’- 4’ bridge”), which restricts or locks the conformation of the ribose ring. These nucleosides are also termed bridged nucleic acid or bicyclic nucleic acid (BNA) in the literature. The locking of the conformation of the ribose is associated with an enhanced affinity of hybridisation (duplex stabilization) when the LNA is incorporated into an antisense oligonucleotide for a complementary RNA or DNA molecule. This can be routinely determined by measuring the melting temperature of the antisense oligonucleotide/complement duplex.

Non limiting, exemplary LNA nucleosides are disclosed in WO 99/014226, WO 00/66604, WO 98/039352, WO 2004/046160, WO 00/047599, WO 2007/134181 , WO 2010/077578, WO 2010/036698, WO 2007/090071 , WO 2009/006478, WO 2011/156202, WO 2008/154401 , WO 2009/067647, WO 2008/150729, Morita et al., Bioorganic & Med.Chem. Lett. 12, 73-76, Seth et al. J. Org. Chem. 2010, Vol 75(5) pp. 1569-81 , and Mitsuoka et al., Nucleic Acids Research 2009, 37(4), 1225-1238, and Wan and Seth, J. Medical Chemistry 2016, 59, 9645-9667, which are incorporated herein in their entirety.

Further non-limiting, exemplary LNA nucleosides are disclosed in Scheme 1.

Scheme 1 :

Particular LNA nucleosides are beta- D-oxy- LNA, 6’-methyl-beta-D-oxy LNA such as (S)-6’- methyl-beta-D-oxy-LNA (ScET) and ENA.

A particularly advantageous LNA is beta- D-oxy- LNA. Modified internucleoside linkage

Advantageously, the antisense oligonucleotide according to the invention comprises one or more modified internucleoside linkages.

The term “modified internucleoside linkage” is defined as generally understood by the skilled person as linkages, other than phosphodiester (PO) linkages, which covalently couple two nucleosides together. The antisense oligonucleotide of the invention may therefore comprise one or more modified internucleoside linkages such as one or more phosphorothioate internucleoside linkages.

In some embodiments at least 50% of the internucleoside linkages in the antisense oligonucleotide according to the invention, or the contiguous nucleotide sequence thereof, are phosphorothioate, such as at least 60%, such as at least 70%, such as at least 75%, such as at least 80%, such as at least 90% or more. In some embodiments all of the internucleoside linkages of the antisense oligonucleotide of the invention, or contiguous nucleotide sequence thereof, are phosphorothioate.

In a further embodiment, the antisense oligonucleotide according to the invention comprises at least one modified internucleoside linkage. It is advantageous if at least 75%, such as all, of the internucleoside linkages within the contiguous nucleotide sequence are phosphorothioate or boranophosphate internucleoside linkages.

Advantageously, all the internucleoside linkages of the contiguous nucleotide sequence of the antisense oligonucleotide according to the invention may be phosphorothioate, or all the internucleoside linkages of the antisense oligonucleotide according to the invention may be phosphorothioate linkages.

Nucleobase

The term nucleobase includes the purine (e.g. adenine and guanine) and pyrimidine (e.g. uracil, thymine and cytosine) moiety present in nucleosides and nucleotides which form hydrogen bonds in nucleic acid hybridisation. In the context of the present invention the term nucleobase also encompasses modified nucleobases which may differ from naturally occurring nucleobases, but which are functional during nucleic acid hybridisation. In this context “nucleobase” refers to both naturally occurring nucleobases such as adenine, guanine, cytosine, thymidine, uracil, xanthine and hypoxanthine, as well as non-naturally occurring variants. Such variants are for example described in Hirao et al (2012) Accounts of Chemical Research vol 45 page 2055 and Bergstrom (2009) Current Protocols in Nucleic Acid Chemistry Suppl. 37 1.4.1.

In some embodiments the nucleobase moiety is modified by changing the purine or pyrimidine into a modified purine or pyrimidine, such as substituted purine or substituted pyrimidine, such as a nucleobase selected from isocytosine, pseudoisocytosine, 5-methyl cytosine, 5-thiozolo- cytosine, 5-propynyl-cytosine, 5-propynyl-uracil, 5-bromouracil 5-thiazolo-uracil, 2-thio-uracil, 2’thio-thymine, inosine, diaminopurine, 6-aminopurine, 2-aminopurine, 2,6-diaminopurine and 2-chloro-6-aminopurine.

The nucleobase moieties may be indicated by the letter code for each corresponding nucleobase, e.g. A, T, G, C or II, wherein each letter may optionally include modified nucleobases of equivalent function. For example, in the exemplified antisense oligonucleotides, the nucleobase moieties are selected from A, T, G, C, and 5-methyl cytosine. Optionally, for LNA gapmers, 5-methyl cytosine LNA nucleosides may be used. 5-methyl cytosine may be denoted as “E”.

High Affinity Modified Nucleosides

A high affinity modified nucleoside is a modified nucleoside which, when incorporated into the antisense oligonucleotide enhances the affinity of the antisense oligonucleotide for its complementary target, for example as measured by the melting temperature (Tm). A high affinity modified nucleoside of the present invention preferably results in an increase in melting temperature between +0.5 to +12°C, more preferably between +1.5 to +10°C and most preferably between+3 to +8°C per modified nucleoside. Numerous high affinity modified nucleosides are known in the art and include for example, many 2’ substituted nucleosides as well as locked nucleic acids (LNA) (see e.g. Freier & Altmann; Nucl. Acid Res., 1997, 25, 4429-4443 and Uhlmann; Curr. Opinion in Drug Development, 2000, 3(2), 203-213).

Sugar Modifications

The antisense oligonucleotide according to the invention may comprise one or more nucleosides which have a modified sugar moiety, i.e. a modification of the sugar moiety when compared to the ribose sugar moiety found in DNA and RNA. Numerous nucleosides with modification of the ribose sugar moiety have been made, primarily with the aim of improving certain properties of antisense oligonucleotides, such as affinity and/or nuclease resistance.

Such modifications include those where the ribose ring structure is modified, e.g. by replacement with a hexose ring (HNA), or a bicyclic ring, which typically have a biradicle bridge between the C2 and C4 carbons on the ribose ring (LNA), or an unlinked ribose ring which typically lacks a bond between the C2 and C3 carbons (e.g. UNA). Other sugar modified nucleosides include, for example, bicyclohexose nucleic acids (WO2011/017521) or tricyclic nucleic acids (WO2013/154798). Modified nucleosides also include nucleosides where the sugar moiety is replaced with a non-sugar moiety, for example in the case of peptide nucleic acids (PNA), or morpholino nucleic acids.

Sugar modifications also include modifications made via altering the substituent groups on the ribose ring to groups other than hydrogen, or the 2’-OH group naturally found in DNA and RNA nucleosides. Substituents may, for example be introduced at the 2’, 3’, 4’ or 5’ positions.

2’ Sugar Modified Nucleosides

A 2’ sugar modified nucleoside is a nucleoside which has a substituent other than H or -OH at the 2’ position (2’ substituted nucleoside) or comprises a 2’ linked biradicle capable of forming a bridge between the 2’ carbon and a second carbon in the ribose ring, such as LNA (2’ - 4’ biradicle bridged) nucleosides.

Indeed, much focus has been spent on developing 2’ sugar substituted nucleosides, and numerous 2’ substituted nucleosides have been found to have beneficial properties when incorporated into antisense oligonucleotides. For example, the 2’ modified sugar may provide enhanced binding affinity and/or increased nuclease resistance to the antisense oligonucleotide. Examples of 2’ substituted modified nucleosides are 2’-O-alkyl-RNA, 2’-O- methyl-RNA (2’oMe), 2’-alkoxy-RNA, 2’-O-methoxyethyl-RNA (MOE), 2’-amino-DNA, 2’- Fluoro-RNA, and 2’-F-ANA nucleoside. For further examples, please see e.g. Freier & Altmann; Nucl. Acid Res., 1997, 25, 4429-4443 and Uhlmann; Curr. Opinion in Drug Development, 2000, 3(2), 203-213, and Deleavey and Damha, Chemistry and Biology 2012, 19, 937. Below are illustrations of some 2’ substituted modified nucleosides. _Q Base

5— z'

O F

J z'J- HN.A

In relation to the present invention 2' substituted sugar modified nucleosides does not include 2' bridged nucleosides like LNA.

In one embodiment, the antisense oligonucleotide according to the invention comprises one or more sugar modified nucleosides, such as 2' sugar modified nucleosides. Preferably the antisense oligonucleotide according to the invention comprises one or more 2' sugar modified nucleoside independently selected from the group consisting of 2'-O-alkyl-RNA, 2'-O-methyl- RNA (2'oMe), 2'-alkoxy-RNA, 2'-O-methoxyethyl-RNA (2'MOE), 2'-amino-DNA, 2'-fluoro-DNA, arabino nucleic acid (ANA), 2'-fluoro-ANA and LNA nucleosides. It is advantageous if one or more of the modified nucleoside(s) is a locked nucleic acid (LNA).

Morpholino Oligonucleotides

In some embodiments, the antisense oligonucleotide of the invention comprises or consists of morpholino nucleosides (i.e. is a Morpholino oligomer and as a phosphorodiamidate Morpholino oligomer (PMO)). Splice modulating morpholino antisense oligonucleotides have been approved for clinical use - see for example eteplirsen, a 30nt morpholino antisense oligonucleotide targeting a frame shift mutation in DMD, used to treat Duchenne muscular dystrophy. Morpholino antisense oligonucleotides have nucleases attached to six membered morpholino rings rather ribose, such as methylenemorpholine rings linked through phosphorodiamidate groups, for example as illustrated by the following illustration of 4 consecutive morpholino nucleotides:

In some embodiments, morpholino antisense oligonucleotides according to the invention may be, for example 8 to 40 morpholino nucleotides in length, such as morpholino 16 to 20 nucleotides in length, such as 18 to 20 nucleotides in length.

Linkers

A linkage or linker is a connection between two atoms that links one chemical group or segment of interest to another chemical group or segment of interest via one or more covalent bonds. Conjugate moieties can be attached to the antisense oligonucleotide directly or through a linking moiety (e.g. linker or tether). Linkers serve to covalently connect a third region, e.g. a conjugate moiety (Region C), to a first region, e.g. an antisense oligonucleotide or contiguous nucleotide sequence complementary to the target nucleic acid (region A).

In some embodiments of the invention the conjugate or antisense oligonucleotide of the invention may optionally comprise a linker region (second region or region B and/or region Y) which is positioned between the antisense oligonucleotide or contiguous nucleotide sequence complementary to the target nucleic acid (region A or first region) and the conjugate moiety (region C or third region). Region B refers to biocleavable linkers comprising or consisting of a physiologically labile bond that is cleavable under conditions normally encountered or analogous to those encountered within a mammalian body. Conditions under which physiologically labile linkers undergo chemical transformation (e.g., cleavage) include chemical conditions such as pH, temperature, oxidative or reductive conditions or agents, and salt concentration found in or analogous to those encountered in mammalian cells. Mammalian intracellular conditions also include the presence of enzymatic activity normally present in a mammalian cell such as from proteolytic enzymes or hydrolytic enzymes or nucleases. In one embodiment the biocleavable linker is susceptible to S1 nuclease cleavage. In some embodiments the nuclease susceptible linker comprises between 1 and 5 nucleosides, such as DNA nucleoside(s) comprising at least two consecutive phosphodiester linkages. Phosphodiester containing biocleavable linkers are described in more detail in WO 2014/076195.

Region Y refers to linkers that are not necessarily biocleavable but primarily serve to covalently connect a conjugate moiety (region C or third region), to an antisense oligonucleotide (region A or first region). The region Y linkers may comprise a chain structure or an oligomer of repeating units such as ethylene glycol, amino acid units or amino alkyl groups. The antisense oligonucleotide of the present invention can be constructed of the following regional elements A-C, A-B-C, A-B-Y-C, A-Y-B-C or A-Y-C. In some embodiments the linker (region Y) is an amino alkyl, such as a C2 - C36 amino alkyl group, including, for example C6 to C12 amino alkyl groups. In some embodiments the linker (region Y) is a C6 amino alkyl group.

The Target

The antisense oligonucleotide of the invention is an oligonucleotide which targets the ELK1- CFP pre-mRNA transcript.

The antisense oligonucleotides of the invention comprise a contiguous nucleotide sequence which is complementary to a transcribed human CFP-ELK1 intergene region.

In some embodiments the target sequence is the human ELK1-CFP pre-mRNA transcript. The human ELK1-CFP pre-mRNA transcript may be referred to as a target sequence.

In some embodiments the target sequence is human ELK1-CFP pre-mRNA transcript, which may be encoded by SEQ ID NO. 1 , or a fragment thereof. An aspect of the present invention relates to an antisense oligonucleotide which comprises a contiguous nucleotide sequence of 8 to 40 nucleotides in length which is complementarity to SEQ ID NO 1 , or a fragment thereof.

In one embodiment the fragment may be 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides in length.

In some embodiments, the antisense oligonucleotide of the invention comprises a contiguous sequence which is at least about 75% complementary, such as at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90% at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or about 100% complementary to SEQ ID NO 1 , or a fragment thereof.

In some embodiments, the antisense oligonucleotide of the invention comprises a contiguous sequence which may comprise one or two mismatches between the contiguous nucleotide sequence and the target nucleic acid (i.e. the human CFP-ELK1 intergene region).

In a preferred embodiment the antisense oligonucleotide of the invention, or contiguous nucleotide sequence thereof, is fully complementary (i.e. about 100% complementary) to SEQ ID NO 1.

An aspect of the present invention relates to an antisense oligonucleotide which comprises a contiguous nucleotide sequence of 8 to 40 nucleotides in length which is complementarity to a sequence selected from the group consisting of SEQ ID NO 95, SEQ ID NO 96, SEQ ID NO 97, SEQ ID NO 98, SEQ ID NO 99, SEQ ID NO 100, SEQ ID NO 101 , SEQ ID NO 102, SEQ ID NO 103, SEQ ID NO 104, SEQ ID NO 105, SEQ ID NO 106, SEQ ID NO 107, SEQ ID NO 108, SEQ ID NO 109, SEQ ID NO 110, SEQ ID NO 111 , SEQ ID NO 112, SEQ ID NO 113, SEQ ID NO 114, SEQ ID NO 115, SEQ ID NO 116, SEQ ID NO 117, SEQ ID NO 118, SEQ ID NO 119, SEQ ID NO 120, SEQ ID NO 121 , SEQ ID NO 122, SEQ ID NO 123, SEQ ID NO 124, SEQ ID NO 125, SEQ ID NO 126, SEQ ID NO 127, SEQ ID NO 128, SEQ ID NO 129, SEQ ID NO 130, SEQ ID NO 131 , SEQ ID NO 132, SEQ ID NO 133, SEQ ID NO 134, SEQ ID NO 135, SEQ ID NO 136, SEQ ID NO 137, SEQ ID NO 138, SEQ ID NO 139, SEQ ID NO 140, SEQ ID NO 141 , SEQ ID NO 142, SEQ ID NO 143, SEQ ID NO 144, SEQ ID NO 145, SEQ ID NO 146, SEQ ID NO 147, SEQ ID NO 148, SEQ ID NO 149, SEQ ID NO 150, SEQ ID NO 151 , SEQ ID NO 152, SEQ ID NO 153, SEQ ID NO 154, SEQ ID NO 155, SEQ ID NO 156, SEQ ID NO 157, SEQ ID NO 158, SEQ ID NO 159, SEQ ID NO 160, SEQ ID NO 161 , SEQ ID NO 162, SEQ ID NO 163, SEQ ID NO 164, SEQ ID NO 165, SEQ ID NO 166, SEQ ID NO 167, SEQ ID NO 168, SEQ ID NO 169, SEQ ID NO 170, SEQ ID NO 171 , SEQ ID NO 172, SEQ ID NO 173, SEQ ID NO 174, SEQ ID NO 175, SEQ ID NO 176, SEQ ID NO 177, SEQ ID NO 178, SEQ ID NO 179, SEQ ID NO 180, SEQ ID NO 181 , SEQ ID NO 182, SEQ ID NO 183, SEQ ID NO 184, SEQ ID NO 185, SEQ ID NO 186 and SEQ ID NO 187, or a fragment thereof.

In some embodiments, the antisense oligonucleotide of the invention comprises a contiguous sequence which is at least about 75% complementary, such as at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90% at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or about 100% complementary to a sequence selected from the group consisting of SEQ ID NO 95, SEQ ID NO 96, SEQ ID NO 97, SEQ ID NO 98, SEQ ID NO 99, SEQ ID NO 100, SEQ ID NO 101 , SEQ ID NO 102, SEQ ID NO 103, SEQ ID NO 104, SEQ ID NO 105, SEQ ID NO 106, SEQ ID NO 107, SEQ ID NO 108, SEQ ID NO 109, SEQ ID NO 110, SEQ ID NO 111 , SEQ ID NO 112, SEQ ID NO 113, SEQ ID NO 114, SEQ ID NO 115, SEQ ID NO 116, SEQ ID NO 117, SEQ ID NO 118, SEQ ID NO 119, SEQ ID NO 120, SEQ ID NO 121 , SEQ ID NO 122, SEQ ID NO 123, SEQ ID NO 124, SEQ ID NO 125, SEQ ID NO 126, SEQ ID NO 127, SEQ ID NO 128, SEQ ID NO 129, SEQ ID NO 130, SEQ ID NO 131 , SEQ ID NO 132, SEQ ID NO 133, SEQ ID NO 134, SEQ ID NO 135, SEQ ID NO 136, SEQ ID NO 137, SEQ ID NO 138, SEQ ID NO 139, SEQ ID NO 140, SEQ ID NO 141 , SEQ ID NO 142, SEQ ID NO 143, SEQ ID NO 144, SEQ ID NO 145, SEQ ID NO 146, SEQ ID NO 147, SEQ ID NO 148, SEQ ID NO 149, SEQ ID NO 150, SEQ ID NO 151 , SEQ ID NO 152, SEQ ID NO 153, SEQ ID NO 154, SEQ ID NO 155, SEQ ID NO 156, SEQ ID NO 157, SEQ ID NO 158, SEQ ID NO 159, SEQ ID NO 160, SEQ ID NO 161 , SEQ ID NO 162, SEQ ID NO 163, SEQ ID NO 164, SEQ ID NO 165, SEQ ID NO 166, SEQ ID NO 167, SEQ ID NO 168, SEQ ID NO 169, SEQ ID NO 170, SEQ ID NO 171 , SEQ ID NO 172, SEQ ID NO 173, SEQ ID NO 174, SEQ ID NO 175, SEQ ID NO 176, SEQ ID NO 177, SEQ ID NO 178, SEQ ID NO 179, SEQ ID NO 180, SEQ ID NO 181 , SEQ ID NO 182, SEQ ID NO 183, SEQ ID NO 184, SEQ ID NO 185, SEQ ID NO 186 and SEQ ID NO 187, or a fragment thereof.

In a preferred embodiment the antisense oligonucleotide of the invention, or contiguous nucleotide sequence thereof, is fully complementary (100% complementary) to a sequence selected from the group consisting of SEQ ID NO 95, SEQ ID NO 96, SEQ ID NO 97, SEQ ID NO 98, SEQ ID NO 99, SEQ ID NO 100, SEQ ID NO 101 , SEQ ID NO 102, SEQ ID NO 103, SEQ ID NO 104, SEQ ID NO 105, SEQ ID NO 106, SEQ ID NO 107, SEQ ID NO 108, SEQ ID NO 109, SEQ ID NO 110, SEQ ID NO 111 , SEQ ID NO 112, SEQ ID NO 113, SEQ ID NO 114, SEQ ID NO 115, SEQ ID NO 116, SEQ ID NO 117, SEQ ID NO 118, SEQ ID NO 119, SEQ ID NO 120, SEQ ID NO 121 , SEQ ID NO 122, SEQ ID NO 123, SEQ ID NO 124, SEQ ID NO 125, SEQ ID NO 126, SEQ ID NO 127, SEQ ID NO 128, SEQ ID NO 129, SEQ ID NO 130, SEQ ID NO 131 , SEQ ID NO 132, SEQ ID NO 133, SEQ ID NO 134, SEQ ID NO 135, SEQ ID NO 136, SEQ ID NO 137, SEQ ID NO 138, SEQ ID NO 139, SEQ ID NO 140, SEQ ID NO 141 , SEQ ID NO 142, SEQ ID NO 143, SEQ ID NO 144, SEQ ID NO 145, SEQ ID NO 146, SEQ ID NO 147, SEQ ID NO 148, SEQ ID NO 149, SEQ ID NO 150, SEQ ID NO 151 , SEQ ID NO 152, SEQ ID NO 153, SEQ ID NO 154, SEQ ID NO 155, SEQ ID NO 156, SEQ ID NO 157, SEQ ID NO 158, SEQ ID NO 159, SEQ ID NO 160, SEQ ID NO 161 , SEQ ID NO 162, SEQ ID NO 163, SEQ ID NO 164, SEQ ID NO 165, SEQ ID NO 166, SEQ ID NO 167, SEQ ID NO 168, SEQ ID NO 169, SEQ ID NO 170, SEQ ID NO 171 , SEQ ID NO 172, SEQ ID NO 173, SEQ ID NO 174, SEQ ID NO 175, SEQ ID NO 176, SEQ ID NO 177, SEQ ID NO 178, SEQ ID NO 179, SEQ ID NO 180, SEQ ID NO 181 , SEQ ID NO 182, SEQ ID NO 183, SEQ ID NO 184, SEQ ID NO 185, SEQ ID NO 186 and SEQ ID NO 187, or a fragment thereof.

In some embodiments the antisense oligonucleotide of the invention, or contiguous nucleotide sequence thereof, is able to reduce the expression of CFP to at least the level of untreated cells (100%).

In some embodiments, the antisense oligonucleotide of the invention comprises a contiguous sequence which is at least about 75% complementary, such as at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90% at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or about 100% complementary to a sequence selected from the group consisting of SEQ ID NO 98, SEQ ID NO 101 , SEQ ID NO 102, SEQ ID NO 105, SEQ ID NO 107, SEQ ID NO 109, SEQ ID NO 110, SEQ ID NO 113, SEQ ID NO 115, SEQ ID NO 116, SEQ ID NO 117, SEQ ID NO 118, SEQ ID NO 119, SEQ ID NO 120, SEQ ID NO 121 , SEQ ID NO 122, SEQ ID NO 123, SEQ ID NO 124, SEQ ID NO 125, SEQ ID NO 126, SEQ ID NO 127, SEQ ID NO 128, SEQ ID NO 129, SEQ ID NO

130, SEQ ID NO 131 , SEQ ID NO 132, SEQ ID NO 133, SEQ ID NO 134, SEQ ID NO 135, SEQ ID NO 138, SEQ ID NO 139, SEQ ID NO 140, SEQ ID NO 141 , SEQ ID NO 142, SEQ ID NO 143, SEQ ID NO 144, SEQ ID NO 145, SEQ ID NO 146, SEQ ID NO 147, SEQ ID NO

148, SEQ ID NO 149, SEQ ID NO 150, SEQ ID NO 151 , SEQ ID NO 152, SEQ ID NO 153, SEQ ID NO 154, SEQ ID NO 155, SEQ ID NO 156, SEQ ID NO 157, SEQ ID NO 158, SEQ ID NO 159, SEQ ID NO 160, SEQ ID NO 161 , SEQ ID NO 162, SEQ ID NO 163, SEQ ID NO

164, SEQ ID NO 165, SEQ ID NO 167, SEQ ID NO 168, SEQ ID NO 169, SEQ ID NO 170, SEQ ID NO 171 , SEQ ID NO 172, SEQ ID NO 173, SEQ ID NO 174, SEQ ID NO 178, SEQ ID NO 179, SEQ ID NO 182, SEQ ID NO 183, SEQ ID NO 185, SEQ ID NO 186 and SEQ ID NO 187, or a fragment thereof.

In a preferred embodiment the antisense oligonucleotide of the invention, or contiguous nucleotide sequence thereof is fully complementary (100% complementary) to a sequence selected from the group consisting of SEQ ID NO 98, SEQ ID NO 101 , SEQ ID NO 102, SEQ ID NO 105, SEQ ID NO 107, SEQ ID NO 109, SEQ ID NO 110, SEQ ID NO 113, SEQ ID NO 115, SEQ ID NO 116, SEQ ID NO 117, SEQ ID NO 118, SEQ ID NO 119, SEQ ID NO 120, SEQ ID NO 121 , SEQ ID NO 122, SEQ ID NO 123, SEQ ID NO 124, SEQ ID NO 125, SEQ ID NO 126, SEQ ID NO 127, SEQ ID NO 128, SEQ ID NO 129, SEQ ID NO 130, SEQ ID NO

131 , SEQ ID NO 132, SEQ ID NO 133, SEQ ID NO 134, SEQ ID NO 135, SEQ ID NO 138, SEQ ID NO 139, SEQ ID NO 140, SEQ ID NO 141 , SEQ ID NO 142, SEQ ID NO 143, SEQ ID NO 144, SEQ ID NO 145, SEQ ID NO 146, SEQ ID NO 147, SEQ ID NO 148, SEQ ID NO

149, SEQ ID NO 150, SEQ ID NO 151 , SEQ ID NO 152, SEQ ID NO 153, SEQ ID NO 154, SEQ ID NO 155, SEQ ID NO 156, SEQ ID NO 157, SEQ ID NO 158, SEQ ID NO 159, SEQ ID NO 160, SEQ ID NO 161 , SEQ ID NO 162, SEQ ID NO 163, SEQ ID NO 164, SEQ ID NO

165, SEQ ID NO 167, SEQ ID NO 168, SEQ ID NO 169, SEQ ID NO 170, SEQ ID NO 171 , SEQ ID NO 172, SEQ ID NO 173, SEQ ID NO 174, SEQ ID NO 178, SEQ ID NO 179, SEQ ID NO 182, SEQ ID NO 183, SEQ ID NO 185, SEQ ID NO 186 and SEQ ID NO 187, or a fragment thereof.

In some embodiments the antisense oligonucleotide of the invention, or contiguous nucleotide sequence thereof, is able to reduce the CFP expression to under 10% of the level in untreated cells. In some embodiments, the antisense oligonucleotide of the invention comprises a contiguous sequence which is at least about 75% complementary, such as at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90% at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or about 100% complementary to a sequence selected from the group consisting of SEQ ID NO 118, SEQ ID NO 119, SEQ ID NO 124, SEQ ID NO 126, SEQ ID NO 128, SEQ ID NO 132, SEQ ID NO 141 , SEQ ID NO 142, SEQ ID NO 145, SEQ ID NO 154, SEQ ID NO 157, SEQ ID NO 159, SEQ ID NO 160, SEQ ID NO 161 , SEQ ID NO 162, SEQ ID NO 163, SEQ ID NO 164, SEQ ID NO 165, SEQ ID NO 168, SEQ ID NO 169 and SEQ ID NO 172, or a fragment thereof.

In a preferred embodiment the antisense oligonucleotide of the invention, or contiguous nucleotide sequence thereof is fully complementary (100% complementary) to a sequence selected from the group consisting of SEQ ID NO 118, SEQ ID NO 119, SEQ ID NO 124, SEQ ID NO 126, SEQ ID NO 128, SEQ ID NO 132, SEQ ID NO 141 , SEQ ID NO 142, SEQ ID NO 145, SEQ ID NO 154, SEQ ID NO 157, SEQ ID NO 159, SEQ ID NO 160, SEQ ID NO 161 , SEQ ID NO 162, SEQ ID NO 163, SEQ ID NO 164, SEQ ID NO 165, SEQ ID NO 168, SEQ ID NO 169 and SEQ ID NO 172, or a fragment thereof.

Antisense Oligonucleotide design

The antisense oligonucleotide of the invention is an oligonucleotide which comprises a contiguous nucleotide sequence of at least 8 nucleotides in length which is complementary to a transcribed human CFP-ELK1 intergene region. The antisense oligonucleotide may be capable of reducing the level of ELF1-CFP pre-mRNA transcript in a cell.

In some embodiments the antisense oligonucleotide comprises the contiguous nucleotide sequence, and may optionally comprise further nucleotide(s), for example a nucleotide linker region which may be used to attach a functional group (e.g. a conjugate group) to the contiguous nucleotide sequence. The nucleotide linker region may or may not be complementary to the target nucleic acid. It is understood that the contiguous nucleotide sequence of the antisense oligonucleotide cannot be longer than the antisense oligonucleotide as such and that the antisense oligonucleotide cannot be shorter than the contiguous nucleotide sequence. Contiguous nucleotide sequence

The term “contiguous nucleotide sequence” refers to the region of the antisense oligonucleotide which is complementary to a target nucleic acid, which may be or may comprise an antisense oligonucleotide motif sequence. The term is used interchangeably herein with the term “contiguous nucleobase sequence”.

The antisense oligonucleotide comprises the contiguous nucleotide sequence, and may optionally comprise further nucleotide(s), for example a nucleotide linker region which may be used to attach a functional group (e.g. a conjugate group) to the contiguous nucleotide sequence. The nucleotide linker region may or may not be complementary to the target nucleic acid.

It is understood that the contiguous nucleotide sequence of the antisense oligonucleotide cannot be longer than the antisense oligonucleotide as such and that the antisense oligonucleotide cannot be shorter than the contiguous nucleotide sequence.

In some embodiments, the entire nucleotide sequence of the antisense oligonucleotide of the invention is the contiguous nucleotide sequence.

The contiguous nucleotide sequence is the sequence of nucleotides in the antisense oligonucleotide of the invention which are complementary to, and in some instances fully complementary to, the target nucleic acid, target sequence, or target site sequence.

In some embodiments, the contiguous nucleotide sequence is 8 to 40 nucleotides in length. In some embodiments, the contiguous nucleotide sequence is 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39 or 40 nucleotides in length.

In some embodiments the contiguous nucleotide sequence is at least 12 nucleotides in length.

In some embodiments the contiguous nucleotide sequence is at least 14 nucleotides in length.

In some embodiments the contiguous nucleotide sequence is at least 16 nucleotides in length.

In some embodiments the contiguous nucleotide sequence is at least 18 nucleotides in length. In a preferred embodiment the contiguous nucleotide sequence is 16 to 20 nucleotides in length.

More preferably, the contiguous nucleotide sequence is 18 to 20 nucleotides in length.

In some embodiments the antisense oligonucleotide of the invention consists of the contiguous nucleotide sequence.

In some embodiments the antisense oligonucleotide of the invention is the contiguous nucleotide sequence.

In some embodiments the contiguous nucleotide sequence comprises a sequence selected from the group consisting of SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11 , SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 16, SEQ ID NO 17, SEQ ID NO 18, SEQ ID NO 19, SEQ ID NO 20, SEQ ID NO 21 , SEQ ID NO 22, SEQ ID NO 23, SEQ ID NO 24, SEQ ID NO 25, SEQ ID NO 26, SEQ ID NO 27, SEQ ID NO 28, SEQ ID NO 29, SEQ ID NO 30, SEQ ID NO 31 , SEQ ID NO 32, SEQ ID NO 33, SEQ ID NO 34, SEQ ID NO 35, SEQ ID NO 36, SEQ ID NO 37, SEQ ID NO 38, SEQ ID NO 39, SEQ ID NO 40, SEQ ID NO 41 , SEQ ID NO 42, SEQ ID NO 43, SEQ ID NO 44, SEQ ID NO 45, SEQ ID NO 46, SEQ ID NO 47, SEQ ID NO 48, SEQ ID NO 49, SEQ ID NO 50, SEQ ID NO 51 , SEQ ID NO 52, SEQ ID NO 53, SEQ ID NO 54, SEQ ID NO 55, SEQ ID NO 56, SEQ ID NO 57, SEQ ID NO 58, SEQ ID NO 59, SEQ ID NO 60, SEQ ID NO 61 , SEQ ID NO 62, SEQ ID NO 63, SEQ ID NO 64, SEQ ID NO 65, SEQ ID NO 66, SEQ ID NO 67, SEQ ID NO 68, SEQ ID NO 69, SEQ ID NO 70, SEQ ID NO 71 , SEQ ID NO 72, SEQ ID NO 73, SEQ ID NO 74, SEQ ID NO 75, SEQ ID NO 76, SEQ ID NO 77, SEQ ID NO 78, SEQ ID NO 79, SEQ ID NO 80, SEQ ID NO 81 , SEQ ID NO 82, SEQ ID NO 83, SEQ ID NO 84, SEQ ID NO 85, SEQ ID NO 86, SEQ ID NO 87, SEQ ID NO 88, SEQ ID NO 89, SEQ ID NO 90, SEQ ID NO 91 , SEQ ID NO 92, SEQ ID NO 93 and SEQ ID NO 94, or a fragment thereof.

In some embodiments the antisense oligonucleotide of the invention, or contiguous nucleotide sequence thereof, is able to reduce the expression of CFP to at least the level in untreated cells (100%).

In a preferred embodiment the contiguous nucleotide sequence comprises a sequence selected from the group consisting of SEQ ID NO 5, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 12, SEQ ID NO 14, SEQ ID NO 16, SEQ ID NO 17, SEQ ID NO 20, SEQ ID NO 22, SEQ ID NO 23, SEQ ID NO 24, SEQ ID NO 25, SEQ ID NO 26, SEQ ID NO 27, SEQ ID NO 28, SEQ ID NO 29, SEQ ID NO 30, SEQ ID NO 31 , SEQ ID NO 32, SEQ ID NO 33, SEQ ID NO 34, SEQ ID NO 35, SEQ ID NO 36, SEQ ID NO 37, SEQ ID NO 38, SEQ ID NO 39, SEQ ID NO 40, SEQ ID NO 41 , SEQ ID NO 42, SEQ ID NO 45, SEQ ID NO 46, SEQ ID NO 47, SEQ ID NO 48, SEQ ID NO 49, SEQ ID NO 50, SEQ ID NO 51 , SEQ ID NO 52, SEQ ID NO 53, SEQ ID NO 54, SEQ ID NO 55, SEQ ID NO 56, SEQ ID NO 57, SEQ ID NO 58, SEQ ID NO 59, SEQ ID NO 60, SEQ ID NO 61 , SEQ ID NO 62, SEQ ID NO 63, SEQ ID NO 64, SEQ ID NO 65, SEQ ID NO 66, SEQ ID NO 67, SEQ ID NO 68, SEQ ID NO 69, SEQ ID NO 70, SEQ ID NO 71 , SEQ ID NO 72, SEQ ID NO 74, SEQ ID NO 75, SEQ ID NO 76, SEQ ID NO 77, SEQ ID NO 78, SEQ ID NO 79, SEQ ID NO 80, SEQ ID NO 81 , SEQ ID NO 84, SEQ ID NO 85, SEQ ID NO 89, SEQ ID NO 90, SEQ ID NO 92, SEQ ID NO 93 and SEQ ID NO 94, or a fragment thereof.

In some embodiments the antisense oligonucleotide of the invention, or contiguous nucleotide sequence thereof, is able to reduce the CFP expression to under 10% of the level in untreated cells.

In a preferred embodiment the contiguous nucleotide sequence comprises a sequence selected from the group consisting of SEQ ID NO 25, SEQ ID NO 26, SEQ ID NO 31 , SEQ ID NO 33, SEQ ID NO 35, SEQ ID NO 39, SEQ ID NO 48, SEQ ID NO 49, SEQ ID NO 52, SEQ ID NO 61 , SEQ ID NO 64, SEQ ID NO 66, SEQ ID NO 67, SEQ ID NO 68, SEQ ID NO 69, SEQ ID NO 70, SEQ ID NO 71 , SEQ ID NO 72, SEQ ID NO 75, SEQ ID NO 76 and SEQ ID NO 79, or a fragment thereof.

In some embodiments the fragment may be at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18 or at least 19 contiguous nucleotides of the contiguous nucleotide sequence preferably at least 10 contiguous nucleotides thereof.

Capable of reducing the levels of ELK1-CFP pre-mRNA transcript

The antisense oligonucleotides of the invention may be capable of reducing the levels of ELK1-CFP pre-mRNA transcript.

The term “reducing the levels” as used herein is to be understood as an overall term to describe an antisense oligonucleotide's ability to reduce the level of ELK1-CFP pre-mRNA transcript in a cell when compared to a control where the cell is not exposed to the antisense oligonucleotide of the invention.

Without wishing to be bound by theory the reduction effected by the antisense oligonucleotide is thought to be related to its ability to reduce, remove, prevent, lessen, lower or terminate the ELK1-CFP pre-mRNA transcript, e.g. by degradation or removal of the ELK1-CFP pre-mRNA transcript or by blockage or prevention of polymerase activity associated with the ELK1-CFP pre-mRNA transcript.

In certain embodiments the antisense oligonucleotides of the present invention may reduce the level of ELK1-CFP pre-mRNA transcript by at least about 10% compared to a control. More preferably the antisense oligonucleotides of the present invention may reduce the level of ELK1-CFP pre-mRNA transcript by at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or about 100% compared to a control.

In some embodiments the antisense oligonucleotide of the invention, or contiguous nucleotide sequence thereof, is able to reduce the CFP expression to under 90%, under 80%, under 70%, under 60%, under 50%, under 40%, under 30%, under 20%, or under 10% of the level of untreated cells.

In some embodiments the antisense oligonucleotide of the invention, or contiguous nucleotide sequence thereof, is able to reduce the CFP expression to under 10% of the level of untreated cells.

Preferably, the antisense oligonucleotides of the invention reduce the levels of ELK1-CFP pre- mRNA transcript in a cell by degradation or removal of the ELF1-CFP pre-mRNA transcript.

Control By the term "control", when used in relation to measurements of the effect of an antisense oligonucleotide, it is generally understood that the control is a cell that has not been exposed to the antisense oligonucleotide.

Alternatively the reduction in ELF1-CFP pre-mRNA transcript levels may be determined by reference to the levels of ELF1-CFP pre-mRNA transcript present in a cell before exposure to the antisense oligonucleotide.

In other embodiments, the control may be a cell treated with a non-targeting oligonucleotide. In some embodiments, the control may be a mock transfection, for example wherein cells are treated with PBS.

Conjugate

The invention encompasses an antisense oligonucleotide of the invention covalently attached to at least one conjugate moiety. In some embodiments this may be referred to as a conjugate of the invention.

The term “conjugate” as used herein refers to an antisense oligonucleotide of the invention which is covalently linked to a non-nucleotide moiety (conjugate moiety or region C or third region). The conjugate moiety may be covalently linked to the antisense oligonucleotide of the invention optionally via a linker group, such as region D’ or D".

Antisense oligonucleotide conjugates and their synthesis has also been reported in comprehensive reviews by Manoharan in Antisense Drug Technology, Principles, Strategies, and Applications, S.T. Crooke, ed., Ch. 16, Marcel Dekker, Inc., 2001 and Manoharan, Antisense and Nucleic Acid Drug Development, 2002, 12, 103, incorporated herein by reference in their entirety.

In some embodiments, the non-nucleotide moiety (conjugate moiety) is selected from the group consisting of carbohydrates (e.g. GalNAc), cell surface receptor ligands, drug substances, hormones, lipophilic substances, polymers, proteins, peptides, toxins (e.g. bacterial toxins), vitamins, viral proteins (e.g. capsids) or combinations thereof.

RNase H Activity and Recruitment The RNase H activity of an antisense oligonucleotide refers to its ability to recruit RNase H when in a duplex with a complementary RNA molecule. WO01/23613 (incorporated herein in its entirety) provides in vitro methods for determining RNase H activity, which may be used to determine the ability to recruit RNase H.

Typically an antisense oligonucleotide is deemed capable of recruiting RNase H if it, when provided with a complementary target nucleic acid sequence, has an initial rate, as measured in pmol/l/min, of at least 5%, such as at least 10%, at least 20% or more than 20%, of the initial rate determined when using an antisense oligonucleotide having the same base sequence as the modified antisense oligonucleotide being tested, but containing only DNA monomers with phosphorothioate linkages between all monomers in the antisense oligonucleotide, and using the methodology provided by Examples 91 - 95 of WO 01/23613 (hereby incorporated by reference). For use in determining RNase H activity, recombinant RNase H1 is available from Lubio Science GmbH, Lucerne, Switzerland.

DNA antisense oligonucleotides are known to effectively recruit RNase H, as are gapmer antisense oligonucleotides which comprise a region of DNA nucleosides (typically at least 5 or 6 contiguous DNA nucleosides), flanked 5’ and 3’ by regions comprising 2’ sugar modified nucleosides, typically high affinity 2’ sugar modified nucleosides, such as 2-O-MOE and/or LNA.

Nuclease Mediated Degradation

In some embodiments, the antisense oligonucleotide may function via nuclease mediated degradation of the target nucleic acid, where the antisense oligonucleotides of the invention are capable of recruiting a nuclease, particularly an endonuclease, preferably endoribonuclease (RNase), such as RNase H. Examples of antisense oligonucleotide designs which operate via nuclease mediated mechanisms are antisense oligonucleotides which typically comprise a region of at least 5 or 6 DNA nucleosides and are flanked on one side or both sides by affinity enhancing nucleosides, for example gapmers, headmers and tailmers.

Complementarity

The term “complementarity” describes the capacity for Watson-Crick base-pairing of nucleosides/nucleotides. Watson-Crick base pairs are guanine (G)-cytosine (C) and adenine (A) - thymine (T)/uracil (II). It will be understood that antisense oligonucleotides may comprise nucleosides with modified nucleobases, for example 5-methyl cytosine is often used in place of cytosine, and as such the term complementarity encompasses Watson Crick base-paring between non-modified and modified nucleobases (see for example Hirao et al (2012) Accounts of Chemical Research vol 45 page 2055 and Bergstrom (2009) Current Protocols in Nucleic Acid Chemistry Suppl. 37 1.4.1).

The term “% complementary” as used herein, refers to the proportion of nucleotides (in percent) of a contiguous nucleotide sequence in a nucleic acid molecule (e.g. antisense oligonucleotide) which across the contiguous nucleotide sequence, are complementary to a reference sequence (e.g. a target sequence or sequence motif). The percentage of complementarity is thus calculated by counting the number of aligned nucleobases that are complementary (from Watson Crick base pairs) between the two sequences (when aligned with the target sequence 5’-3’ and the antisense oligonucleotide sequence from 3’-5’), dividing that number by the total number of nucleotides in the antisense oligonucleotide and multiplying by 100. In such a comparison a nucleobase/nucleotide which does not align (form a base pair) is termed a mismatch. Insertions and deletions are not allowed in the calculation of % complementarity of a contiguous nucleotide sequence. It will be understood that in determining complementarity, chemical modifications of the nucleobases are disregarded as long as the functional capacity of the nucleobase to form Watson Crick base pairing is retained (e.g. 5’- methyl cytosine is considered identical to a cytosine for the purpose of calculating % identity).

Within the present invention the term “complementary” requires the contiguous nucleotide sequence to be at least about 75% complementary, or at least about 80% complementarity, or at least about 85% complementarity, or at least about 90% complementary, or at least about 95% complementarity to a human ELK1-CFP pre-mRNA transcript. In some embodiments the contiguous nucleotide sequence may be at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% complementary to a human ELK1-CFP pre-mRNA transcript.

Put another way, in some embodiments, the contiguous nucleotide sequence of the antisense oligonucleotide according to the invention may include one, two, three, four, five or more mis- matches, wherein a mis-match is a nucleotide within the contiguous nucleotide sequence which does not base pair with its target.

The term “fully complementary”, refers to 100% complementarity.

In some embodiments the contiguous nucleotide sequence is fully complementary to a human ELK1-CFP pre-mRNA transcript. In some embodiments the human ELK1-CFP pre-mRNA transcript may have the sequence of SEQ ID NO 1 , or a fragment thereof.

It will be understood that the target ELF1-CFP pre-mRNA transcript nucleic acid may be an allelic variant of SEQ ID NO 1 , such as an allelic variant which comprises one or more polymorphism in the human ELF1-CFP pre-mRNA transcript nucleic acid sequence.

Identity

The term “identity” as used herein, refers to the proportion of nucleotides (expressed in percent) of a contiguous nucleotide sequence in a nucleic acid molecule (e.g. antisense oligonucleotide) which across the contiguous nucleotide sequence, are identical to a reference sequence (e.g. a sequence motif).

The percentage of identity is thus calculated by counting the number of aligned nucleobases that are identical (a Match) between two sequences (in the contiguous nucleotide sequence of the compound of the invention and in the reference sequence), dividing that number by the total number of nucleotides in the antisense oligonucleotide and multiplying by 100. Therefore, Percentage of Identity = (Matches x 100)/Length of aligned region (e.g. the contiguous nucleotide sequence). Insertions and deletions are not allowed in the calculation the percentage of identity of a contiguous nucleotide sequence. It will be understood that in determining identity, chemical modifications of the nucleobases are disregarded as long as the functional capacity of the nucleobase to form Watson Crick base pairing is retained (e.g. 5-methyl cytosine is considered identical to a cytosine for the purpose of calculating % identity).

It is therefore to be understood that there is a relationship between identity and complementarity such that a contiguous nucleotide sequence within an antisense oligonucleotide of the invention that is complementary to a target sequence also shares a percentage of identity with said complementary sequence. Hybridisation

The terms “hybridising” or “hybridises” as used herein are to be understood as two nucleic acid strands (e.g. an antisense oligonucleotide and a target nucleic acid) forming hydrogen bonds between base pairs on opposite strands thereby forming a duplex. The affinity of the binding between two nucleic acid strands is the strength of the hybridisation. It is often described in terms of the melting temperature (Tm) defined as the temperature at which half of the antisense oligonucleotides are duplexed with the target nucleic acid. At physiological conditions Tm is not strictly proportional to the affinity (Mergny and Lacroix, 2003, Oligonucleotides 13:515-537). The standard state Gibbs free energy AG° is a more accurate representation of binding affinity and is related to the dissociation constant (Kd) of the reaction by AG°=-RTIn(Kd), where R is the gas constant and T is the absolute temperature. Therefore, a very low AG° of the reaction between an antisense oligonucleotide and the target nucleic acid reflects a strong hybridisation between the antisense oligonucleotide and target nucleic acid. AG° is the energy associated with a reaction where aqueous concentrations are 1 M, the pH is 7, and the temperature is 37°C. The hybridisation of antisense oligonucleotides to a target nucleic acid is a spontaneous reaction and for spontaneous reactions AG° is less than zero. AG° can be measured experimentally, for example, by use of the isothermal titration calorimetry (ITC) method as described in Hansen et al., 1965, Chem. Comm. 36-38 and Holdgate et al., 2005, Drug Discov Today. The skilled person will know that commercial equipment is available for AG° measurements. AG° can also be estimated numerically by using the nearest neighbor model as described by SantaLucia, 1998, Proc Natl Acad Sci USA. 95: 1460-1465 using appropriately derived thermodynamic parameters described by Sugimoto et al., 1995, Biochemistry 34:11211-11216 and McTigue et al., 2004, Biochemistry 43:5388-5405.

In some embodiments, antisense oligonucleotide of the present invention hybridises to a target nucleic acid with estimated AG° values below -10 kcal for antisense oligonucleotides that are 10-30 nucleotides in length.

In some embodiments the degree or strength of hybridisation is measured by the standard state Gibbs free energy AG°. The antisense oligonucleotides of the invention may hybridise to a target nucleic acid with estimated AG° values below the range of -10 kcal, such as below -15 kcal, such as below -20 kcal and such as below -25 kcal. In certain embodiments the antisense oligonucleotide of the invention hybridises to a sub-sequence of the target nucleic acid of SEQ ID NO: 1 with a AG° below -10 kcal, such as with a AG° between -10 to -60 kcal, such as -12 to -40, such as from -15 to -30 kcal or-16 to -27 kcal such as -18 to -25 kcal. In some embodiments the degree or strength of hybridisation is measured by the standard state Gibbs free energy AG°. The antisense oligonucleotides of the invention may hybridise to a target nucleic acid with estimated AG° values below the range of -10 kcal, such as below -15 kcal, such as below -20 kcal and such as below -25 kcal. In certain embodiments the antisense oligonucleotide of the invention hybridises to a sub-sequence of the target nucleic acid of SEQ ID NO: 1with a AG° below -10 kcal, such as with a AG° between -10 to -60 kcal, such as -12 to -40, such as from -15 to -30 kcal or-16 to -27 kcal such as -18 to -25 kcal.

Delivery of antisense oligonucleotide

The invention provides for antisense oligonucleotides according to the invention wherein the antisense oligonucleotide is encapsulated in a lipid-based delivery vehicle, covalently linked to or encapsulated in a dendrimer, or conjugated to an aptamer.

This may be for the purpose of delivering the antisense oligonucleotide of the invention to the targeted cells and/or to improve the pharmacokinetics of the antisense oligonucleotide of the invention.

Examples of lipid-based delivery vehicles include oil-in-water emulsions, micelles, liposomes, and lipid nanoparticles.

Salts

The term “salts” as used herein conforms to its generally known meaning, i.e. an ionic assembly of anions and cations.

The invention provides for pharmaceutically acceptable salts of the antisense oligonucleotide according to the invention, or the conjugate according to the invention.

The invention provides for antisense oligonucleotides according to the invention wherein the antisense oligonucleotides are in the form of a pharmaceutically acceptable salt. In some embodiments the pharmaceutically acceptable salt may be a sodium salt or a potassium salt.

The invention provides for a pharmaceutically acceptable sodium salt of the antisense oligonucleotide according to the invention. The invention provides for a pharmaceutically acceptable potassium salt of the antisense oligonucleotide according to the invention.

Pharmaceutical Composition

In a further aspect, the invention provides pharmaceutical compositions comprising an antisense oligonucleotide of the invention and a pharmaceutically acceptable diluent, carrier, salt and/or adjuvant. A pharmaceutically acceptable diluent includes phosphate-buffered saline (PBS) and pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts.

The invention provides for a pharmaceutical composition according to the invention, wherein the pharmaceutical composition comprises the antisense oligonucleotide of the invention, and an aqueous diluent or solvent.

The invention provides for a solution, such as a phosphate buffered saline solution of the antisense oligonucleotide of the invention. Suitably the solution, such as phosphate buffered saline solution, of the invention is a sterile solution.

WO 2007/031091 provides suitable and preferred examples of pharmaceutically acceptable diluents, carriers and adjuvants (hereby incorporated by reference). Suitable dosages, formulations, administration routes, compositions, dosage forms, combinations with other therapeutic agents, pro-drug formulations are also provided in W02007/031091.

Oligonucleotides of the invention may be mixed with pharmaceutically acceptable active or inert substances for the preparation of pharmaceutical compositions or formulations. Compositions and methods for the formulation of pharmaceutical compositions are dependent upon a number of criteria, including, but not limited to, route of administration, extent of disease, or dose to be administered.

In some embodiments, the antisense oligonucleotide or antisense oligonucleotide conjugate of the invention is a prodrug. In particular, with respect to antisense oligonucleotide conjugates, the conjugate moiety of the antisense oligonucleotide is cleaved once the prodrug is delivered to the site of action, e.g. the target cell.

Target Cell The term “target cell” as used herein refers to a cell which is expressing the target nucleic acid. In some embodiments the target cell may be in vivo or in vitro. In some embodiments the target cell is a mammalian cell such as a rodent cell, such as a mouse cell or a rat cell, or a primate cell such as a monkey cell or a human cell.

Applications

The antisense oligonucleotides of the invention may be utilised as, for example, therapeutics and prophylactics, and research reagents.

Research Reagents

The antisense oligonucleotides of the invention may be used as research reagents. In research, such antisense oligonucleotides may be used to specifically reduce the levels of ELF1-CFP pre-mRNA transcript in cells (e.g. in vitro cell cultures) and experimental animals, thereby facilitating functional analysis of the target or an appraisal of its usefulness as a target for therapeutic intervention.

Method for modulating ELF1-CFP pre-mRNA transcript levels

The invention provides for a method for reducing, downregulating the levels of, or removing ELF1-CFP pre-mRNA transcript in a cell, such as a cell which is transcribing ELF1-CFP pre- mRNA transcript or a cell which is TDP-43 deficient or TDP-43 depleted, said method comprising exposing an antisense oligonucleotide of the invention, or the pharmaceutical composition of the invention in an effective amount to said cell.

In some embodiments the method is an in vitro method.

In some embodiments the method is an in vivo method.

In some embodiments, the cell is either a human cell or a mammalian cell.

In some embodiments, the cell is part of, or derived from, a subject suffering from or susceptible to a disease associated with increased levels of ELF1-CFP pre-mRNA transcript. Such diseases include but are not limited Amyotrophic lateral sclerosis (ALS).

Treatment The term ’treatment’ as used herein refers to both treatment of an existing disease (e.g. a disease or disorder as herein referred to), or prevention of a disease, i.e. prophylaxis. It will therefore be recognised that treatment as referred to herein may, in some embodiments, be prophylactic.

The invention provides methods for treating or preventing a disease, comprising administering a therapeutically or prophylactically effective amount of an antisense oligonucleotide or a pharmaceutical composition of the invention to a subject suffering from or susceptible to the disease.

The invention provides for a method for treating or preventing a disease associated with increased levels of ELF1-CFP pre-mRNA transcript, comprising administering a therapeutically or prophylactically effective amount of an antisense oligonucleotide of the invention or a pharmaceutical composition of the invention to a subject suffering from or susceptible to a disease associated with increased levels of ELF1-CFP pre-mRNA transcript.

In some embodiments the disease may be a disease associated with reduced levels of TDP- 43.

In one embodiment the disease is Amyotrophic lateral sclerosis (ALS).

In some embodiments, the subject is an animal, preferably a mammal such as a mouse, rat, hamster, or monkey, or preferably a human.

The invention provides for an antisense oligonucleotide of the invention or a pharmaceutical composition of the invention, for use as a medicament.

Within the invention an antisense oligonucleotide of the invention or a pharmaceutical composition of the invention is typically administered in an effective amount.

The invention provides for an antisense oligonucleotide of the invention or a pharmaceutical composition of the invention, for the preparation of a medicament.

The invention provides an antisense oligonucleotide of the invention or a pharmaceutical composition according to the invention for use in therapy. The methods of the invention are preferably employed for treatment or prophylaxis against diseases caused by abnormal levels of ELF1-CFP pre-mRNA transcript. The disease may in particular be caused by increased levels of ELF1-CFP pre-mRNA transcript.

The invention further relates to use of an antisense oligonucleotide of the invention or a pharmaceutical composition of the invention as defined herein for the manufacture of a medicament for the treatment of abnormal levels ELF1-CFP pre-mRNA transcript, in particular high levels of ELF1-CFP pre-mRNA transcript.

The invention provides for the use of an antisense oligonucleotide of the invention or a pharmaceutical composition of the invention, for the preparation of a medicament for the treatment or prevention of Amyotrophic lateral sclerosis (ALS).

Administration

The antisense oligonucleotide or pharmaceutical composition of the invention may be administered topically (such as, to the skin, inhalation, ophthalmic or otic) or enterally (such as, orally or through the gastrointestinal tract) or parenterally (such as, intravenous, subcutaneous, intra-muscular, intracerebral, intracerebroventricular or intrathecal).

In a preferred embodiment the antisense oligonucleotide of the invention is administered by a parenteral route including intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion, intrathecal or intracranial, e.g., intracerebral or intraventricular, administration. In one embodiment the antisense oligonucleotide is administered intracerebrally or intracerebroventricularly. In another embodiment the antisense oligonucleotide of the invention is administered intrathecally.

Combination therapies

In some embodiments the antisense oligonucleotide or pharmaceutical composition of the invention is for use in a combination treatment with another therapeutic agent.

Method of Manufacture In a further aspect, the invention provides methods for manufacturing the antisense oligonucleotides of the invention comprising reacting nucleotide units and thereby forming covalently linked contiguous nucleotide units comprised in the antisense oligonucleotide. Preferably, the method uses phosphoramidite WO 2017/081223 PCT/EP2016/077383 chemistry (see for example Caruthers et al, 1987, Methods in Enzymology vol. 154, pages 287-313), incorporated by reference in their entirety.

In a further embodiment the method further comprises reacting the contiguous nucleotide sequence with a conjugating moiety (ligand). In a further aspect a method is provided for manufacturing the composition of the invention, comprising mixing the antisense oligonucleotide or conjugated antisense oligonucleotide of the invention with a pharmaceutically acceptable diluent, solvent, carrier, salt and/or adjuvant.

EXAMPLES

Example 1

TDP-43 has been shown to affect mRNA splicing. In order to identify new genes whose mRNAs are regulated by the presence of TDP-43, a TDP-43 knockdown was carried out in a neuronal cell model. RNA sequencing was performed on the cells, and de novo transcript analysis performed to identify affected genes with new splice patterns.

Human glutamatergic neurons (Fujifilms) were plated at 60,000 viable cells, together with 10,000 viable Astrocytes (Fujifilms), in 96-well plates coated with Laminin and Poly(ethyleneimine) solution (Sigma Aldrich) in 200 pl culture medium (day -1).

To knockdown TDP-43, 5 μM of compound A (SEQ ID 2) was added to the culture medium on day 0, with PBS added in other wells instead as a control. Half the cell culture medium (100 pl) was changed three times a week throughout the experiment (days 2, 5, 7, 10, 12, 14 & 17). The cells were harvested on day 20 using Magnapure lysis buffer (Roche) and RNA was isolated using the MagNA pure 96 system (Roche) according to the manufacturer's instructions, including the DNase treatment step. NGS libraries were prepared from 100 ng of total RNA using the KAPA mRNA HyperPrep Kit Illumina Platforms (Roche). Libraries were subjected to paired-end sequencing on a NovaSeq6000 sequencer (Illumina) with 150-bp read length. Data analysis was carried out using CLC Genomics Workbench 21. Data was first analyzed by running a large gap-mapping analysis using hg38 genome assembly, followed by transcript discovery. Predicted novel transcript changes were examined by visual inspection to identify real changes.

In the analysis, one of the predicted novel transcript changes as a result of TDP-43 depletion was a novel transcript just downstream of the gene ELK1 on the X chromosome (Figure 1 , grey arrow). However, closer manual inspection revealed that the increased expression of RNA from this region of the chromosome was due to the lack of proper polyadenylation of the ELK1 mRNA transcript, causing the RNA polymerase to continue transcription of the DNA. Interestingly, it was noticed that this lack of proper polyadenylation of ELK1 because of TDP- 43 depletion had previously been described in a conditional mouse TARDBP knockout model (Wu et al. 2019). This conservation of mechanism across species is notable since it is generally thought that mRNA targets for TDP-43 are not highly conserved between species.

Additionally, the apparent 10-fold upregulation of the CFP gene was observed as a result of TDP-43 depletion in neuronal cells. The CFP gene is located downstream of ELK1 in the same orientation (Figure 1). It is hypothesized that increased expression of CFP mRNA could be caused by read-through of the RNA polymerase II starting from the ELK1 promoter, thereby producing a long fusion transcript with two open reading frames. Normally it would be expected that such a mRNA would undergo non-sense mediated (NMD) decay, due to the presence of exon-exon splice junctions more than 50 bases downstream of the stop codon. However, since increased expression of the spliced CFP mRNA and even a 2-fold upregulation of the ELK1 mRNA is observed, it is hypothesized that this long fusion transcript is being translated in both open reading frames, thereby causing the potential increased expression of CFP in neurons lacking TDP-43 expression in their nucleuses.

Example 2

In order to show that a single mRNA containing both the ELK1 and CFP reading frame appears as a consequence of TDP43 depletion in a neuronal cell we designed a tiling gapmer ASO (antisense oligonucleotide) library complementary to the region containing the ELK1 gene through the intergenic region and into the first exons of CFP. If this region is expressed as a single mRNA then any working ASO in this region should result in downregulation of both genes ELK1 and CFP.

Here we show that ASOs are able to downregulate the combined pre-mRNA ELK1-CFP transcript that are thought to be a consequence of missed poly-adenylation of ELK1 in TDP- 43 depleted cells. Human glutamatergic neurons (Fujifilms) were plated at 60,000 viable cells, together with 10,000 viable Astrocytes (Fujifilms), in 96-well plates coated with Laminin and Poly(ethyleneimine) solution (Sigma Aldrich) in 200 pl culture medium (day -1).

To knock down TDP-43, 5 μM of compound A (SEQ ID NO 2) was added to the culture medium on day 0, except for four control wells per plate with PBS. Half the cell culture medium (100 pl) was changed three times a week throughout the experiment (days 2, 5, 7, 9, 12, 14, 16 & 19). 1 μM of each ASO targeting the ELK1-CFP fusion transcript was added to the culture medium on day 5.

92 different ASOs (SEQ ID NOs 3-94) were added in total. 88 wells received only compound A (SEQ ID NO 2) to serve as a baseline reference. The Compound Table provides the ASOs used, their HELM sequences and natural analogue sequences, and their target sequences.

The cells were harvested on day 20 using Magnapure lysis buffer (Roche) and RNA was isolated using the MagNA pure 96 system (Roche) according to the manufacturer's instructions, including the DNase treatment step. The purified RNA was denatured for 30 s at 90 °C before cDNA synthesis. cDNA was created using the iScript Advanced cDNA Synthesis Kit for RT-qPCR (Biorad) according to the manufacturer's instructions.

Measurement of the expression levels of the target genes was done by droplet digital PCR using the QX1 system (Bio-Rad) together with the QX1 software stand edition. PCR-probe assays were used to measure expression of the two genes ELK1 and CFP. This was normalized to the expression of the HPRT 1 mRNA, and finally the expression of TSRBP was measured to validate the effect of the knockdown.

The following PCR probe assays (obtained from Integrated DNA technologies) were used:

TARDBP:

Primer 1 : CAGCTCATCCTCAGTCATGTC (SEQ ID NO. 188),

Primer 2: GATGGTGTGACTGCAAACTTC (SEQ ID NO. 189),

Probe: /5Cy5/CAGCGCCCCACAAACACTTTTCT/3IAbRQSp/ (SEQ ID NO. 190)

ELK1(exon 3-4):

Primer 1 : TCAGGGTAGGACACAAACTTG (SEQ ID NO. 191),

Primer 2: GACCAACATGAATTACGACAAGC (SEQ ID NO. 192), Probe: /5HEX/CAAGAACAT/ZEN/CATCCGCAAGGTGAGC/3IABkFQ/ (SEQ ID NO. 193)

CFP (exon 9-10):

Primer 1 : CCTTGTAGCTCCTCACACC (SEQ ID NO. 194), Primer 2: GCCTCTGCACACCCTTG (SEQ ID NO. 195),

Probe: /56-FAM/CTTCTCGCC/ZEN/CTGACCTTCGACC/3IABkFQ/ (SEQ ID NO. 196)

The following CY5.5-labelled HPRT1 probe (obtained from BioRad) was used: dHsaCPE13136107

Data shown in Table 1 were normalized to the expression of the house keeping gene HPRT1 , and finally normalized to the average expression value of the four control (PBS) wells per plate that did not receive any TDP-43 knock-down or CA-repeat ASO.

TABLE 1 - CFP and ELK1 expression reduction by ASOs.

The data confirmed the observations described in Example 1 , since a 13-fold increase in CFP and 3-fold increase in ELK1 expression were measured in the wells with TDP-43 knockdown only.

All of the tested gapmer ASOs were able to downregulate both the ELK1 and the CFP transcript from the average starting point of ELK1 305% and CFP 1332% seen in the TDP-43- depleted cells. 72 of the 88 tested ASOs were able to reduce the expression of CFP to at least the level of the untreated cells (100%) and 21 ASOs were able to reduce CFP expression to under 10% of that seen in untreated cells.

The data validate that increased expression of CFP mRNA is caused by transcription from the ELK1 promoter, since even ASO gapmers targeting intron 1 of ELK1 were able to dramatically decrease the expression of CFP (Figure 2). Likewise, ASOs targeting regions inside the CFP mRNA were able to reduce the expression of ELK1 mRNA from the 305% observed in TDP- 43-depleted cells (Figure 3).

Taken together these data demonstrate that an ASO targeting the intergenic space between ELK1 and CFP could enable the selected knockdown of CFP and ELK1 in only sick cells, where TDP-43 has become depleted due to protein aggregation, and not in the remaining ‘healthy’ cells of the body where normal expression of ELK1 and CFP are needed.

REFERENCES

Wu et al. (2019) Transcriptomopathies of pre- and postsymptomatic frontotemporal dementialike mice with TDP-43 depletion in forebrain neurons, Acta Neuropathologica Communications 7(50) https://doi.Org/10.1186/S40478-019-0674-X

SEQ ID NO 1 is the sequence of the ELK1-> CFP fusion transcript. This sequence is presented below and is based on Ensamble Havana v 17 gene annotation:

HG 38 Chr X: 47.650.604 ->47.623.172, Minus strand orientation.

CTCGGGCCCACGTGAGCTGTAGGGAAACGCAGGGGCGGCTTCTAGGTGCTGCCGCCG

CCACCGCCACCACCACCTCCACCGCCGCCTCGGAACCCAGGCCTGGGGGGCGGTGG

GGCCGCGTATGGAGCCCCCGCCCCCCGGAGCTGCCAACATTGCCAACGCCACCGCCA

CGCTACACACAGGTGAGCTCTGGGCCTGGAGGGTGGAGGGCCCAGTCCGTGACCCCA

CGTATCCTTCCCGCCCCCGCGCAGAGGATGTGGCTTGGCCGGTGGCCTGCTGGTGTT

CGACTCCCCGCCGCCACCACCACGGCTGGTGGACCTGCGTGTGGCATTGCTCAAGCC CTTCGTCCCTTATAGTGGATCTGACCGTGGCCTAACCTCCCCCTCCCGTTGTATAATGG

ATCGGTCTGCGTGCTTATGTTTTTCCCCACGCCAACTTAGGGTGGACTCGTCCATAGGC

TTTCCCCCTACCCCTACCCCCACCCGCCCCCCACCCACCCCCACTTATAGGGAGCTAG

CCTGTGACAGTGTTCAGCCCCCTTAATAGTAGGTGTATCTGAGTGTTTGGATTTCTCCT

AGCCTCAACTTTCAGGAGACCCGTCCGTGGCCTTATTTATTCCACCCTTCCTGTACATC

GTAGCGAATCAATCCGTGGCGCCGCACTCCTCCGCATCCCTCTTTAACAGTGAGTCTA

CCTGAGTTTGTATTCCTGCCTCCTCCAGTCCCCCAATGCATCAGTCCATGGCTTTTTTCA

AAACCACCCCCCCCCCCCACCACCACCACCACCATTTTATAGTGGATCAGTCTTCATGT

TTGGATTCACGTCCCTTTAGCTATCAGTCCATTGCCCTGTCTCCTCTTTTTCGTAGACTG

TCGCTCTAGCGCTTGGGGTTCTCTCCTTTTTAATAGTGGGTTAGTTTAGTGCTTTACATC

TCCCTATTTTCAGTAGCTCAGTCCATAGCCTTTTCCCTCTCTGCTTTGAATCAGTCTGTG

TGATGGCTTTTCTCTCCTCTCACTGTGGTGCAGGAGTCTGTAAGGTTTTCTGCTCCCCT

TCCCCTCAGACGATGAGCCTCATCACCCCTCCCTTTGCAGTGGATCTGTTCATTGGCCT

TCCTCCCCCACACCCCTGTATGCTTGCACAGTCCCCCGCATACCCTCCCGTGAGTCCA

GTGCTTTGTAATTGGGGGAAGATCGGCCGTGTACTTTCAATTTCCTTCTCTTTTTTTTTC

CCTTCCCAGAAGAGAACGTGCTGATCCTCTTTTCCTTGTGATGGAGCACTGTACGGCCT

TCCCTGTCCTCCACCTCTTAATAGTGGGTCAGCCTGGTCACACTCGTAACCACAAAGAC GTTCTGCCTCACTACAATAAGTGAATACATTAGCGCCAGTGATGATAGTAATGCCAATA

GGAGCTAGCGTTTATTGAGCACCTGCCGTATATCAGGCAGAGTGTTTCATATGAATGAG

TTCCTTTAATCCTCACCACAACCCCATGAGATAGGTAGTGAGGGAATTGGGCCATAGGG

AGGTTAAATTCCTTACTCAAAGGTGGCCCAGGAAGTACTCAGGACACAGGATAATTTTT

GAGGCTCCCCTCTTAGATCTCATCCCACCCCACGTGAGGCCTCAGTCCTTAAAAACCAA

ATTAGTTCTCACCTCCAAGCTTTTGTTCTTGCTGTTCCCTCTGCCTGCAGAGCAGTCTTC

ATTCCTCACCTTCTCTCAGCACCCACCCACCCCCCAATCCCAGCAGCTGTGTGACCTTA GATGAGTGATTTAACCACTTCCTCTCTGTGCCTTTTTCTTCATTTGTGAAATGGGGGATC

AGAACTAGAATCCCTTCCAGTGTAAATAAATATTAACTGTGAATTAATGGTAAAACAAGT AACCTTTCTAGCATTACCGCCTATACCTTCCCACCCCCTTACTGTGCTCCAACCACACT GGTTCCACCACACAGCTCCTCAAACATAGCAGGCATGGGCCAGCCTCAGGGCCTTTGC

ATTGGCTGTTCTTTGTGCCTTGAACACCACCCCCAGATCTCTGCATGGTTCCTTTCAGC

ACTCTGTTCAAATACCAACCCCCAAAGGATGTTTACCACACTGTGGTGTGTGGAATGCA

GTATAAAAAGATACATGTATGAAAGATGTTTCTAAGAATGCCAGTTTATATATGAGTAAG

GGTTTTCCTCCTTTTCAAAAGGATGTGTTCACAAGACTGAGGTACGTAATATGCTCTGTA

AAGGCATAGGTATAAAAGCTGTTTCTAAGAGTGCTAGTTGTATATGTGAATAAGGACTGT

GTTGTTGCATTGAAAGAATGTGTTCACAAAACTGTGGTGTGTAGCTGGGCACAGGGGT

GTGCACTTATAGTCCCAGCTACTCGGGAGGCTGAGGTGGGAGGATAGCTTGAGCCCA

GGAGTTCCAGGCCAGCCTGGGCAATATAGCGAGATCCCTTTTTCTGAAAAAAAAAAACC

CAAAAAACAACCACTGTGGTGTGTGTAATACACTCTCTTAAAGGCACAGGCGTAAAAGA

TGTTTCTAAGAGTGATTTGATTTTGTTGTACCTGGGTAGTGTTATTTCATTAAAGGTTGAT

TGTCACACCATGGTACTTAGAATATACTATACAAAGGCACATGTATAAAAAAGAAGTTCC

TAACAATGCTACTTGTAAAAACAAAAAAAGTCAACCCCTCCCTGACCATCTGAAGCTGC

ACACCCAGCAGGGCTCAAGCCAGGTCCCTGATTTGTCCCGTTGCCCTTTATCACTGTCT

GACACACTGTGTGGGTTACTTGTTTGTTGTCTGTCTCTCCTCATTAGAATGTGAGTCCCA

TGAGGACTCAGTTTTTGTCTGCTCATTCACCGCTATGTCTCCAGCAACTGAAAATGTAC

CAGGTAAACAGCAGATGCTCAATAAGAATTTATCCCATGAATAAATGGGAAAAAGATAAT

TTGAGACACCAGTGTAGGGGTCACATGAGGAAGAAGAAGGGATATGAGAGCTAATTAG

TTGGGACTGTTCCTGCAACCAACCTTTACTGAGCCCCTTGTGAGTACCAGGCACTCTTC

TAGGTGCTGGAATATGGCAAGGAACGAAACAGGGCGAAATATTTGTGCTTGTGGAGCT

GACATTTATTAAAAGTTGGGGAGGCTGGGCACGGTGGCTAACACCAGTAATCCTAGCA

CTTTGGGAGGCTGAGGTGGGTGGATTGCCTGAGCTCAGGAGTTCAAGACCAGCCTGG

GCATCACGGTGAAACCCCATCTCTACTAAAATACAAAAAAATTAGCTGGGTGTGGCGGC

GTGCGCCTGTAATCCCAGTTACTTGGGAGGCTGAGGCAGGAGAATTGCTTGAACCCAG

GAGGCGGAGGTTGCAGTGAGCCAAGATTGCACCATGCACTCCAGCATGGGTGACAGA

GCGAGACTCTGTCTCAAAAAAAAAAAAAAAAAAACCCAAGCTGGGGAGACAGACTATGT

GAACAAAATAAATAAGTAAAATTAGCATAATAGACAAATGCTGAGAAGAAATAATGAAAT

AGGGCAGGTAGGAATCGAGTATTGGGGGTGGTTGAAATCTTAGGCATAGGGAAGGTGA

CTCTTGAATTAGGGCCTCACGGTAGAGACGGAGAGAGCCTTAATGTCTACGGGAAGAG

CATACCAGGCAGAAGGAAGTGCCAGTTCAGAGGCCCTGAGGTGGGATGGTGCCTGCC

ATGTTCAAGGAACATCAAGGCGGCCAGTGTGGCTGGAGCAGAGTGCGGGGAAGGGTA

GAAGGTGAGGTCACACAGGTGATGGGAGCCAGATAGTGCAGGTGATACATAGGATAG

GATTGCAGGGGCAGGGGGAGAAGGATGACACACTAGCTAATAGCTAATATCTGTGGAG

TATCTGCCACACATCAGAGGCACTTTTCTGTTCAAATACCAACCCCCAAAGGATGTTTAC

CACACGGTGGTGCCTGGAATGCACTACAAAAAGATACATGTGTCAAAGATATTTCTAAG

AATGCCAGTTTATATATGAATAAGGATTTTCCTCCTTTTCAAAAGGATATGTTCACAAAAC

TGAGGTATGTAATATGCTCTATGGAGGCACAGGTATAAAAGCTGTTTCTAAGAGTGCTA GTTGTATATGTGAATAAGGACTGTGTTGTTGCATTGAAAGAATGTGTTCACAAAACTGTG

GTGTATAGCTGGGCACAGAGGCTGAGGTGGGAGGATCACTTGAGCCCAGGAGTTCCA

GGCCAGCCTGTGCAACATAGTGAGATCCCCGTCTCCGAAAAAAAACCCAAAAAATAACC

ACTGTGGTGTGCATAATACACTCTCCTAAAGGCACAGGCATAAAAGATGTTTCTAAGAG

TGATTTGATTTTCTGGCACCTGGGTAGTGTTATTTCAGTAAATATTCATTGTTAAACACTA

TGGTGCTTGGAATATACTATACAAAGGCACATGTATAAAAAAGAAGTTCCTAACAATGCT

ACTTGTAAAAACAAAAAAAAAAGTCAACCCCTCAAGTCTTTTACAGAAGTATTTTACAGA

ATTGACACAGTTAATCCTCACAGAAGCCTTGGAGGGTAGGTGTACTTTTGAGAATATTT

GACTTTGAGAGTAAAATATCCCCATTTTACAGATGAGGAAACAAGCAAAGAGAGGCTGA

GTAACTTGCTCAGGGTAACCTTGAGCCAGAATAGCCAGAACAGGGGCAGAGGAACATA

AGGCTGAAGACTCAGAAAGAAGAACAGATTTGCAAGGGCAGGCAGGTTATTTGGCACC

TTTGATCACGCAGAGGAAGAATGAATTCTTCCTGGAATGCGATGATTGTGTAATTGAGT

CCATGAAGTGAGGGAAGAGCTCTCCCTGCGGGGAGAGCAGCCCGTGCTTACCCTCAG

AGGTTGGAACTGTCTCGGGAACGGTAGATAGTTCAGGGTGGCTGCCTCAGAGCCTTTG

TACACGCCGTTCGCTCCGCCTGAAGGCCCTCCCCCACAGAGGGCAGCCTGGCTTGCT

ACCTCCCCTCCTTCAGGCCAGGTGTCACTTCCTTGGGGAAGAACTCCTTGTACACCCT

GTGTAAAAACAGCATTCCTGTCACTCTCTAGGATCTTACCCTTCTCTGTTTCTCTTCATG

GAGAACTGGTGGTGTAAGGCGATTGCTCACTAGAGCCAAACCACTTAGGTTTTGAATTC

CGGCTCTGCCAACTTCCAGCTGTGTGACTTGGGGCAGGTTACTTCTCTGTACCTCCATT

GCCTCATGTGGAAAATGGGGGAAATGATAATCCCCACCCTTTAACGGTGGTTTGAGGAT

TCAATGAGTTAAATGATTCAAAGAGTGTGCAGCCCACGGTAAGGATCGTATGCCTGGCT

TGCATTTTTAAATTATTAAAATGTATTCATAGCATACCCGTAGATGTCTGACAGGCCTCT

GAAACTGACCATCTCCAAAACTGGTGTGACCCCCCTCAAAACTTACTCTCCTGCAAGGC

TCCCACCTCAGCTGACCACAATGCTGTCTTTGTGGGTGATTAGGCCACAAACCTAGACT

CACCCTGGACCCTTCTCTCACCTCCCCCGGGCATGCTATCTGTCAGCAAGTCCTATTCT

GTGTTCAGGCTTTCTCCAGACCCCAGCTACTTCTCGCCAGCCCCACTGCTACCACCATC

ATCTACCGCAGTGGCTTCTTCAGCCCCAACAGTCTGTTCTTCTCACAACCGCCAGGGG

GATCCTGTTGAAATCTCAGTCACAGCCTGTCATCCCTCTGTTCACAACCCTCTTGTGGC

TCCTGTCTCACTCAACATCCTGACAGCACCCGAAAAAGCCTGGCTCAGTCTGGCCCCC

GTCACTCTGTTCTCCTCTCCCGCTGCCTCCCTCCTCTCCCCCGAACCCATGTCAGCCTC

CTCTCCCCATGTCACCTGCCAGGCACTCTCCAGCCTTGGGCCCTTCACACCGGCTGCC

GCTTCCACCTGGCTTGCTGCTCTCAGATACTCACGAGCTCAGTTCCTTCACCTCCTTTT

GGTCTCTGCCCAGTTGTCCCCTCCTTGGCGACTACTCTCCCCTGACTCTCCATGTAAAA

GTAACAGCTCTCCCCTGCTTGATTTTTTTCCCATAACCCTTACCACCATCTGACGCACTC

TTGTACATGTTTGCCTTTTTTCTCATCTGCCTTTCCTGCTAAGATGTGAGCTCCTCTGGA

GCAGGGATTGTTGTCTGTTGCGTTCTCGACTGTATCCGCAGCACCTGGCCCACAGTAT

GCACTCACCAAATGCTTGCTAAGTGAAGGAGATGGAAAGAAGTAAACATATCTAAGCAA GGCAGATTACTAGAACTCTTATGAGGGGTCAAGTGGGAGAAATAACAAAATACAAAGCC

CTGTGTGCTGTTGAAGCTGGAAGCCCACCGTCACCTTTCACTCAGCTGATAGGTTCTGT

TGCAGCTGAAAGAAGAACTGGTAAAATTGCCTGTCTTTGCCACCAGGAGTCACTCTGAA

CTCTTTGGACTTCTGAAACAAGCCTGCCTGCAAAACAAATTCAGTGAGGTCTCTGGTGA

CAGGACTCTCTAATCGGACACTTCATGGCTTTGTGACTTGTATAATTGAACAGGAAGCT

CATGTTTATTTCCAGTTTGCTCTCAGGAAAGAAAGCAGTAATGGGGTGACAGGGAGGAT

CCTGTTCTTTGAAGGACATGCTGAGAGGGAATAGTCGGTGTGAGGGGCCGTTTAGCCT

AATGGTGAAATGCTTAGTCAACTGGAACCTGACTGCCTGGCTCCAAATGCTGGCTTTGC

CACATGTGGCCTGTGGAAGGTACGTAGACATTTGTGCCTCCGCTGCCTCATTTATAAGT

TAATGACAGGAAGACTACCTACTTCTCAGAGGCATGATCAGGGATAAATAAGTCAATGC

CCATTAAAAAAAAAAATAACAGTACAGGTGTACATGCCCTTACCCAAAACCCCTGGGGC

CAGATGTGTTTCAGAATTTCCATTTCTTCACATTTCAGAAAGGTAACATGATGCATATAC

GATGTTACATAACACCCCTTAGTAAGCTCTGTAATCGGCTGTGTTCATATTTCTGCAGCA

AAATGTATGAACACTCGCAATAAGTGAGATTCAATAAAGACCAGAAAGGGCCTTGTTTC

ATTTCAAATCAGGTTTTGCCACCAAAAGAGTTAAAAGAAGCTTTCTGCCCTTAGAGCATT

TTGGATGTTGGAATGGCAGATAAGGGACTATGAACAAATCAGAAACTAATATTTAGAGC

TTATCATACACCTCAGGCATATAATATTTAATCTGTACAATATCTCTGTGAACTAGGTATT

GTTATCCCCATTTTACTAATGGGCATATCATTATCCCTGTTTTACACATGAGAAAATTGA

GGTACAAAGAGGTTAAAGAATATCAGCCAAGCGTGGTGCCTCATGCCTGTAATCCTAGC

ACTTTAGGAGGCCAGGGAGAGAGGATTGCTTGAGCCCAGGAGTTCAAGAACAGCCTG

GGCAACATAGTGAGACCCCATTTCTTAAAAAAAGAAAAAGAATATGCCCAGGGTCACAC

AGCTAATAAGTGGCAGAACCAGGATTCAAACACTGGCAATCTGGCCCTAGTGCCTGGG

CAGTTGTCCAGCAGGCCATATTGCCTCTCTGCTTAGAAGAGTGCCTGATGTGTATTAAG

CTCTCAATAGATGTTAATTATTGCTGTCATCAAGAAAGAGAATATTCAGGGCCGAGCAC

GGTATCTCACACCTGTAATCCTAGCACTTTGGGAAGCCAAGGTGGGAGGTGGGTGGAT

CACTTGAGGTCAGGAGTTCAAGACCAGTCTGGCCAACATGGTGAAACCATGTCTCTACT

AAAAATACAAAAATTAGCCGGGCGTGGTCGTGGGTACCTGTAATCCCAGCTATGCGGG

AGGCTGAGGCATGAGAATCGCTTGAACCTGGGAGGCAGAGGTTGCAGTGAGTCAAGAT

TGTGCCACTATACTGCGCTCCAGCCTGGGCAACAGAGAGAGACTCCATCTCAAAAAAA

AAGAAAAAAAAAAAAAGAACGAGAACATTCAGGGACAGTAACAGGACAGAGAGATCAG

GCATTCATTCAGTTTTAGGACGGCTTCATAAAGGAAAGGACCTTTGGGCTGGGACTTGA

AAGATAAGAAAGACTGCTAGGCGAAAGAAGGAAGCCCCACGTGGAGTGAGCAGCCTTT

GCAAGGGTCTGGCAAAAAAAAAACCTCCTGAAAAACTTGACATTGATATGGTAGAAGAT

ACAGATAATATTCAAATGAGGAGGATGGAACTAGTTTTCTATTCTTGCTGTAACAAATAA

TCACAAATTTAGCTGCTTAAAACAACACATTTGTTATCTTACAGTTCTGTAGGTCAGAAG

TCCCACACTGGTCTCAGCTGAAATCAAGGTATTGGCAGGGTTGCGTTCCTTCTGGAGG

CTCTAGGGGAAAATCCATTTCCTGCTCATTCAAGTTGTTGGCAGAATCCAATTCCTTGA GATTGTAGGACTGAAGTCCCTGTTTCCTTACTGTCTGTCAGCTGACAGCCATTCCCAGC

TTTAAGAGGCTGCCCACAGTCCTTGATTCTGAGCCTCCTTCCTCCATCTTCAAAGCCAA

CAATGGCAGGTTGAGTCCCTCTCACATTTTGAATCTCCCCTGCCTCTTCTGTCATCACAT

CTCTGAGCTACCTTTCTTTCTCCATCATCTGCTTTTCAGAGCTCATATGATTAGATTGAAT

TTCATCAAATAATCCAGAAATAATCTCCCTGTCTTAGGGTTTGTAACCTTAATTTCATCTG

CAAAGTTCCTTTCTGCTATGTAGTGTAAATAACATACAGGTTCCGAGGATTAGGATGCG

GAGCAGGGTTGGCATCATTTGGCCTACCACAGTCTGCCCTCTGACCCCCACAGATTCA

CATTTGTTTCACATTCAAAATACATTCACACCATCCTAAGATACCCATGAGTTGTATCCC

ATCACAGGGAGCATCCCAAGCAAGACTGGGGAGCTAGAAGTTAGTCTGGGAAGGTTTA

GGGAATGATGGTAGGGTTTGAAGACAGAGATGGAGAAAAGATGACAAAAAAAGGAATC

CTCCTCCCGCTGACCTCTGCCCCTCCTATGTCCACAGCCTCTCAACTCAGCTGTTTGCT

CTCCAGGTACCCCTGGGATGGCGTGAGCACTCCCCCAGCGATGGACCCATCTGTGAC

GCTGTGGCAGTTTCTGCTGCAGCTGCTGAGAGAGCAAGGCAATGGCCACATCATCTCC

TGGACTTCACGGGATGGTGGTGAATTCAAGCTGGTGGATGCAGAGGAGGTGGCCCGG

CTGTGGGGGCTACGCAAGAACAAGACCAACATGAATTACGACAAGCTCAGCCGGGCCT

TGCGGTACTACTATGACAAGGTACAGTCTCTCAGACCAGGGCTGGAACCCCCCCCTGA

CATTTATATCAGAGATTTTATTTTGTTTTAATTGCCATAGCTATGACTTTGAAAGCAATGT

TAAAATTTATCAGTTTTTAACCCATCCATTGGTACCCTAAGACCAGTATTGGGCCCCTAA

ACACTATCTCACCTACTTTCTTGTCTTACTGCTTTGGCGAAAACCACCACTGCATTATTA

ATACTGACTCTTTCTACTCCCACTCTGAGATCCCCAAAGTGATGCTGCCCTGCCATGCA

CAACGTTTCATTTTCCTGCCGTATTCCTATAGCCAACATCTTGAAAACACGGTTAATAAT

GGCTGGTCCTCATTCTGTCCTCTGAAACCCCTGGACCAGTGCTGGGCTTCCCTAGTTAT

TTCATAGTCTTCTCTTATTCCAGAATATTTCATATTCCTAGAATCAAAACCTCCCTGACAG

TATTATACCAACTTTTGCTTACTTCGTCCTCTGGGACTCCCAACATTTTTAACTAATTATT

TTCTTCTCTTATTCCTTAAGCTAAAACCTCTGAAACAACATGTTTAATGCCAACTTCCACA

CACTGTCTTCTTAAATTCCCAGAGGAGCACTGGTCTACCCCAATATTTATATCAATTGTT

TTCATTATCTTATTCCTACAGCTAAAATTTAAAATTTCCCCCATCCTGCCCTCTCAGACCC

CCAGGCCACTGCTGGGTTTCTCCTCCCCCCAATATTTATACCAGTTATTTCATTTTCTTA

ATCCTTCTACTGAAATTTCCCAAACAATATTCATGAATATCCCTTTTTATTCCTCTTCCGA

GAAACACAGGCCCACCCTGTGACATATCTGACCAGCACTAAGACTTCCACAGAATGCC

CTAGTGCCTCCACACTCCCAGAACTGCCCTGGTCATATGCTGCACACCCTTCATAGTTT

ATGGCATTTATTCCATTTTCATATTTTATTCCTTGAGCTCAACCCTCCAGAACAATATTAG

TAATGAGGCTATCTCACTCTGCCCTCTGGGAACACTAGGTCATCTCTGAGACCCACCAG

ACCTGAAAAACCCCAAACATGCTCTGTGACCCAGCAAAGCCAGCCTTGATATAGGCCC

CCTCCCAATATTTATACCAGTATTTTTAATATTCTTGTCTAATTGCTGTTAGTTACTTCATT

TTCTTACCATTTTAGCTTCAACCTCCACAGTGGTATTAATAATGAGGCCTCCTCACCCTC

TCCTCTGCAGTGACCTTGCCCTGTTCCACTTCTGTATCATTAGCTTCAAATAATCCTCTG TATTAATAATTCATCACCCCATCCATCTACATTAATTTACTTTCTTCCAACATTTATATCAG

TTACTTCATTTTCTTGTCTTATTGCATTTGCTAGTCTCCAAAACTGTGTCCTGACAGTCAC

CCACAAGGCCTGGCCCTGTGAACCTCTGGTTCTTGGAGCCAGGAAGAAGACCTGCTCC

CACACCTACTGCCCTTCATGGGCCCCATTGTGAAGAGGAGCTACAGTGTGGAGGGAGT

GGGAAAATCAGATAGACGGACCTGGAGAGTGACAAAGGGAGGCTTGGAAAGGGGTGG

ATGGGAAGGGGCTTAAAGAAAAAATTATTGTGGAGAGAAGAGAGAAAGGGGAGGAACT

GGGGAGGGATATAGATAAGGGGAATCTGGCGGGAGAGGAAAGAGGTAGAAACCAATG

GGAGAGGAAATGGGCTGAGAGAGAAACTTACAGAGGAGGGACGGGAAAGCCGGAGAG

GAGCAAGTGGGAAGAAAGCCACTAGGAGAAGAGAAATGCCACTCCACCCCCCTCCTCC

CCCCTGACACCCTGCCTCCAGCCTGCCCCTTTCCTCTAAGGCCCTCTGTTCCTCTCCTC

CCTTCCTAGAACATCATCCGCAAGGTGAGCGGCCAGAAGTTCGTCTACAAGTTTGTGTC

CTACCCTGAGGTCGCAGGGTGCTCCACTGAGGACTGCCCGCCCCAGCCAGAGGTGTC

TGTTACCTCCACCATGCCAAATGTGGCCCCTGCTGCTATACATGCCGCCCCAGGGGAC

ACTGTCTCTGGAAAGCCAGGCACACCCAAGGGTGCAGGAATGGCAGGCCCAGGCGGT

TTGGCACGCAGCAGCCGGAACGAGTACATGCGCTCGGGCCTCTATTCCACCTTCACCA

TCCAGTCTCTGCAGCCGCAGCCACCCCCTCATCCTCGGCCTGCTGTGGTGCTCCCCAG

TGCAGCTCCTGCAGGGGCAGCAGCGCCCCCCTCGGGGAGCAGGAGCACCAGTCCAA

GCCCCTTGGAGGCCTGTCTGGAGGCTGAAGAGGCCGGCTTGCCTCTGCAGGTAAGGA

CTGGAATATAGAAATTCAGTAAGAGGAGGCAGTAAAGAGGAGGGATGTGTAATCCCCG

GATGACGAGGGAGGCTGGTAGAAGAGACATAGCTGTCCCGTTAAAGGAGATAATGGTG

GAAGGAAGACATCCTCTTAGAAGGAGGGGTGGGGCAGAGAGCTACACAAATCCTGAGA

CAGAAAGGGGAGAATACTGTGCTACAGTTAGTAGCTGCCGAACATGTAGGTGCCTGGA

ATATGTTTAATGCACTGGTTCTCGCACTCTAGTGCTCATCAGAATCACCTGTAGGTCTTG

TTAAACCACAGATTGCCAGGCACACCCCCAGAGTTTCTGAATTTGTGGGGCTGGGGAG

GGGCCCAATAATTTGCATTTTTAACTTGTTACCAAATGAGGCAGATATTGCTGGTCCAGA

GGAAGGTAAATTTTTGAGAACCACTGTTTTAATGAATGAGGATGGTATCCCTCAGATTCC

TACCCGCTTCAGGTTGTCTCACCCCAAGTCCCCTGTCCCATAGGAGGACCTGCCCCAG

AGGAAGCCCTACTTCCTTGACGAAGTCCTATGTCCTGTTGGAGGACTTGCTCTGATGTG

CAGTCATGCCCTGAGTACCAAGTGCCACCTTCCCGGGGAAATCACTGGGTGGCCCACA

GGAGAATCCTGTGGTCCCAACAATCCTTACACACCCTCTTTGTGCCTGCTCCTCTACCA

GGTCATCCTGACCCCGCCCGAGGCCCCAAACCTGAAATCGGAAGAGCTTAATGTGGAG

CCGGGTTTGGGCCGGGCTTTGCCCCCAGAAGTGAAAGTAGAAGGGCCCAAGGAAGAG

TTGGAAGTTGCGGGGGAGAGAGGGTTTGTGCCAGAAACCACCAAGGCCGAGCCAGAA

GTCCCTCCACAGGAGGGCGTGCCAGCCCGGCTGCCCGCGGTTGTTATGGACACCGCA

GGGCAGGCGGGCGGCCATGCGGCTTCCAGCCCTGAGATCTCCCAGCCGCAGAAGGG

CCGGAAGCCCCGGGACCTAGAGCTTCCACTCAGCCCGAGCCTGCTAGGTGGGCCGGG

ACCCGAACGGACCCCAGGATCGGGAAGTGGCTCCGGCCTCCAGGCTCCGGGGCCGG CGCTGACCCCATCCCTGCTTCCTACGCATACATTGGTGAGTGCCTGCGGAGGGGTGG

GGTGTGTGGGCGCCAGCACCAATGGGATTGGCATGTGGCGGGTGACTGACAGGAATC

AGAGGAGGGTGAGTGTGGTTGTTCCTGAAGAGGGTGGAGCCTTTGAGTGTGATTGACT

GGGCTGATGCTGGCTGGAGGGATGGGGTTTGATTAGTGAAGGGCAGGGCTGGGGACT

TTTGTGGGTGGGACCGAGGGTCTTTCTGTGGGACTGTTTCTAGGTTGGGGTGTGGCTT

AAGGAACCTGAGCAGGGCTTTTTGGTCTGGGATGTGCATATGGGGTTTCATGGGTGGG

ACATGAGTTGGAAGTTGACCCTGAGCTGTTGGGATGGTGACTTGAGAGGTTTGAGAGC

AGCATTTGGATAGGGTCTTGTGGGTGGTCTTGACCAATGAGGTCTCAGCAACTGAGGG

TGGGCTCTCGGGAGTGGGGAGTGGGGGGTGTCTGCAGAGTTTGAGAGATCATAGGCA

TGGCCTCCATGGTTACCATCTAGGTGTGTGCACTTTGGGGAGTGACCCTGGGAGTGTG

AGTGGGAAATCTGTGGTGGGGCCGGGACCAAGTCCCTTCCAGCATCCCACATCTACTT

TCTACTCACTTGAGCTCCGACCACCCTCTCCCCACAGACCCCGGTGCTGCTGACACCC

AGCTCGCTGCCTCCTAGCATTCACTTCTGGAGCACCCTGAGTCCCATTGCGCCCCGTA

GCCCGGCCAAGCTCTCCTTCCAGGTAGGATCCTCTTTCCCCGTTTTGGACAGGGTGAG

AGACCCAAGAATGGCCCCCATCTGTGACCTCTCCCTCTTTTTGCCCCCAGTTTCCATCC

AGTGGCAGCGCCCAGGTGCACATCCCTTCTATCAGCGTGGATGGCCTCTCGACCCCC

GTGGTGCTCTCCCCAGGGCCCCAGAAGCCATGACTACTACCACCACCACCACCACCCC

TTCTGGGGTCACTCCATCCATGCTCTCTCCAGCCAGCCATCTCAAGGAGAAACATAGTT

CAACTGAAAGACTCATGCTCTGATTGTGGTGGGGTGGGGATCCTTGGGAAGAATTACT

CCCAAGAGTAACTCTCATTATCTCCTCCACAGAAAACACACAGCTTCCACAACTTCTCTG

TTTTCTGTCAGTCCCCCAGTGGCCGCCCTTACACGTCTCCTACTTCAATGGTAGGGGC

GGTTTATTTATTTATTTTTTGAAGGCCACTGGGAGGAGCCTGACCTAACCTTTTAGGGTG

GTTAGGACATCTCCCCCACCTCCCCACTTTTTTCCCCAAGACAAGACAATCGAGGTCTG

GCTTGAGAACGACCTTTCTTTCTTTATTTCTCAGCCTGCCCTTGGGGAGATGAGGGAGC

CCTGTCTGCGTTTTTGGATGTGAGTAGAAGAGTTAGTTTGTTTTGTTTTATTATTCCTGG

CCATACTCAGGGGTCCAGGAAGAATTTGTACCATTTAATGGGTTGGGAGTCTTGGCCAA

GGAAGAATCACACCCTTGGAATAGAAATTTCCACCTCCCCAACCTTTCTCTCAGACAGC

TTATCCTTTTCAACCAACTTTTTGGCCAGGGAGGAATGTCCCTTTTGTTCTTCCCCCTGA

GAAGCCATTCCTTTGTCTGCCAACCTCCCTGGGGTCCTGCCTGTTTCCTCCCAATGGAG

GGTTTTTTTGGGGGGTGGTCCCCGTCTGGGGGGCCCCTCCAGCCAGTACTCCAGGTC

TCCCTGTCTCTCCCCCGCTGCCATTTTGATAGTATAATCTATTTTTAAATGGGGCTTTTC

AATAGGGGAGAGGGAGTCATCTCTTCCTATATTTGGTGGGGTGGGTGGGAAGGAAGG

GATTTGGGGGGGAATCTTCCTGCCGCCTCCCCCACTCCAAGTGTTTATTTTTGATACCA

AACATGAATTTTCAGTTCCCTCCCTCCCAGCCCCCCAATTTCCTGCGGGCGGGTACAAA

GGACCCTTTCAATGTCCCTGGAGTTGGGAGGGAGGAATGGGGGACATAAAGCCTGTCC

TGTCTCTATTCTAGGCAAGAGAGAGTGGGTTCAAAAGACTCCTGGGCTCACCTGTTAGC

GCTGGCCCAGCCCAGGCCTTGGGACCTGGGGGTTGGTGATTTGGGGGACAGTGCTAC ACTCGTCTCCACTGTTTGTTTTACTTCCCCAAAATGGACCTTTTTTTTTTCTAAAGAGTCC

CAGAGAATGGGGAATTGTTCCTGTAAATATATATTTTTCAAAGTGATGCTGGAGGCTGC

TGGCACTTTTTCTCTGATGTTGTCAGGACTAGCTGAGCGTGACACGCATGACTGTGTGT

GGAGGGTGTGAGGCAGAGGTCGATTGTGAGAGAAACGGTGTGATGGCGTGTGTGAGA

AACAATAGTAGGGACTGCATATCGGTAACCATGTGTGTGTCAGAGACGTGGAAAGCTG

TGATTGTGTGTGAGAGTGTGCAGAAGTTGTGACCACAGACTTTTGATGGTGTGTCTATG

TGGGAAATTGTGGCTGTGTTTCAGAAAATAACTGAGCCTGGATGAATAGTCAAGGTACT

GTACAAGAACCTGTGATGGTATGCACAAAATAATGTCTACGAGAAGGGAACGGGGACT

GCAGCCCTGTCACTGTTTGATGGCGAGCAAGACAGACTGGGTGACCAAAACCACATGT

GAGAGACTACGTGGGCATGAAACTACAGTAGAGGTTGTCAGGGATTGTGGCAAAGATT

GTGTAGGTGACAAACCTCACTGTAGGCGACTCAACGTTTGAGAAATAGATGATGTGCAA

AACTGTTTACTTGAGGCTGTAGTGTCAGCTAAAGCTGTAAGACTGTATGGGTGCGAACT

TGATTGTACGGGAGAGAATGGCTGGGAGACCAAACCAGTGACCGAGACCAAAGATACA

AGAAGGTGCAATTGTGTGTACGAAAGCAGGCGTGAGAGACTGCAGCAAAGACTGAGAA

AAACGAATGGCTGTGACTGGGAGAGTGTGACAGGCTGTGTGTGTGTGTGTGTACACCT

GAAACTAGTTGCAAATCGATTTACCATTACTACCACAGTCTATCCCACAGGTTCAGGGA

CTGTGAGAAAGTAGGAGAGAAAATGACGGTGAGCCGGACGGAGCCACTGACATGGTG

TGAGAGACCACATCACTGGGCATGCAAGGGAGAGGGACAAGGAACTGTTTATGAGAGA

CTTGGCAGTAGCAGTAACTGGAAGTCAGAAACTGTGGGAGAGGAACTTGACAACTGAG

TGATGTCAGAGCACCAGGGGGTCTGTGAAACAGTTCATAAGAAACCATAAATCAGGGG

CCATACAAAAGAGCTGAGCAAATGAGACATGCCAAGTGACCACGTGACAGTGTGTGAG

AAACTTGTAAAGCTGTGCACGTGTGTCCGTGATGAGCTGACCATATGAAGAAAATGACG

GTGTGCACGACTGAGAGGGCATGGGTGACTGTGGGTGACTTATCGAGGGCCTAGTGG

TGATTGTGAAATACTGACCGAGTATATCAGAAGCTGACTGAGGGACAGTGATGACTGAG

ACAGTGGCTGAGAAACGGATTGTGTGTCCATGGGTAAACCCTTTGCAAGAGACTTTATG

AAAGGCTGTAAATCAAGGACTGAGGGTACTGGAAGCTGATTGAGAAGAAACGTGAGAC

TAGGTGACGACAGTGTCTGAGATACTGTGTGATTGTGTATGCGTGTGAATGTCTTTGTG

GGAGAGGCTGTAATCAGGGATCGTGGGTGTGTGAAAAAGTGGCTTGAGTGAGAAAGG

GGTAGGAGAGGTGATTTTTGTGCATGTGTGGGAAACTTCATGAGAGACTAGTTAGTGGT

GACTATAAATCAGTGACTATTTGTGTATGTGAGAAAGTAACAGGCCAAATGAGTGCCTA

AACGTTTCATGAGATTGTGTGACTGCACGTGTATGTAAGGAACTGACTGGTGAGAGAAA

CTATTGGCAATATTGACTGAATGAGACAGGCTGTTAAACATGATTGTGTGTGTGAAACT

GTTAGGAGACTGTAGTAAGGCTGGAATCCATGACACAGAAAATGACTATGAGTGACAGA

ATGTCTGAGAGGCTGTGTGATTAAGAGAATATAGTAGAGGCTGTAAATCCGGACTGGGT

GCGGTGGCTCATGCTTGTAATCCCAGCACTTTGGGAGGCCAAGGTGGGAGGATCACTT

GAGGCCAGGAGCTTGAGACCAGCCTGGGCCACATGGTGAGACCCCGTCACTACTAAA

AATTTAAAAAATTAGCATAACGTAGTGTTGTGCACCTGTAATCCCAGCTACATGGGAGG CTGATGTGGGAGGACTACTTGAGGCCAGGATTTTGAGTTTGAGACCAACCTGGGCAAC

ATAGAAGGACCCTGCCGCTACCAAAACAAAAAACAAAATTACCCAGGTATAGTGGTGCA

TGCCTGTAGTCCCAGCTACTGAGGACACTGAGTTGGGAGGATCGCTTCAGCCCAGGAG

TTTGAGTTTGAGACCAGCCTGGGCAATGTGGCAAAACCCTGTCTCTCCAAATAGTTTAA

AAAATTAGCCAGGTGTGGCGTGTGCCTGTAGTCCCAGTTACTCGGGAGGCTGAGGTGG

GAGGATGGTTTGAGCCCAGGAGGTTGAGGCTGTAGTCTGCGATCACGCCACTGCACTC

CAGCCTGGGTAACAGAACCAGACCCTGTCTCGAAAAGAAAAGAAAGAAAAGAAAAACAT

AACTGTGTAGATAGGTTTAAAAAAAAAAAGAGAGAGAGAGAAAATAACAGACCAAATGA

GACTGTGTGAGAGGTCTAATGCCCTGCAGACCCTCGAATGTTCCAGGCCCTGGAGAAA

AACCCTCTTCTGTTTTTCCTGTTCCTCCACAGGCCCCTCCTGCTCAGTCCGCTGGGCTG

GCTTCTCCTACTTCTCTAAAACTTGAAATGCTGGGGTTCCCCAGGGCAAAGCCCTGGGA

TTCCCTTCTCTCTTCTCACGCATCCTCTCTAGGTCATCTTATCCAGGTCTTCACACTTGA

AATACTACTTATCCTCCATGGGATGCCCACATTTTTACCTCTAACTCAGACTGTCCTTCT

GGCATTTTATCGTAATCTCTAAAGTTTTGTTTATTTCATTGATTGATTCATTTAGCAGATG

TTTATTGAGCATCCACTGTTTCCTGGTGCCTATCTAATGCCCTGCAGACCCAGCAGTTA

ACAAGCCAGACAAAAATCCCTGCCCTTGTGGAACTGATATTTAGGGAGTGAGAATAACA

AGAAGCAGGCAAATACATAGTAGCAGGTAGGGATGTAATAATTAAATAAGACAAGACTC

TCACTCTTTGTTTAAGGAATCTGCAGTCATTTTATTGCCTAAACGTGTGGGAAAAGATAG

AGTGCTGACAGTCCTCTTCCACAGGGAATCCTATGAGTATTTATACAGTTTAGAAACAAA

GGAAATATTGTTAATATTATTACCAGGGCAACAGGTGGTTAAATAAAGAAGCCAGCCAG

AATTTCAGGTTTCTTTCTTTTTTTTTTTTTTTTTTTTTGGGACAGAGTCTCACTCTGTCGCC

CAGGCTGGAGTGCAGTGGCACGATCTCAGCTCACTAGAGCCTCTGCCTCCCGAGTTCA

AGCGATTCTCCTGCCTCAGCCTCCCAAGTAGCTGGGATTACAGGCATCCGCCACCACG

CCTGGCTAATTTTTGTATTTTTAGTAGAGACGGCGTTTCACCATGTTGACCAGGCTGGT

CTCGAACTCCTGACGTCAGGTGATCCACCTGCCTCAATCTCCCAAAGTGTTGGGATTAC

AGGCGTGAGCCACCATGCCTGGCCAGTTTCAGGTCTCTTGGAGAGTGTTTTCACAGGA

TAACACAGCAATGATTGTCTTGAAGTCGTAGAATCTTAATAAGACTCCTTTGTTCAAAGC

ACATTTTCAAGGATAGCCTTATGTTATCAGAGAACAGAGTATGTCTGTAATGGATACAGA

TATTAACACATACTTTACTGCCCATAAAACAAGTGTTCCTGAAGAACTTCTGTTTTAACA

GCTGACCAAATTATTACATTTGAATTTGCAGAATTCCTCTGCCTCCCTGAGAGCAGAGG

AAATTAGGCATTGCCTTCCTATCTTTCTTGTGCTTCTTTTTGTTAATTAAAAGCAAAATAA

AAATTTTAAAGTGGGCCCTGGAGTTTCATTAGGGTTTATAAAAAAAAAATAAAAAAATAA

AAAAAAAAGAAGGGCTGGTGTGATGGCTCATGCCTGTAATCCCAGCACTTTGGGAGGC

TGAGGTTGGGAGAAAATATTGAAGCCAGGAGTTTGAGACCAGCCTGGGCAACACAACG

AGACCCCATCTATACAGAAAATAAAAACATTAGTTGTGTGGTGGTGTGTGCCTGTAGTC

CCAGCTACTTGGGGACGCTAAGGTGGGAAAATCACTTGAGCCCAGGAGTTTGAGGCTG

CAATGAGCTATGATCAGACCACTGCACTCCAGCCCAGGTGACAGCAAGACTCTGTCTC TTTAAAACAAAACAAACAAAAAAACAGGTTTGGAAGCTACTGGGCTAGAGAGTGACAGG

GATGCCAGACAAGGGTATGGGGAAGGCCTCTGTGAGGAGGGGCAACATTTGTTATCCA

TCTTCTCGAGGGCCAGGATTTTTGTCTGTCCTATTCTCTGCTCTAATGCTAAGAACCGC

ATCTGGCATCCAGTAGGTGCTCAATAAATGTTGAATAAATTTATCAAACACAGAGCACCT

ACTATATGCCAGGCACTGTTCTAAAGACTTTCCAAAAGCTTAACTCCTGGCCGGGCACG

GTGACTCACGCCTGTAATCCTAGCACTTTGGGAGGCCAAGGCAGGTGGATAACCTGAG

GTCAGGAGTTCGAGACCAGCCTGGCCAACATGGTGAAACCCTGTCTCCACTAAAAATA

CAAAAATTAGCTGGACATGGTGGTACACGGCTGTAATCCCAGCTACTCAGGAGGCTGA

GGCAGGAGAATTGGTTGAAATCGGGAGGCAGAGGTTGCAGTGAGCCGAGATCGTGAC

ACTGCACGCCAGCCTGGGCGACGGAGTGAGACTCCATCTCGACAAAACAAACAAAAGC

TTAACTCGCAATCTTTGCAACACACCCTACCTATGTTGTAGGTGCTGTCTGTGAGCACA

TTTTACAGAGGGGAAACAGGCTCCAGAAGGGGGGTGTTATCTGCCTGAGGGTTCTCTG

ATGGAATATGGCCCAGCTTATTCCCTCCTAATCACCCCAGACCCTGGTGTTGTCCCCAG

CTGGGCCCATGGAACAATGGATAGACCCCATTCCCCTCCTGAATCCCTCCCCAGGCTC

CTGCCACTTCCCTTTCTCACTTCCCTTGTGCTGACCTCCCCGACCACAAGCTGACCACA

GGGAACCTCTCAGGAAAGGAGGGCCGAGGTTAAGGGAGGGTGGAAGGTGGAGCAACT

GACTCGATGCTCCCTCCACCCCCACGAGCAGGAACATACTGAGCACCGCACACTCACT

TCACCCTGGTTCAACACCCCCACGAGGTTGACCCCGTCATTATGTTAGAGATTATGCAT

TTTCCACATAGGGAAACTGAGGCTCAGGGGTGTTAAGTGACTCACCCAAGGTCACACG

GCTAGGAAGTTGCTGCACGCTCCTATGCTCCATTTCCTCTGGGTGAGAATTTGTCCTAG

GAGCTTCTATTGAACACTGTGAGGTTGCTGGGAGGAGCGCGAGGTGCTGGGGATATAG

TAACAAACAGAACAAGACCAAGAGTAAGACAGGAAATGGGGAGGGGAGAGTAGAGAAG

GAGGCAGAGGGGACAGGGTGGGGTCAAAGCAGGATGCCCTCCCAGTTCCTCCTGCCT

CTAGGTTCCTCTTTCCCTGCTGATTCCAGGAGCCTATCAACCCAGATAAAGCGGGACCT

CCTCTCTGGTAGAGGTGCAGGGGGCAGTACTCAACATGATCACAGAGGGAGCGCAGG

CCCCTCGATTGTTGCTGCCGCCGCTGCTCCTGCTGCTCACCCTGCCAGCCACAGGTGA

GGGGGTGAGGGCCCAGCTGGCCCCAGGGGTGTCCAGGCCCACCACATCCCTGACCG

AGCCCCACCCACCCATCCCCTGGCCCTGTTACAGGCTCAGACCCCGTGCTCTGCTTCA

CCCAGTATGAAGAATCCTCCGGCAAGTGCAAGGGCCTCCTGGGGGGTGGTGTCAGCG

TGGAAGACTGCTGTCTCAACACTGCCTTTGCCTACCAGAAACGTAGTGGTGGGCTCTG

TCAGCCTTGCAGGTTAGGGGGAGCCTGGGGTGGGGGATGGGGGGGAGGGAAGGACT

GTCTGTGGGGGTCACCCAAAACTGGGCACTTTCTTTCTCTGGGACTTCCAAGGGTGGC

CCTGAGGCCCTGATCGTGACCATACCTCAGGGTTTCTCAACCTTGTTACTATTGACATT

TGGGGCTGGAAATTCTTGGCTGGGGGAGGGGATTGCTTACCTGTGTATTGTGGGATGT

TTTGTAACACCCCTAGCCTTCCACTAGATGCCAGTAGCAATTCTCCCCACCAGCTGTGA

CAACCAAACATGTCAGCAGACATTGCCAAATGTCCCCTGGGGGTCAAAATCACCCCTG

ATTGAGAACCACTGACATAGCTCACACATATTGAGCACCTCCTGCACACTGGAAGCATT GCAGGTGGGGTGCAGCTTGGTATTTCCCTAATTTTGATGACAGCTCATGTTTATTGAGT

GCTTACTGTATATGGACACACTGTGGGGGCGGGAAAGTTTGGGGAATTCAAAGGGCAG

CTCTAGGCCCCTAATTATGATGACACTTCACATTCATTGAGCACTCCCTGTATACCAGCT

GCATGGCAGGGTTGGGAGATGAGCTTGAGGATTTCTGAGGGTAGCTCTAGTCTCCTGA

TCATGATTTCCAGCTTGCATGGATTGAGCCCTTACCGTATGCCAGACCTGTTTTCTACTT

GTTTACTATATATCCCAGATACACCGTGGGCATGGGGAGCTTGGGGATTTTTGAGGGC

AGCTCTGGGCCCCAAGTCATGTCAACAGTTCATCACATCTATTTGGCATGAACAGCATA

CCTAGGGCTATGCAGTGGGTTGGGGATAGATCTGGGGTTATTTCTGAGGAGGCCTCTG

AGGTCATAGGGATGATGGCTCAGGTTTATTAAGCACTTTCTATTTACCTACTACCGTGTA

GTGTGGTGATCTGGTGGGTTTCTGACAGCAGCCCTTGGTTTCTAGTCCTTACCTTTGCT

TACTGCGGACCTGGTGTTGGCCATGCAGCCTTCCAATGGTAACTCCAGATGCCTGATT

GTGAAGGTAAATGACATTATCAAGCACTTATTGTATATCCACTGCTTCCCATCAGTATCC

CAATATCCCTAAAAGGTAAGGACTTATCAATCCATCGCACACGCACTTGCCTAGCAACC

CTTCGTGTCTGCCCACACTCTGGAGTCCCACATGCCATTCTTGTTAGCTCATGCCAGGA

TGGGATGTGTGTGCTCTTCTCCACAGGTCCCCACGATGGTCCCTGTGGTCCACATGGG

CCCCCTGTTCGGTGACGTGCTCTGAGGGCTCCCAGCTGCGGTACCGGCGCTGTGTGG

GCTGGAATGGGCAGTGCTCTGGAAAGGTGGCACCTGGGACCCTGGAGTGGCAGCTCC

AGGCCTGTGAGGACCAGCAGTGCTGTCCTGGTGAGGAGGATGAGCAAGGCGGGCAAG

CACTCCCTGTCACCCAGCACTGGTGGGAACGATCGGTACAGAGGTCCTCAGAGTGGG

CCCGTCCCCAGTGTTTGTAAGGACCTAAGGAGGAGAGTGGTGGGAGGTGAGGTCAGA

GAGGTGAGGAGGGTCCATTGCCTGGGGGCCACAGAGAGGACGTTGGCTTTTCATCTG

AGTAATTTGGACTGTGGGCAGAGGAGGAACGTGATCTGACTGGGGTTGGGAGGAGAA

CAGACTGTGCACGGTGAGGGTGAGAGGGGAAGCGGAGGCACCCATGTGAAGGTTGCT

GCGGCAGGCCCGGTGAGCGGGGATGGTGGGCCCAGAATGGTGGCAGAGGATATAAG

GACAGGATTAGCTCATGTGGGGCACTCAAGGAAGCAAAGAGCCCTGAATGACACCCCA

CCTCCATCCCATTACCCTCTCTTCTCTCCCACAGAGATGGGCGGCTGGTCTGGCTGGG

GGCCCTGGGAGCCTTGCTCTGTCACCTGCTCCAAAGGGACCCGGACCCGCAGGCGAG

CCTGTAATCACCCTGCTCCCAAGTGTGGGGGCCACTGCCCAGGACAGGCACAGGAAT

CAGAGGCCTGTGACACCCAGCAGGTCTGCCCCAGTGAGTGAGGGAAGCCACAGGATG

CTGATGGGTGCACCCAGCGTGGGTCTGCGGGGTTACAGCAGGGAACTGCCAGGCTGA

GAGGACTTTAGCATGCATGACCACACCTGCATCCCTCCACCTCCCACCCCACCACAGC

ACACGGGGCCTGGGCCACCTGGGGCCCCTGGACCCCCTGCTCAGCCTCCTGCCACG

GTGGACCCCACGAACCTAAGGAGACACGAAGCCGCAAGTGTTCTGCACCTGAGCCCTC

CCAGAAACCTCCTGGGAAGCCCTGCCCGGGGCTAGCCTACGAGCAGCGGAGGTGCAC

CGGCCTGCCACCCTGCCCAGGTACACCAGGATGAGGCTGCTGATGTTGCTGGTGGGG

CTCTTGATTGAGGGCAGAGATGCTCTGCCATTTCTAGAGTTCCTAGGCCATGTGACCAT

GCAGCCCTTCATTTGGGGACAGTCTGCTTCAAGGGTCTAGGGGCTGAGAGGAAGGATT GAGGAGGCCTTTCTCCTCACTCCCTTTCCTCCTCCAACAGTGGCTGGGGGCTGGGGGC

CTTGGGGCCCTGTGAGCCCCTGCCCTGTGACCTGTGGCCTGGGCCAGACCATGGAAC

AACGGACGTGCAATCACCCTGTGCCCCAGCATGGGGGCCCCTTCTGTGCTGGCGATG

CCACCCGGACCCACATCTGCAACACAGCTGTGCCCTGCCCTGGTCAGCATCTCAGGGT

TCACGATTTGCATGCCTAAGTCCCCCTTGCCCTTCTCTGCTGCCCAGCTCCTGCTGCTG

AGGCCTTGTTCCTGTCTCTGATTCCTCCTTTCCTGGCCCTGATACCTTGTTTCCACCCCT

GGTCCCCCATTCACACATCTGATCACCTCTACTCCCTCCTACCGCCCTCATTCCTTCCT

CTGAACCCCCTTGCTGATTCCCTGCTTTGGTCCAATCCCCTGTTGCCCTGTCTCTGCAG

TGGATGGGGAGTGGGACTCGTGGGGGGAGTGGAGCCCCTGTATCCGACGGAACATGA

AGTCCATCAGCTGTCAAGAAATCCCGGGCCAGCAGTCACGCGGGAGGACCTGCAGGG

GCCGCAAGTTTGACGGACATCGATGTGCCGGGCAACAGCAGGATATCCGGCACTGCTA

CAGCATCCAGCACTGCCCCTGTGAGTGTCCCACAGACTGTGCTCTGAGGGTGGGGTG

GCCCATGCAGGATAAGGGGTTTCCAACTCTCTGCTGTGGACCTCACGTCTCTGCAGCC

TCCCTCTCACTTTCCCACCAAGACCTCCAGTTCTGACTCTGTGACCCCTACCCCTCATT

GCAGTGAAAGGATCATGGTCAGAGTGGAGTACCTGGGGGCTGTGCATGCCCCCCTGT

GGACCTAATCCTACCCGTGCCCGCCAGCGCCTCTGCACACCCTTGCTCCCCAAGTACC

CGTGAGTGAGAGGGCAAAGTATGCCTGGGAGGGGGTCATTATATTACAGGCGGGAGA

GTCGGCTACCCCAGGAAACCAGGGTTTCCACTGCTGAATCTAAGGTATCTGCCTTGCC

GAGTGCCACCTCCCAGCTGCAGGAAGTGGGGAGAAACTAAGAAGATGAGAAAGAAGTT

CTTGGAGGATAATTCGTAGCCTCTGTGCCCCTCAGTCACCTGTTCAGTCACTCATCCAC

TTAGTCCTTCATGTGTCATTGACTCCGACCTTCAGTTACCCATGCTTTTAGCTGCCATCA

CTCAGGCCTCCATTCACTGCCTGCCTCCCACAGTCACTCATGCCTCCATCCACTTTGTC

AGCATCTCACTGAGCCAAGGCACTCCAAGGCTTTTTCAGCAACAGTTGCAGATACACAC

AGCCCAGATCCTGTGGATCCTCCAAGGGACCCAGCTGAGCAAGCCCCTGACAGGAGG

CATATGAGGAACAGCTGGTGCATGCAACAGGTGCTGCCACCTCAACCTAGGTCCAGGA

AGCTGACTGGGCCTCCCAGGGCAGGTAGGGGTTGGGAAAGGCATTCCTGGCATCAAG

ACTGTGAACGGAGCCTTGGAGCTAGGTGATTAGCCGCTATAAGTGTTTCCAGGTGCCA

GAGGGGCCCAGGTGTTAGAGAGGGTTAAAATGTTCATCCCTTTTCTTCCTTCCACATAC

ATCAAGCTACTAGATTAAGTGAACTATTCCCAGCTCGTGGAAGGGCTGCCATCTGGGAA

GGGATCTCCCGGCTGAGGGGGTTAGAGTGGGGCCTGAAGATTAGAGGTGTTGAATGG

AGGCACAAAGGACTTGAGTGAGCGAGAGACTGAGTGGAGGAAGAAGCAATTGAGAGA

ACTGGGATGGGAGTGAATGGAGGGCTTTCCTAGTGGGACAAATAACCCAGGTAAAGAC

CTTTCTGGACAGGGACTGAGCTTGAATAAAGTGGAGGAGACAGAGAGAGAGATTGGCT

TGGGGCAGGGATTTGGGCCCAACAATAAGGCCTTGAATGTCAGGACAAGGCATTGGGT

TTTTTCCTGGAGGCAGTGGAAAGAGATATAAAGGGTATATATTTCGGAGGTAAAAGAAA

GTAGCTCATGCCAACAAGTAACATTCACCAGCATTTATTGAGCACTTGCTGTGTGCCAG

GCACTGCTCTGAGCACTTTACCTGAATTAACTCATTTATCCTCATTCAACCCCGTGAGGT GAGTATTAATATTACTATTCCTACCATTCTCATTTTGCAGATGAGGGAAACCGAGGCACA

GAGAAGCTAAAGAAATTGCCCAATATCCTATAGCTAAAAAGTAGTGTTGGGAGGCCAGG

CACAGTGGCTCACGCCTGTAATCCCAGCAACACTTTGGGAGGCTGAGGCAGGAGCATC

ACTTGAGCCCAGGAGATTGAGGCTGCAGTGAGCTATGATTGTGCCACTGCACTGCAGC

CTGGGCAACAGAGTGAGACCCTGCCTCAAAAAAAAAAAAGGAAAAAAAAAAAAAAAAAA

AGTAGCCCTCGGCATATAGCAGGCACTCAATAAGAATTGAATGCATTCTTGCCTTCCCT

GAGATTCTCCCTTCCGTTCCTCCCCACCCCTAATGCCTCAGGCCCACCGTTTCCATGGT

CGAAGGTCAGGGCGAGAAGAACGTGACCTTCTGGGGGAGACCGCTGCCACGGTGTGA

GGAGCTACAAGGGCAGAAGCTGGTGGTGGAGGAGAAACGACCATGTCTACACGTGCC

TGCTTGCAAAGACCCTGAGGAAGAGGAACTCTAACACTTCTCTCCTCCACTCTGAGCCC

CCTGACCTTCCAAACCTCAATAAACTAGCCTCTTCGAGTTCGTCTACGATTCCTTAAAGG

AGGAAAAACAACCTATCCCCTTCCCCAAAAGTAGGGTGATAGCATCTCATAGGGCAAAC

AGCACAGCACGGGCCAAGACACAGCAGCCTGTTGCGGAAAACAGTATTTTCCACTGTG

GTCGCAGGTTGGCAGGAGTGAAAGCGAGTGTGCAGTCCCAGCCTCTCCCCGCCGCTG

CCATAACCCCAATGTCTGGGCCCTGCGCAGCCCCCTTAGAGCCTCAGGGCCGCCCGG

CACGTTCCTCTGCAGAGCTCCGCGTTACGGGTTTCCTGATTGGCTACTTGTCCTGGCA

GGCACTTCTTGATTGGCCGGCGTGCTTGTTCGTTGTCTCCCCGTCTCCTGGACCTCCC

TAGATTCCCACCAGCCGGAACCCTGGAGGAGCCTCTGCCGGCCGACTCCGACTCCGC

GTCAGTCGAGGATGAACGGCAGAGGCGTGGCCGAGGCTGGCACCCTGCCGCCCGAG

CACCTGCACCCACCCACTCCCGACAGCCCAGCTCCAATGCCACAATTACCAGCGACAA

ATAGCTCGGCGAGGCGGGGGGCGGGCCGAGGAAGTGCCTGTGTTGCGCTCTGAGCT

GAGGAGGGGCAAAGAAGCTAGAGTCAGTATCCGCCCGGAGGAGCCCCCAAGACAAAT

CCAGACACTTTCCTCTGGTAATTTGTTCATTCATTCAACACTTATTAGCGCCTGCTGTAT

GCTAAAGCTGAGTCTGAGCATCTAGGGTGGAGGAGGGCTGGTAGAAAGAGGACATCT

GAGTGAGGGAACAGACTCAGAAGGTATTCCTTTCGTGTGAACATCTATTCAACACTTAA

CGCCTACTGTATACTAACAATAGTGCTGGACATCACGGAGAATAGAGAAGAAGAGGAAT

CAGAGCGTTACGTGTCTCCGCAGTAAAGGGAACAAAGCCAGACACCAGTGCTTTTAGC

ATTTGCTCATTCATTTACCACTTACTGAATGCCGGGCCCCTTGTGAAGAGACGCATCAT

AAGAATCATAATAATCATGGTAATAGCTACTACTT

Compound Table

Helm Annotation Key:

[LR](G) is a beta-D-oxy-LNA guanine nucleoside

[LR](T) is a beta-D-oxy-LNA thymine nucleoside

[LR](A) is a beta-D-oxy-LNA adenine nucleoside

[LR]([5meC]) is a beta-D-oxy-LNA 5-methyl cytosine nucleoside [dR](G) is a DNA guanine nucleoside [dR](T) is a DNA thymine nucleoside [dR](A) is a DNA adenine nucleoside [dR](C) is a DNA cytosine nucleoside

Claims

1. An antisense oligonucleotide, wherein said antisense oligonucleotide is 8 to 40 nucleotides in length and comprises a contiguous nucleotide sequence of at least 8 nucleotides in length which is complementary to a transcribed human CFP-ELK1 intergene region.

2. The antisense oligonucleotide of claim 1 , wherein the contiguous nucleotide sequence is 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides in length.

3. The antisense oligonucleotide of claim 1 or claim 2, wherein the contiguous nucleotide sequence is the same length as the antisense oligonucleotide.

4. The antisense oligonucleotide of any one of claims 1 to 3, wherein the contiguous nucleotide sequence is at least 75% complementary to the transcribed human ELK1-CFP intergene region.

5. The antisense oligonucleotide of claim 4, wherein the contiguous nucleotide sequence is at least 80%, at least 85%, at least 90% or at least 95% complementary the transcribed human ELK1-CFP intergene region.

6. The antisense oligonucleotide of claim 5, wherein the contiguous nucleotide sequence is fully complementary to the transcribed human ELK1-CFP intergene region.

7. The antisense oligonucleotide of any one of claims 1 to 6, wherein the transcribed human ELK1-CFP intergene region comprises SEQ ID NO 1 , or a fragment thereof.

8. The antisense oligonucleotide of any one of claims 1 to 7, wherein the transcribed human ELK1-CFP intergene region consists of SEQ ID NO 1 , or a fragment thereof.

9. The antisense oligonucleotide of any one of claims 1 to 8, wherein the transcribed human ELK1-CFP intergene region is SEQ ID NO 1 , or a fragment thereof.

10. The antisense oligonucleotide of any one of claims 7-9, wherein the fragment is 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides in length.

11. The antisense oligonucleotide of any one of claims 1 to 10, wherein the transcribed human ELK1-CFP intergene region is within a human ELK1-CFP pre-mRNA transcript.

12. The antisense oligonucleotide of any one of claims 7 to 11 , wherein the contiguous nucleotide sequence is complementary to a sequence selected from the group consisting of SEQ ID NO 95, SEQ ID NO 96, SEQ ID NO 97, SEQ ID NO 98, SEQ ID NO 99, SEQ ID NO 100, SEQ ID NO 101 , SEQ ID NO 102, SEQ ID NO 103, SEQ ID NO 104, SEQ ID NO 105, SEQ ID NO 106, SEQ ID NO 107, SEQ ID NO 108, SEQ ID NO 109, SEQ ID NO

110, SEQ ID NO 111 , SEQ ID NO 112, SEQ ID NO 113, SEQ ID NO 114, SEQ ID NO

115, SEQ ID NO 116, SEQ ID NO 117, SEQ ID NO 118, SEQ ID NO 119, SEQ ID NO

120, SEQ ID NO 121 , SEQ ID NO 122, SEQ ID NO 123, SEQ ID NO 124, SEQ ID NO

125, SEQ ID NO 126, SEQ ID NO 127, SEQ ID NO 128, SEQ ID NO 129, SEQ ID NO

130, SEQ ID NO 131 , SEQ ID NO 132, SEQ ID NO 133, SEQ ID NO 134, SEQ ID NO

135, SEQ ID NO 136, SEQ ID NO 137, SEQ ID NO 138, SEQ ID NO 139, SEQ ID NO

140, SEQ ID NO 141 , SEQ ID NO 142, SEQ ID NO 143, SEQ ID NO 144, SEQ ID NO

145, SEQ ID NO 146, SEQ ID NO 147, SEQ ID NO 148, SEQ ID NO 149, SEQ ID NO

150, SEQ ID NO 151 , SEQ ID NO 152, SEQ ID NO 153, SEQ ID NO 154, SEQ ID NO

155, SEQ ID NO 156, SEQ ID NO 157, SEQ ID NO 158, SEQ ID NO 159, SEQ ID NO

160, SEQ ID NO 161 , SEQ ID NO 162, SEQ ID NO 163, SEQ ID NO 164, SEQ ID NO

165, SEQ ID NO 166, SEQ ID NO 167, SEQ ID NO 168, SEQ ID NO 169, SEQ ID NO

170, SEQ ID NO 171 , SEQ ID NO 172, SEQ ID NO 173, SEQ ID NO 174, SEQ ID NO

175, SEQ ID NO 176, SEQ ID NO 177, SEQ ID NO 178, SEQ ID NO 179, SEQ ID NO

180, SEQ ID NO 181 , SEQ ID NO 182, SEQ ID NO 183, SEQ ID NO 184, SEQ ID NO

185, SEQ ID NO 186 and SEQ ID NO 187, or a fragment thereof.

13. The antisense oligonucleotide of claim 12, wherein the contiguous nucleotide sequence is complementary to a sequence selected from the group consisting of SEQ ID NO 98, SEQ ID NO 101 , SEQ ID NO 102, SEQ ID NO 105, SEQ ID NO 107, SEQ ID NO 109, SEQ ID NO 110, SEQ ID NO 113, SEQ ID NO 115, SEQ ID NO 116, SEQ ID NO

117, SEQ ID NO 118, SEQ ID NO 119, SEQ ID NO 120, SEQ ID NO 121 , SEQ ID NO

122, SEQ ID NO 123, SEQ ID NO 124, SEQ ID NO 125, SEQ ID NO 126, SEQ ID NO

127, SEQ ID NO 128, SEQ ID NO 129, SEQ ID NO 130, SEQ ID NO 131 , SEQ ID NO

132, SEQ ID NO 133, SEQ ID NO 134, SEQ ID NO 135, SEQ ID NO 138, SEQ ID NO

139, SEQ ID NO 140, SEQ ID NO 141 , SEQ ID NO 142, SEQ ID NO 143, SEQ ID NO

144, SEQ ID NO 145, SEQ ID NO 146, SEQ ID NO 147, SEQ ID NO 148, SEQ ID NO

149, SEQ ID NO 150, SEQ ID NO 151 , SEQ ID NO 152, SEQ ID NO 153, SEQ ID NO 154, SEQ ID NO 155, SEQ ID NO 156, SEQ ID NO 157, SEQ ID NO 158, SEQ ID NO

159, SEQ ID NO 160, SEQ ID NO 161 , SEQ ID NO 162, SEQ ID NO 163, SEQ ID NO

164, SEQ ID NO 165, SEQ ID NO 167, SEQ ID NO 168, SEQ ID NO 169, SEQ ID NO

170, SEQ ID NO 171 , SEQ ID NO 172, SEQ ID NO 173, SEQ ID NO 174, SEQ ID NO

178, SEQ ID NO 179, SEQ ID NO 182, SEQ ID NO 183, SEQ ID NO 185, SEQ ID NO 186 and SEQ ID NO 187, or a fragment thereof.

14. The antisense oligonucleotide of claim 13, wherein the contiguous nucleotide sequence is complementary to a sequence selected from the group consisting of SEQ ID NO 118, SEQ ID NO 119, SEQ ID NO 124, SEQ ID NO 126, SEQ ID NO 128, SEQ ID NO 132, SEQ ID NO 141 , SEQ ID NO 142, SEQ ID NO 145, SEQ ID NO 154, SEQ ID NO 157, SEQ ID NO 159, SEQ ID NO 160, SEQ ID NO 161 , SEQ ID NO 162, SEQ ID NO 163, SEQ ID NO 164, SEQ ID NO 165, SEQ ID NO 168, SEQ ID NO 169 and SEQ ID NO 172, or a fragment thereof.

15. The antisense oligonucleotide of any one of claims 12-14, wherein the fragment is 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides in length.

16. The antisense oligonucleotide of any one of claims 1 to 15, wherein the antisense oligonucleotide is single stranded.

17. The antisense oligonucleotide of any one of claims 1 to 16, wherein the contiguous nucleotide sequence comprises a sequence selected from the group consisting of SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11 , SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 16, SEQ ID NO 17, SEQ ID NO 18, SEQ ID NO 19, SEQ ID NO 20, SEQ ID NO 21 , SEQ ID NO 22, SEQ ID NO 23, SEQ ID NO 24, SEQ ID NO 25, SEQ ID NO 26, SEQ ID NO 27, SEQ ID NO 28, SEQ ID NO 29, SEQ ID NO 30, SEQ ID NO 31 , SEQ ID NO 32, SEQ ID NO 33, SEQ ID NO 34, SEQ ID NO 35, SEQ ID NO 36, SEQ ID NO 37, SEQ ID NO 38, SEQ ID NO 39, SEQ ID NO 40, SEQ ID NO 41 , SEQ ID NO 42, SEQ ID NO 43, SEQ ID NO 44, SEQ ID NO 45, SEQ ID NO 46, SEQ ID NO 47, SEQ ID NO 48, SEQ ID NO 49, SEQ ID NO 50, SEQ ID NO 51 , SEQ ID NO 52, SEQ ID NO 53, SEQ ID NO 54, SEQ ID NO 55, SEQ ID NO 56, SEQ ID NO 57, SEQ I D NO 58, SEQ ID NO 59, SEQ ID NO 60, SEQ ID NO 61 , SEQ ID NO 62, SEQ ID NO 63, SEQ ID NO 64, SEQ ID NO 65, SEQ ID NO 66, SEQ ID NO 67, SEQ ID NO 68, SEQ ID NO 69, SEQ ID NO 70, SEQ ID NO 71 , SEQ ID NO 72, SEQ ID NO 73, SEQ ID NO 74, SEQ ID NO 75, SEQ ID NO 76, SEQ ID NO 77, SEQ ID NO 78, SEQ ID NO 79, SEQ ID NO 80, SEQ ID NO 81 , SEQ ID NO 82, SEQ ID NO 83, SEQ ID NO 84, SEQ ID NO 85, SEQ ID NO 86, SEQ ID NO 87, SEQ ID NO 88, SEQ ID NO 89, SEQ ID NO 90, SEQ ID NO 91 , SEQ ID NO 92, SEQ ID NO 93 and SEQ ID NO 94, or at least 10 contiguous nucleotides thereof.

18. The antisense oligonucleotide of claim 17 wherein the contiguous nucleotide sequence comprises a sequence selected from the group consisting of SEQ ID NO 5, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 12, SEQ ID NO 14, SEQ ID NO 16, SEQ ID NO 17, SEQ ID NO 20, SEQ ID NO 22, SEQ ID NO 23, SEQ ID NO 24, SEQ ID NO 25, SEQ ID NO 26, SEQ ID NO 27, SEQ ID NO 28, SEQ ID NO 29, SEQ ID NO 30, SEQ ID NO 31 , SEQ ID NO 32, SEQ ID NO 33, SEQ ID NO 34, SEQ ID NO 35, SEQ ID NO 36, SEQ ID NO 37, SEQ ID NO 38, SEQ ID NO 39, SEQ ID NO 40, SEQ ID NO 41 , SEQ ID NO 42, SEQ ID NO 45, SEQ ID NO 46, SEQ ID NO 47, SEQ ID NO 48, SEQ ID NO 49, SEQ ID NO 50, SEQ ID NO 51 , SEQ ID NO 52, SEQ ID NO 53, SEQ ID NO 54, SEQ ID NO 55, SEQ ID NO 56, SEQ ID NO 57, SEQ ID NO 58, SEQ ID NO 59, SEQ ID NO 60, SEQ ID NO 61 , SEQ ID NO 62, SEQ ID NO 63, SEQ ID NO 64, SEQ ID NO 65, SEQ ID NO 66, SEQ ID NO 67, SEQ ID NO 68, SEQ ID NO 69, SEQ ID NO 70, SEQ ID NO 71 , SEQ ID NO 72, SEQ ID NO 74, SEQ ID NO 75, SEQ ID NO 76, SEQ ID NO 77, SEQ ID NO 78, SEQ ID NO 79, SEQ ID NO 80, SEQ ID NO 81 , SEQ ID NO 84, SEQ ID NO 85, SEQ ID NO 89, SEQ ID NO 90, SEQ ID NO 92, SEQ ID NO 93 and SEQ ID NO 94, or at least 10 contiguous nucleotides thereof.

19. The antisense oligonucleotide of claim 18 wherein the contiguous nucleotide sequence comprises a sequence selected from the group consisting of SEQ ID NO 25, SEQ ID NO 26, SEQ ID NO 31 , SEQ ID NO 33, SEQ ID NO 35, SEQ ID NO 39, SEQ ID NO 48, SEQ ID NO 49, SEQ ID NO 52, SEQ ID NO 61 , SEQ ID NO 64, SEQ ID NO 66, SEQ ID NO 67, SEQ ID NO 68, SEQ ID NO 69, SEQ ID NO 70, SEQ ID NO 71 , SEQ ID NO 72, SEQ ID NO 75, SEQ ID NO 76 and SEQ ID NO 79, or at least 10 contiguous nucleotides thereof.

20. The antisense oligonucleotide of any one of claims 1 to 19 comprising one or more modified nucleosides.

21. The antisense oligonucleotide of claim 20, wherein the one or more modified nucleoside is independently selected from 2'-O-methyl-RNA and LNA nucleosides.

22. The antisense oligonucleotide of claim 20 or claim 21 , wherein the antisense oligonucleotide comprises any number of LNAs at the 5’ end.

23. The antisense oligonucleotide of claim 20 or claim 21 , wherein the antisense oligonucleotide comprises any number of LNAs at the 3’ end.

24. The antisense oligonucleotide of claim 20 or claim 21 , wherein the antisense oligonucleotide comprises any number of LNAs at the 5’ end and any number of LNAs at the 3’ end.

25. The antisense oligonucleotide of any one of claims 1 to 24, wherein the antisense oligonucleotide is capable of recruiting RNase H1.

26. The antisense oligonucleotide of any one of claims 1 to 25, wherein the antisense oligonucleotide is a gapmer.

27. The antisense oligonucleotide of any one of claims 1 to 26, where the antisense oligonucleotide comprises at least one modified internucleoside linkage.

28. The antisense oligonucleotide of any one of claims 1 to 27, wherein one or more, or all, of the modified internucleoside linkages comprise a phosphorothioate linkage.

29. The antisense oligonucleotide of claim 28, wherein all the internucleoside linkages present within the antisense oligonucleotide are phosphorothioate internucleoside linkages.

30. The antisense oligonucleotide of any one of claims 1 to 29, wherein the antisense oligonucleotide is capable of reducing the level of the ELK1-CFP pre-mRNA transcript by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% in a cell, compared to a control.

31. The antisense oligonucleotide of claim 30, wherein the control is a cell that has not been exposed to the antisense oligonucleotide.

32. The antisense oligonucleotide of any one of claims 1 to 31 , wherein the antisense oligonucleotide is covalently attached to at least one conjugate moiety.

33. The antisense oligonucleotide of any of claims 1 to 32, wherein the antisense oligonucleotide is in the form of a pharmaceutically acceptable salt.

34. The antisense oligonucleotide of claim 33, wherein the salt is a sodium salt or a potassium salt.

35. The antisense oligonucleotide of any one of claims 1 to 34, wherein the antisense oligonucleotide is encapsulated in a lipid-based delivery vehicle, covalently linked to or encapsulated in a dendrimer, or conjugated to an aptamer.

36. A pharmaceutical composition comprising the antisense oligonucleotide of any one of claims 1 to 35 and a pharmaceutically acceptable diluent, carrier, salt and/or adjuvant.

37. The pharmaceutical composition of claim 36, wherein the pharmaceutical composition comprises an aqueous diluent or solvent, such as phosphate buffered saline.

38. An in vivo or in vitro method for reducing the level of ELK1-CFP pre-mRNA transcript in a target cell, the method comprising exposing said cell to an effective amount of an antisense oligonucleotide of any one of claims 1 to 31 or a pharmaceutical composition of claim 36 or claim 37.

38. The method of claim 37, wherein the cell is either a human cell or a mammalian cell.

40. The method of claim 38 or claim 39, wherein the level of human ELK1-CFP pre-mRNA transcript is decreased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%, compared to a control.

41 . The method of claim 40, wherein the control is a cell that has not been exposed to the antisense oligonucleotide.

42. A method of treating or preventing a disease comprising administering a therapeutically or prophylactically effective amount of the antisense oligonucleotide of any one of claims 1 to 35 or a pharmaceutical composition of claim 36 or claim 37 to a subject suffering from or susceptible to the disease.

43. The antisense oligonucleotide of any one of claims 1 to 35 or a pharmaceutical composition of claim 36 or claim 37 for use as a medicament for the treatment or prevention of a disease in a subject. 44. Use of the antisense oligonucleotide of any one of claims 1 to 35 or a pharmaceutical composition of claim 36 or claim 37, for the preparation of a medicament for treatment or prevention of a disease in a subject.

45. The method of claim 42, the antisense oligonucleotide or pharmaceutical composition for use according to claim 43 or the use according to claim 44, wherein the disease is associated with increased levels of human ELK1-CFP pre-mRNA transcript.

46. The method of claim 42, the antisense oligonucleotide or pharmaceutical composition for use according to claim 44 or the use according to claim 45, wherein the disease is Amyotrophic lateral sclerosis (ALS).