Antisense oligonucleotides and their uses

Abstract

The present invention relates to exon-including antisense oligonucleotides targeting a region in the pre-mRNA of human ASPP2, as well as their uses in the therapy in diseases and conditions related to ASPP2K-variant expression.

Description

Antisense oligonucleotides and their uses

[0001] The present invention generally relates to antisense oligonucleotides for use in research, diagnostics, and / or therapeutics, in particular concerning cancer and stress-induced inflammation conditions.BACKGROUND

[0002] Cancer is a disease in which some of the body’s cells grow uncontrollably and spread to other parts of the body. Cancer can start almost anywhere in the human body, which is made up of trillions of cells. Normally, human cells grow and multiply to form new cells as the body needs them. When cells grow old or become damaged, they die, and new cells take their place. Sometimes this orderly process breaks down, and abnormal or damaged cells grow and multiply when they shouldn’t. These cells may form tumors, which are lumps of tissue. Tumors can be cancerous or not cancerous (benign). Cancerous tumors spread into, or invade, nearby tissues and can travel to distant places in the body to form new tumors (a process called metastasis). Cancerous tumors may also be called malignant tumors. Many cancers form solid tumors, but cancers of the blood, such as leukemias, generally do not. Benign tumors do not spread into, or invade, nearby tissues. When removed, benign tumors usually don’t grow back, whereas cancerous tumors sometimes do. Benign tumors can sometimes be quite large, however. Some can cause serious symptoms or be life threatening, such as benign tumors in the brain.

[0003] It is generally acknowledged that tumorigenesis is a multistep progress driving the transformation of a normal cell to malignancy.

[0004] The prevalence of cancer has revealed some key players in the tumor progression responsible for the initiation of increased mutability. One of the most prominent regulators is the p53 tumor suppressor protein (also known as Tumor protein P53, TP53, cellular tumor antigen p53, or transformation-related protein 53 / TRP53), encoded by the TP53 gene, responsible for monitoring the integrity of the genome. The p53 proteins are crucial in vertebrates, where they prevent cancer formation. As such, p53 has been described as "the guardian of the genome" because of its role in conserving stability by preventing genome mutation. Hence p53 is classified as a tumor suppressor.

[0005] However, the p53 pathway is a complex cellular stress response network with multiple diverse inputs and downstream outputs relevant to its role as a tumor suppressor pathway; as such, the p53 pathway remains challenging in the realm of clinical translation.

[0006] Despite the many different approaches for the treatment of cancer and related conditions such as stress-induced inflammation, death rates remain high in many cancers and there still is the need for an efficient, different and improved approach for treating these diseases or conditions. Accordingly, it is an object of the invention to provide for such approaches.BRIEF SUMMARY OF THE INVENTION

[0007] According to the invention, this and other objects are solved by the provision of an antisense oligonucleotide comprising or having a targeting sequence (or sequence) with at least 90% sequence complementarity to a target nucleic acid sequence within a region of a pre-mRNA of human ASPP2, wherein the region of pre-mRNA of human ASPP2 is transcribed from a region -200 bases to +200 bases of exon 17 of human ASPP2, wherein the antisense oligonucleotide is exon-including.

[0008] In other words, the present invention is directed to an antisense oligonucleotide comprising or having a targeting sequence (or sequence) with at least 90% sequence complementarity to a target nucleic acid sequence within a region of a pre-mRNA of human ASPP2, wherein the region has the SEQ ID No. 16, wherein the antisense oligonucleotide is exon-including. SEQ ID no. 16 represents the pre-mRNA Region -200 bases to +200 bases of exon 17.

[0009] The invention also provides for the use of an exon-including antisense oligonucleotide comprising a targeting sequence with at least 90% sequence complementarity to a target nucleic acid sequence within a region of a pre-mRNA of human ASPP2, wherein the region of pre-mRNA of human ASPP2 is transcribed from a region the region -200 bases to +200 bases of exon 17 of human ASPP2, for use in the determination of prevalence, or treatment or prevention of diseases and conditions related to ASPP2K- variant expression, preferably in diseases and conditions in which proliferative signaling is sustained, growth suppressors are evaded, cell death is resisted, replicative immortality is enabled, vasculature is induced and / or accessed, invasion and metastasis is activated, cellular metabolism is reprogrammed, and / or an immune destruction is avoided; and also preferably, in diseases and conditions in which the induction of apoptosis is disrupted, halted, lowered or inhibited.

[0010] The invention also provides for the use of an exon-including antisense oligonucleotide comprising a targeting sequence with at least 90% sequence complementarity to a target nucleic acid sequence within a region of a pre-mRNA of human ASPP2, wherein the region of pre-mRNA of human ASPP2 is transcribed from a region the region -200 bases to +200 bases of exon 17 of human ASPP2, for use in the treatment or prevention of a cancer or cell-stress-induced inflammation.

[0011] The invention also provides for pharmaceutical compositions comprising the exon-including antisense oligonucleotide of the invention and a pharmaceutically acceptable carrier. The pharmaceutical compositions can be used together with an apoptosis-inducing therapeutics and / or within an apoptosis-inducing therapy. The pharmaceutical compositions can be used, alone or together with an apoptosis-inducing therapeuticsand / or within an apoptosis-inducing therapy, for use in the treatment or prevention of a cancer or cell-stress-induced inflammation.

[0012] The above mentioned uses and pharmaceutical compositions also apply to an antisense oligonucleotide comprising a targeting sequence with at least 90% sequence complementarity to a target nucleic acid sequence within a region of a pre-mRNA of human ASPP2, wherein the region has the SEQ ID No. 16, wherein the antisense oligonucleotide is exon-including.

[0013] Other features of the invention are set out in the appended claims.DETAILED DISCLOSURE OF THE INVENTION

[0014] As mentioned above, disclosed herein are antisense oligonucleotides comprising or having a targeting sequence with at least 90% sequence complementarity to a target nucleic acid sequence within a region of a pre-mRNA of human ASPP2, wherein the region of pre-mRNA of human ASPP2 is transcribed from a region the region -200 bases to +200 bases of exon 17 of human ASPP2. According to the invention, the antisense oligonucleotide is exon-including.

[0015] As also mentioned above, disclosed in the present invention are antisense oligonucleotides comprising a targeting sequence with at least 90% sequence complementarity to a target nucleic acid sequence within a region of a pre-mRNA of human ASPP2, wherein the region has the SEQ ID No. 16, wherein the antisense oligonucleotide is exon-including.

[0016] "Targeting sequence", within the present invention, means a sequence that targets a target sequence as defined herein.

[0017] "Exon-including", within the present invention and in relation to an anti- sense-oligonucleotide, means that the antisense-oligonucleotide has or comprises a sequence that promotes the inclusion of exon 17 during the splicing of ASPP2 pre-mRNA. Inother words, "Exon-including" refers to a splicing outcome in which a targeted exon is retained or included in the mature mRNA transcript, as opposed to being skipped or excluded. In the context of antisense oligonucleotides (AONs), the term denotes a strategy where the AON is designed to promote the inclusion of a specific exon during pre-mRNA splicing.

[0018] Within the present invention, the expression "-200 bases to +200 bases of exon 17" means the 200 bases upstream of the sequence of exon 17 plus the sequence of exon 17, plus the 200 bases downstream of exon 18 of human ASPP2. In other words, the notation “-200 bp to +200 bp” describes a 400 base pair (bp) region flanking a exon17 as genomic reference point, wherein specifically “-200 bp” indicates the sequence region extending 200 nucleotides upstream (i.e., 5’ direction) from exon 17, and “+200 bp” indicates the sequence region extending 200 nucleotides downstream (i.e., 3’ direction) from exon 17

[0019] Generally, Apoptosis-stimulating of p53 proteins (ASPP) are a family of proteins, which were originally described as direct interactors with the tumorsuppressor p53. These proteins share an evolutionary conserved C-terminus including four-ankyrin repeats, a SH3-domain and a poly-proline-rich domain, which directly interacts with the p53 core domain.

[0020] ASPP2 is a haploinsufficient tumor suppressor gene and was originally described as a direct binding partner of p53 by enhancing the DNA binding and transactivation function of p53 at the promoters of proapoptotic genes. More recently, however, ASPP2 has been ascribed a more central role in the control of cellular homeostasis through direct interaction with other binding partners such as BCL-2 and TN Fa. Tumor Protein P53 Binding Protein 2 (alias ASPP2, BBP, 53BP2, P53BP2, PPP1 R13A, tumor protein p53-binding protein, 2 ) is a protein that in humans is encoded by the TP53BP2 Gene (NCBI Genbank GenelD 7159).

[0021] Multiple transcript variants encoding different isoforms have been found for this gene. ASPP2 plays a central role in regulation of apoptosis and cell growth via itsinteractions. ASPP2 regulates p53 by enhancing the DNA binding and transactivation function of p53 on the promoters of proapoptotic genes in vivo. While ASPP2 binds to wild-type p53, it fails to bind to mutant p53.

[0022] More generally, the ASPP family comprises so far three members, two pro-apoptotic (ASPP1 , ASPP2) and one inhibitory (iASPP). ASPP proteins regulate apoptosis through interacting with p53 either by enhancing its functions on the promoters of proapoptotic genes (ASPP1 , ASPP2), or by inhibiting the apoptotic response (iASPP) through antagonizing the ASPP2-p53 interaction.

[0023] Both ASPP1 and ASPP2 have tumor-suppressing character and are dysregulated in many cancers.

[0024] Proceeding from the ASPP2 (TP53BP2) gene, which comprises 19 exons altogether, various transcripts are produced by alternative splicing. The database (GenBank) of the National Center for Biotechnology Information (NCBI) stores two transcripts of the ASPP2 gene, ASPP2 transcript variant 1 (database No. NM_001031685) and ASPP2 transcript variant 2 (database No. NM_005426). The numbering of the exons corresponds hereinbelow to the numbering according to the abovementioned GenBank entries with respect to ASPP2 transcript variant 2.

[0025] ASPP2 transcript variant 2 comprises all the exons, 1 to 19, of the ASPP2 gene and has a length of 3405 bases (according to the NCBI Consensus CDS [CCDS] Project, CCDS ID: CCDS44319.1). In contrast, in the case of ASPP2 transcript variant 1, exon 3 is removed by splicing, producing a shortened sequence, with respect to transcript variant 2, of 3018 bases altogether (CCDS ID: CCDS1538.1).

[0026] While exon 3 is removed by splicing in case of ASPP2 transcript variant 1, the numbering of the (remaining) exons of this variant remains the same as in variant 2 (which comprises all exons 1 to 19), this means that in variant 1, the exons are numbered 1, 2, 4, 5, 19, thus, simply skipping number “3” in the numbering of the exons.

[0027] Accordingly, as generally and as presently, i.e. in this invention, understood, the numbering and expression "Exon 17", in both transcript variants 1 and 2, designate the same sequence.

[0028] Thus, within the present invention, the referenced "exon 17" formally corresponds to isoform 2 and would be exon 16 in isoform I .Both transcripts encode isoforms of the ASPP2 protein: transcript variant 1 for isoform 1 and transcript variant 2 for isoform 2.

[0029] ASPP2 isoform 1 (database No. NP_001026855) is, at 1134 amino acids (aa), the larger of the two proteins. ASPP2 isoform 2 (database No. NP_005417) is, despite the integration of exon 3 into transcript variant 2, an N-terminally truncated isoform (having an alternative start codon situated further C-terminally compared to isoform 1) of 1005 aa in length, which, however, is otherwise identical in sequence to isoform 1.

[0030] The C-terminal region contains the binding domains required for binding to known binding partners, for example p53, NFkB or bcl-2. Both known isoforms are, fundamentally, expressed in both healthy tissue and tumor tissue.

[0031] ASPP2 is frequently undergoing alternative splicing, producing multiple splice variants with varying function. Alternative splicing occurs by rearranging the pattern of intron and exon elements to alter the mRNA coding sequence, a process that enables the mRNA to direct the synthesis of different protein variants (isoforms). In this regard, and as generally understood, while the primary form of most eukaryotic genes is the pre- mRNA state, which comprises a conglomerate of alternating exons (protein coding segments) and introns, the mature mRNA, i.e., the mRNA which encodes the necessary information for the transcriptional product, is created during the splicing process. In this connection, while the term "constitutive splicing" is used to describe the normal exon ligation in the order they appear in a gene, "alternative splicing" occurs when a deviation from the preferred sequence occurs and a new product is being sewed together resulting in various forms of mature mRNA. Through alternative splicing, one single gene can create mRNAvariants for multiple proteins. As an effect, the alternative mRNA isoforms can have distinct properties but also translate into functional proteins with divergent functions.

[0032] Also known is an oncogenic stress-inducible dominant-negative alternative splicing variant, which is truncated at its C-terminus, designated as ASPP2K. ASPP2 isoform K has lost the ability to induce apoptosis via p53 following cell stress / damage.

[0033] The splicing variant ASPP2K arises by missplicing with the omission of the whole of exon 17. Owing to said missplicing, transcription of exon 18 follows directly after exon 16. This results in a reading frame shift with a distinctly shortened transcript having a sequence characteristic of this variant. Said reading frame shift, in turn, causes ASPP2K to have a modified C-terminus with respect to the ASPP2 wild type. In keeping with the N-terminally differentially encoded transcription variants ASPP2 isoform 1 and isoform 2, corresponding isoforms for ASPP2K (ASPP2K isoform 1 and ASPP2K isoform 2) are also to be found.

[0034] With the formation of the ASPP2 transcript variant K as part of an alternative splicing process, the formation of the functionally intact ASPP2 isoforms 1 and 2 is completely or at least largely lost in the tumor cells, and this is associated with missing or at least greatly reduced activity of the ASPP2 protein in tumor cells. In other words, in the tumor cells, there is no apoptosis induction, or only inadequate apoptosis induction, via the p53 signal transduction pathway. Apoptosis induction, however, is an important basis for the (a) maintenance of cell and tissue integrity following cell stress, and (b) for the control of cancer by means of chemotherapeutics.

[0035] ASPP2K can thus be understood to be a precancerous condition or early aberration in the context of tumorigenesis. Moreover, in the case of ASPP2K-positive cancers, increased therapy refractoriness has been demonstrated.

[0036] Within the present invention, the inventors have targeted the splicing process by switching the alternative splicing process through the splice switching, exon-including antisense oligonucleotides as presently claimed, which are useful not only in diagnostic or prognostic applications, but also in therapeutic appliances.

[0037] Splice switching, exon-including antisense oligonucleotides (also abbreviated as "AONs"), generally, and as presently understood, are small synthetic molecules comprising of nucleotides or nucleotide analogues that bind complementary to RNA through base-pairing. They are typically 15-40 nucleotides long and have a higher gua- nine / cytosine ratio to increase their stability. The antisense oligonucleotides, or rather their sequences, can be chemically modified, e.g., to escape RNA-cleaving from RNAse H, or to generally increase their stability. Antisense oligonucleotides are used in therapies to correct pre-mRNA splicing.

[0038] Presently, and as generally understood, AONs are, thus, defined as short sequences of single-stranded (or double-stranded) or RNA (or DNA) or chemically modified versions thereof. This definition also encompasses virally delivered small nuclear RNAs (snRNAs) and AONs encoded by plasmids. Depending on their chemistry, AONs can either sterically block (e.g. to modulate splicing) or cause degradation of the target mRNA through RNase H activity.

[0039] Presently and as generally understood, the expression "exon-including" antisense oligonucleotide means a type of synthetic nucleic acid designed to modify the splicing of pre-messenger RNA (pre-mRNA) during the process of gene expression. Unlike other antisense oligonucleotides that typically aim to block mRNA translation or alter splicing to exclude certain exons, exon-including AONs are specifically engineered to promote the inclusion of exons in the mature mRNA transcript. These oligonucleotides function to bind to specific sequences in the pre-mRNA that are involved in splicing regulation, and by binding to their target sequences, exon-including AONs can change the splicing machinery's behavior, leading to the inclusion of an exon that might otherwise be skipped in the mature mRNA. The inclusion of an exon can restore the reading frame of the mRNA or add important functional domains to the encoded protein, which is particularly crucial in cases where genetic mutations cause disease by disrupting these elements. Throughout this invention, whenever it is referred to an antisense oligonucleotide of the invention, an exon-including antisense oligonucleotide is meant, unless otherwise defined.

[0040] It is noted that the feature “exon-including” is a mandatory feature of the antisense oligonucleotide of the invention, since according to the invention, the inclusion of exon 17 in the mRNA is crucial in order to generate wild-type ASPP2. Accordingly, antisense oligonucleotides not having this property, are not covered by the invention.

[0041] Specifically, the inventors of the present invention have found that the exon-including antisense oligonucleotides of the invention (i.e., comprising a targeting sequence with at least 90% sequence complementarity to a target nucleic acid sequence within a region of a pre-mRNA of human ASPP2, wherein the region of pre-mRNA of human ASPP2 is transcribed from a region the region -200 bases to +200 bases of exon 17 of human ASPP2) can efficiently block ASPP2K isoform-specific cis-active regulatory splicing sequences in the ASPP2 pre-mRNA, thereby shifting the reading-frame towards the wild-type isoform.

[0042] In the context of the present invention, and as generally understood, the term "antisense oligonucleotide(s)" refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), and may be used to refer to unmodified oligonucleotides or oligonucleotide analogs. The term "unmodified oligonucleotide" refers generally to oligonucleotides composed of naturally occurring nucleobases, sugars, and covalent internucleoside linkages. The term "oligonucleotide analog" refers to oligonucleotides that have one or more non-naturally occurring nucleobases, sugars, and / or internucleoside linkages. Such non-naturally occurring oligonucleotides are often selected over naturally occurring forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for other oligonucleotides or nucleic acid targets, increased stability in the presence of nucleases, or increased inhibitory activity.

[0043] In an embodiment, morpholinos can be used as “antisense oligonucleotide”, which comprise a backbone made of morpholine rings, which are different from the ribose or deoxyribose sugars found in DNA and RNA, which rings are linked by non-ionic phosphorodiamidate groups instead of the negatively charged phosphodiester linkages in natural nucleic acids. Attached to these backbones are the nucleic acid bases according to the invention that allow the morpholinos to hybridize with complementary RNA sequences as mentioned herein.

[0044] The exon-including antisense oligonucleotides / oligomers are capable of hybridizing to at least a target region of a pre-RNA molecule. Generally, an oligonucleotide is "antisense" to a target nucleic acid when, written in the 5' to 3' direction, it comprises the reverse complement of the corresponding region of the target nucleic acid. Such oligonucleotides are known as "antisense compounds", which include, without limitation, oligonucleotides (i.e. , antisense oligonucleotides), oligonucleosides, oligonucleotide analogs, oligonucleotide mimetics and combinations of these. In general, an antisense oligonucleotide comprises a backbone of linked monomeric subunits (sugar moieties) where each linked monomeric subunit is directly or indirectly attached to a heterocyclic base moiety. Modifications to antisense compounds / oligonucleotide encompass substitutions or changes to internucleoside linkages, sugar moieties, or heterocyclic base moieties, such as those described below. As used herein, the term "modification" includes substitution and / or any change from a starting or natural nucleoside or nucleotide. Modified antisense compounds are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for nucleic acid target, increased stability in the presence of nucleases, or increased inhibitory activity.

[0045] Exon-including antisense oligonucleotides / compounds are routinely prepared linearly but can be joined or otherwise prepared to be circular and may also include branching. Separate antisense compounds can hybridize to form double stranded compounds that can be blunt-ended or may include overhangs on one or both termini.

[0046] The exon-including antisense oligonucleotides / compounds in accordance with the present invention comprise from 15 to 40 nucleosides in length, preferably from 20 to 40 in length, i.e., from 15 to 40 linked nucleosides. One of skill in the art will appreciate that this embodies antisense compounds of 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 nucleosides in length.

[0047] In one embodiment, the exon-including antisense oligonucleotides of the invention are 19 to 25 nucleosides in length, as exemplified herein, or from 19 to 23.

[0048] In preferred embodiments, the exon-including antisense oligonucleotides of the invention are 19, 20, 21, 22 or 23 nucleosides in length.

[0049] "Complementary," or “sequence complementarity” as used herein, refers to the capacity for hybridization between nucleobases. An exon-including antisense oligonucleotide (comprising or consisting of the targeting sequence) and the target nucleic acid are "fully complementary" to each other when each nucleobase of the antisense compound is complementary to an equal number of nucleobases in the target nucleic acid. For example, an antisense oligonucleotide 23 nucleosides in length targeted to a sequence of -200 bases to +200 bases of exon 17 of a pre-m RNA of human ASPP2 is fully complementary to that sequence within -200 bases to +200 bases of exon 17 of a pre-m RNA of human ASPP2 when each of the 23 nucleobases in the antisense oligonucleotide is complementary to the nucleic acid sequence within -200 bases to +200 bases of exon 17 of a pre-mRNA of human ASPP2. The antisense oligonucleotide and the target nucleic acid sequence are "essentially fully complementary" to each other when the degree of precise base pairing permits stable and specific binding between the antisense oligonucleotide and a target nucleic acid sequence, so that the antisense oligonucleotide inhibits or blocks or lowers the regulatory splicing sequences for ASPPSK, thus shifting the reading-frame towards the wild-type isoform, as mentioned above.

[0050] In the context of this invention, and as generally understood, "hybridization" means the pairing of nucleobases of an antisense oligonucleotide with corresponding nucleobases in a target nucleic acid. In the context of the present invention, the mechanism of pairing involves hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between corresponding nucleobases. For example, adenine and thymine are complementary nucleobases that pair through the formation of hydrogen bonds. Both natural and modified nucleobases are capable of participating in hydrogen bonding. Hybridization can occur under varying circumstances.

[0051] It is understood in the art that the sequence of the antisense oligonucleotide need not be fully complementary to that of its target nucleic acid to be active in blocking the target nucleic acid. In some embodiments there are "non-complementary" po-sitions, also known as "mismatches", between the antisense oligonucleotide and the target nucleic acid, and such non-complementary positions may be tolerated between an antisense oligonucleotide and the target nucleic acid provided that the antisense oligonucleotide remains specifically hybridizable to the target nucleic acid. A "non-complementary nucleobase" means a nucleobase of an antisense oligonucleotide that is unable to undergo precise base pairing with a nucleobase at a corresponding position in a target nucleic acid. As used herein, the terms "non-complementary" and "mismatch" are interchangable. Up to 3 non-complementary nucleobases are often tolerated in an antisense oligonucleotide without causing a significant decrease in the ability of the antisense oligonucleotide to modulate the activity, level or function of a target nucleic acid.

[0052] In preferred embodiments, the exon-including antisense oligonucleotide contains no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1, non-complementary nucleobases with respect to a ASPP2 pre- mRNA target nucleic acid. For example, an antisense oligonucleotide 23 nucleosides in length, 22 of which are able to undergo precise base pairing with nucleobases in corresponding positions in a target nucleic acid, and one of which is not able to undergo such base pairing, is considered to have one non-complementary nucleobase. The location of such a non-complementary nucleobase at the 5' end or 3' end of the antisense oligonucleotide is preferred, however, the non-complementary nucleobase may be at any position in the antisense oligonucleotide. When two or more non-complementary nucleobases are present, they may be contiguous (i.e. linked) or non-contiguous.

[0053] According to the invention, the exon-including antisense oligonucleotide comprises a targeting sequence with at least 90% sequence complementarity to a target nucleic acid sequence within a region of a pre-mRNA of human ASPP2, wherein the region of pre-mRNA of human ASPP2 is transcribed from a region the region -200 bases to +200 bases of exon 17 of human ASPP2.

[0054] In further embodiments of the invention, the exon-including antisense oligonucleotide comprises at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence complementarity to a pre-mRNA of human ASPP2 target nucleic acid, which has been transcribed from theregion within -200 bases to +200 bases of exon 17. For example, an antisense oligonucleotide in which 22 of 23 nucleobases of the antisense oligonucleotide are complementary (i.e. , one nucleobase is non-complementary) to a pre-mRNA of human ASPP2 target nucleic acid as disclosed would represent 95.6 % complementarity. Likewise, an antisense compound in which 21 of the 23 nucleobases are complementary (i.e. two nucleobases are non-complementary) to a pre-mRNA of human ASPP2 target nucleic acid as disclosed would represent 91.3 % complementarity. Percent complementarity of an antisense oligonucleotide with a region of a target nucleic acid can be determined routinely by those having ordinary skill in the art, and may be accomplished, e.g., using BLAST programs (basic local alignment search tools) and PowerBLAST programs known in the art ( Altschul et al., J. Mol. Biol., 1990, 215, 403-410 ; Zhang and Madden, Genome Res., 1997, 7, 649-656 ).

[0055] In the present invention the phrase "stringent hybridization conditions" or "stringent conditions" refers to conditions under which an antisense oligonucleotide of the invention will hybridize to its target nucleic acid sequence, but to a minimal number of other sequences. Stringent conditions are sequence-dependent and will vary with different circumstances and in the context of this invention; "stringent conditions" under which antisense oligonucleotides hybridize to a target sequence are determined by the nature and composition of the exon-including antisense oligonucleotides and the assays in which they are being investigated. One having ordinary skill in the art will understand variability in the experimental protocols and be able to determine when conditions are optimal for stringent hybridization with minimal non-specific hybridization events.

[0056] Exon-including antisense oligonucleotides may have a defined percent identity to target sequences within the RNA sequence that corresponds to SEQ ID No. 1 (representing the coding strand of the region -200 bases to +200 bases of exon 17). This identity may be over the entire length of the antisense oligonucleotide, or over less than the entire length of the antisense oligonucleotide. It is understood by those skilled in the art that an antisense oligonucleotide need not have an identical sequence to those described herein to function similarly to the antisense oligonucleotide specifically described herein. Shortened or truncated versions of antisense oligonucleotide taught herein have one, two or more nucleosides deleted, and fall within the scope of the invention. When an antisense oligonucleotide has two or more deleted nucleosides, the deleted nucleosidesmay be adjacent to each other, for example, in an antisense oligonucleotide having two nucleosides truncated from the 5' end (5' truncation), or alternatively from the 3' end (3' truncation), of the antisense oligonucleotide. Alternatively, the deleted nucleosides may be dispersed throughout the antisense, for example, in an antisense oligonucleotide having one nucleoside deleted from the 5' end and one nucleoside deleted from the 3' end.

[0057] Also falling within the scope of the invention are lengthened versions of exon-including antisense oligonucleotides taught herein, i.e. , antisense oligonucleotides having one or more additional nucleosides relative to an antisense oligonucleotide disclosed herein. When two are more additional nucleosides are present, the added nucleosides may be adjacent to each other, for example, in an antisense oligonucleotide having two nucleosides added to the 5' end (5' addition), or alternatively to the 3' end (3' addition), of the antisense oligonucleotide. Alternatively, the added nucleosides may be dispersed throughout the antisense oligonucleotide, for example, in an antisense oligonucleotide having one nucleoside added to the 5' end and one nucleoside added to the 3' end.

[0058] "Targeting" an antisense oligonucleotide to a particular nucleic acid molecule, including a pre-mRNA of human ASPP2 target nucleic acid within the region -200 bases to +200 bases of exon 17, in the context of this invention, can be a multistep process. The process usually begins with the identification of a target nucleic acid whose levels, expression or function is to be modulated.

[0059] In the context of the present invention, the target nucleic acid is a pre- mRNA sequence of human ASPP2 within the region -200 bases to +200 bases of exon 17. It is to be understood that the targeting sequence of the antisense oligonucleotide of the invention, thus, is also complementary (with the change of thymine with uracil) to a DNA sequence (the coding strand) within the DNA region -200 bases to +200 bases of exon 17. “-200 bases to +200 bases of exon 17”, in this connection, shall mean and encompass the 200 bases upstream (-200) and the 200 bases downstream (+200) of the sequences of exon 17; while the sequence of exon 17 is represented in SEQ ID No. 3, the region +200 to -200 bases of exon 17, including exon 17, is represented in SEQ ID No. 1.

[0060] In the attached Fig. 1 , the DNA sequence (double-stranded) of this region, i.e. the region of about -200 base pairs (bases) to +200 bases of exon 17 is shown, with the sequences of exon 17 being depicted in bold letters. It is to be understood that, as commonly understood, the upper strand (SEQ ID No. 1) represents the coding (or informational) strand, and the lower strand (SEQ ID No. 2) represents the non-coding (or antisense) strand. SEQ ID No. 3 represents the DNA sequence (coding strand) of exon 17. The RNA formed from the DNA is complementary to the lower, i.e., non-coding strand; consequently, the RNA sequence is - apart from the replacement of thymine with uracil - identical with the upper strand of the DNA, i.e., with the coding strand. During transcription, the sequence of the non-coding strand or template strand is transcribed to synthesize an RNA transcript with complementary bases. By convention, the coding strand is the strand used when displaying a DNA sequence, and is presented in the 5’ to 3’ direction.

[0061] Accordingly, the exon-including antisense oligonucleotide of the invention, that comprises a targeting sequence with at least 90% sequence complementarity to a target nucleic acid sequence within the region -200 bases to +200 bases of exon 17 of a pre-mRNA of human ASPP2, has a sequence that is complementary - or antisense - to the pre-mRNA.

[0062] For purposes of understanding, it is noted that the sequence of the pre- mRNA of ASPP2 and the sequences of the splice-variant ASPP2k are identical; ASPP2k is generated from missplicing of the pre-mRNA.

[0063] The targeting process usually also includes determination of at least one target segment sequence within the region of -200 bases to +200 bases of exon 17 of a pre-mRNA of human ASPP2 for the interaction to occur such that the desired effect, e.g., modulation of levels, activity, or expression, will result. As used herein, a "target sequence" means a sequence within the region of -200 bases to +200 bases of exon 17 of a pre-mRNA of human ASPP2 to which one or more antisense oligonucleotides are complementary. Multiple antisense oligonucleotides complementary to a given target segment sequence may or may not have overlapping sequences. For example, the sequence of SEQ ID NO: 11 (designated with 340-362) is complementary to target sequence 140 to 162 (SEQ ID No. 5) of exon 17 (SEQ ID NO: 3), and the sequence of SEQ ID NO: 12 iscomplementary to target sequence 142 to 164 (SEQ ID No. 6) of exon 17 (SEQ ID NO. 3). Nucleosides 140 to 162 (SEQ ID No. 5) thus represent a target segment of SEQ ID NO. 1.

[0064] Exon-including antisense oligonucleotides of the invention may be also be described as complementary to a portion of a target nucleic acid sequence. A "portion" is defined as at least 18 contiguous nucleosides of a target sequence. In other embodiments, a portion is 19 or 20 contiguous nucleosides of a target site. In preferred embodiments, a portion is 21 , 22, or 23 contiguous nucleosides of a target sequence.

[0065] Once one or more target sequences have been identified, exon-includ- ing antisense oligonucleotides are designed to be sufficiently complementary to the target sequences, i.e. , hybridize sufficiently well and with sufficient specificity, to give the desired effect, i.e., the blocking of ASPP2K isoform-specific cis-active regulatory splicing sequences, and the shifting of the reading-frame towards the wild-type isoform.

[0066] The exon-including antisense oligonucleotides of the invention may be in the form of single-stranded, double-stranded, circular or hairpin antisense compounds and may contain structural elements such as internal or terminal bulges or loops.

[0067] Once introduced to a system, the exon-including antisense oligonucleotides of the invention may block or inhibit the splicing towards the splicing variant ASPP2 , thus shifting the reading frame towards the wild-type isoform. While the variant ASPP2 , as discussed above, has lost the ability to induce apoptosis via p53 following cell stress / damage, the wild-type isoform performs its central role in apoptosis.

[0068] Thus, in another aspect of the invention, the exon-including antisense oligonucleotide of the invention is for use in the treatment and / or prevention of conditions and / or diseases related to ASPP2K-variant expression, in particular / preferably of diseases or conditions, in which the induction of apoptosis is disturbed, reduced or inhibited.

[0069] A disease or condition related to ASPP2K-variant expression, is a disease or condition in which due to the expression of the ASPP2K-variant, proliferative signaling issustained, growth suppressors are evaded, cell death is resisted, replicative immortality is enabled, vasculature is induced and / or accessed, invasion and metastasis is activated, cellular metabolism is reprogrammed, and / or and immune destruction is avoided; according to one aspect, a disease or condition related to ASPP2K-variant is a disease or condition in which due to the expression of the ASPP2K-variant the p53 protein cannot be activated anymore, thereby preventing induction of apoptosis.

[0070] According to one aspect of the invention, at least one, preferably at least two, at least three or more exon-including antisense oligonucleotides are used in combination with one another / simultaneously according to the invention. The combined use of the exon-including antisense oligonucleotides of the invention can lead to an improved or more efficient expression of wild-type ASPP2.

[0071] As discussed above, with the ability of the exon-including antisense oligonucleotides of the invention, to efficiently block the splicing of the ASPP2 pre-mRNA into ASPP2K, and increase the level of the wild-type ASPP2 isoforms, fully functional ASPP2 proteins are generated, e.g., in a target cell to be treated with the antisense oligonucleotide; by the direct binding of p53, ASPP2 triggers or intensifies the induction of programmed cell-death mechanisms, i.e. , apoptosis, following cell stress, and / or in tumors. Also, by administering the antisense oligonucleotides of the invention to persons or human patients that have been identified to express the variant ASPP2K, the increased tumor incidence for these patients can be lowered or even abolished.

[0072] Accordingly, the exon-including antisense oligonucleotide of the invention can be used for treating diseases or conditions such as cancer or cell-stress-induced inflammation. As presented herein, the cancer can be selected from hematological neoplasms, solid tumors, precancerous conditions, and in particular acute or chronic myeloid or acute lymphoblastic leukemia.

[0073] The aforementioned diseases or conditions have been shown to be related to the expression of the ASPP2K variant, in particular in both, in hematologic malignancies and solid tumors (see, e.g., for leukemia and lymphoma: Schittenhelm, M.M., etal., “Alternative splicing of the tumor suppressor ASPP2 results in a stress-inducible, oncogenic isoform prevalent in acute leukemia”. EBioMedicine, 2019. 42: p. 340-351; for Colon: Rieger, I., et al., “ASPP2kappa Is Expressed In Human Colorectal Carcinoma And Promotes Chemotherapy Resistance And Tumorigenesis”. Front Mol Biosci, 2021. 8: p. 727203; for sarcoma: Tsintari, V., et al., “Alternative splicing of Apoptosis Stimulating Protein of TP53-2 (ASPP2) results in an oncogenic isoform promoting migration and therapy resistance in soft tissue sarcoma (STS)”. BMC Cancer, 2022. 22(1): p. 725; for breast tumors: Kampa-Schittenhelm K et al., DGHO-Jahrestagung 2021; Plenary Session, Abstract V686; for glioma: Marlon Hafner et al. AACR Annual Meeting, Cancer Res (2023) 83 (7_Supplement): 2535), and ASPP2K has been used as a respective marker for these diseases. By blocking the expression of ASPP2K, these conditions / diseases can be treated or prevented.

[0074] As already discussed above, in a preferred embodiment of the exon-in- cluding antisense oligonucleotide, the region -200 bases to +200 bases of exon 17 of a human ASPP2 is transcribed from SEQ ID No. 1. This sequence corresponds to a region spanning the 200 bases upstream and downstream of the exon 17 sequence, including exon 17.

[0075] According to an embodiment of the invention, and as discussed above, the exon-including antisense oligonucleotide is single stranded, preferably a modified or an unmodified RNA or DNA oligonucleotide.

[0076] In a preferred embodiment, the exon-including antisense oligonucleotide is essentially fully complementary to a ASPP2 target nucleic acid. Alternatively, the antisense compound is fully complementary to a pre-mRNA ASPP2 target nucleic acid. The antisense oligonucleotide can comprise a plurality of sugar modified nucleosides. The plurality of sugar modified nucleosides may, in some embodiments, comprise 1, 2, 3, 4, 5, 6, 7, 8, or more modifications. In some embodiments, each nucleoside of the AON can be modified. In some embodiments, the antisense oligonucleotide does not comprise modified nucleosides.

[0077] The exon-including antisense oligonucleotides of the invention may also contain one or more modified or substituted sugar moieties. The base moieties (natural, modified or a combination thereof) are maintained for hybridization with an appropriate nucleic acid target. Sugar modifications may impart nuclease stability, binding affinity or some other beneficial biological property to the antisense compounds. Representative modified sugars include carbocyclic or acyclic sugars, sugars having substituent groups at one or more of their 2', 3' or 4' positions, sugars having substituents in place of one or more hydrogen atoms of the sugar, and sugars having a linkage between any two other atoms in the sugar. Antisense compounds of particular use in the instant invention may comprise a sugar substituent group selected from: OH; halo; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted Ci to Cw alkyl or C2to Cw alkenyl and alkynyl. Particularly suitable are O((CH2)nO)mCH3, O(CH2)nOCH3, O(CH2)nNH2, O(CH2)nCH3, O(CH2)nNH2, and O(CH2)nON((CH2)nCH3)2, where n and m are from zero to about 10. Some oligonucleotides comprise a sugar substituent group selected from: Ci to C lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, F, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocyclo- alkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties.

[0078] One modification that imparts increased nuclease resistance and a very high binding affinity to nucleosides is the 2'-methoxyethoxy (2'-MOE or 2'-OCH2CH2OCH3) side chain (Baker et a / ., J. Biol. Chem., 1997, 272,11944-12000).

[0079] Additional modifications include 2'-dimethylaminooxyethoxy, i.e. , a O(CH2)2ON(CH3)2-group, also known as 2'-DMAOE, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylamino-ethoxy-ethyl or 2 -DMAEOE), i.e., 2'-O-CH2- O-CH2-N(CH3)2.

[0080] Specifically, in some embodiments, the exon-including antisense oligonucleotide comprises modified nucleotides selected from O-methyl RNA nucleotides,phosphorothioate 2'0-methyl RNA nucleotides, phosphorothioate DNA nucleotides, locked-nucleic-acid nucleotides and natural RNA nucleotides.

[0081] In some embodiments, each nucleoside of the plurality of sugar modified nucleosides may comprise a 2'-MOE sugar modification. The antisense oligonucleotide may comprise at least one phosphorothioate internucleoside linkage. The antisense compound may further comprise a least one 5-methylcytosine.

[0082] In one embodiment of the exon-including antisense oligonucleotide of the invention, the 1, 2, 3, 4, and / or 5 or more terminal nucleosides are modified, i.e. , at either or both of the 3’ or 5’- terminal.

[0083] Preferably, in one embodiment, 3, 4 or 5 consecutive nucleosides at the 5’ terminal end and 5 consecutive nucleosides at the 3’ terminal end are modified according to the invention. In a preferred embodiment, the 5’ and 3’ terminal nucleosides, which can be 2, 3, 4, or 5 in number, represent phosphorothioated 2’-O-methoy RNA, or locked nuclei acids being phosphorothioated DNA bases.

[0084] In addition, also the nucleosides flanked by the modified terminal nucleosides can be modified, while the modification can be identical to the ones of the terminal nucleosides or different. In a preferred embodiment, at least one of the nucleosides flanked by the modified terminal nucleosides is a phosphorothioated DNA base. In a preferred embodiment, each of the nucleosides flanked by the (eventually) modified terminal nucleosides is a phosphorothioated DNA base.

[0085] In a preferred embodiment, the exon-including antisense oligonucleotides of the invention are 15 to 40 nucleosides in length. In another preferred embodiment, the exon-including antisense oligonucleotides of the invention are from 20 to 24 nucleotides in length, preferably from 21 to 23 nucleotides in length.

[0086] Further, according to another aspect of the invention of the exon-includ- ing antisense oligonucleotide, in the targeting sequence, the proportion of guanine and cytosine nucleic bases is, either naturally of artificially modified, at least 40% and at most 60% of all nucleic bases.

[0087] A higher G / C content in the antisense oligonucleotide of the invention, e.g., an increased thermal stability can be achieved, since guanine and cytosine pairs form three hydrogen bonds between them, compared to two in adenine-thymine pairs. This higher number of bonds contributes to a greater thermal stability of the nucleic acid duplexes. Therefore, ANOs with higher G / C content will have a higher melting temperature (Tm), meaning they can remain hybridized to their target RNA at higher temperatures, enhancing their stability under physiological conditions. Further, with a higher G / C content, the binding affinity can be improved, since the stronger interactions between G and C bases result in a higher affinity for the target nucleic acid sequence. This can lead to more effective binding and consequently, more efficient inhibition or alteration of the target mRNA function.

[0088] Also, nucleoside sequences rich in G and C are generally more resistant to enzymatic degradation by nucleases. This resistance can enhance the longevity and effectiveness of ANOs in biological environments, such as within cells, where nuclease activity is prevalent. Further, the increased stability and binding affinity can also influence the pharmacokinetic properties of antisense oligonucleotides, such as their distribution, metabolism, and excretion, potentially leading to better therapeutic outcomes.

[0089] As a consequence, preferably target sequences in the region of -200to +200 bases of exon 17 can be selected for generating antisense oligonucleotides according to the invention, which have a G / C content of at least 40%, 50% or 60%.

[0090] In one aspect of the invention, the exon-including antisense oligonucleotide has a sequence that is selected from the group consisting of SEQ ID No. 11 (“340- 362’), SEQ ID No. 12 (“342-364"), and SEQ ID No. 14 (“471-492’).

[0091] As can be seen from Fig. 2 depicting the target sequences and the targeting sequences of the above mentioned specific AONs, the AON having SEQ ID No. 11 has 5 modified nucleosides at each of its termini, i.e., 5 modified nucleosides at its 5’ terminus and 5 modified nucleosides at its 3’ terminus, wherein each of the modified nucleosides represents modified hosphorothioated 2’-O-methyl RNA. The remaining nucleosides flanked by the 5’ and 3’ terminal modified nucleosides represent phosphorothioated DNA bases.

[0092] In the AON having the SEQ ID No. 12, 4 of the 5’ terminal nucleosides and of the 3’ terminal nucleosides represent locked nucleic acids that are phosphorothioated DNA bases, while the remaining nucleosides flanked by the 5’ and 3’ terminal modified nucleosides represent phosphorothioated DNA bases.

[0093] In the AON having the SEQ ID No. 14, 3 of the 5’ terminal nucleosides and of the 3’ terminal nucleosides represent locked nucleic acids that are phosphorothioated DNA bases, while the remaining nucleosides flanked by the 5’ and 3’ terminal modified nucleosides represent phosphorothioated DNA bases.

[0094] The inventors of the present invention have found that with the three exon-including antisense oligonucleotides as specified above the ASPP2k junction site, i.e., the cis-active regulatory splicing sequence towards ASPP2k, can be blocked, while simultaneously increasing the expression of exon 17. Via the increased expression of exon 17, the wild-type ASPP2 isoform is generated, which in turn can efficiently bind to tumor suppressor protein p53, thereby stimulating it to promote apoptosis.

[0095] According to another aspect, the invention also concerns a pharmaceutical composition comprising or consisting of a) at least one, or at least two or at least three or more exon-including antisense oligonucleotide of the invention and as outlined above, and b) a pharmaceutically acceptable carrier.

[0096] The exon-including antisense oligonucleotide of the invention can be formulated together with any suitable auxiliaries and excipients, which may be selected onthe intended use or administration. Accordingly, a pharmaceutical composition of the invention can, in addition to one or more exon-including antisense oligonucleotides, also comprise a range of excipients and / or auxiliaries which, for example, allow a longer shelf life or more effective administration. Such excipients and auxiliaries are known comprehensively from the prior art; cf. Row et al. (2006), Handbook of Pharmaceutical Excipients, 5th edition, Pharmaceutical Press. The content of the present publication is, by way of reference, part of the present application.

[0097] Such a pharmaceutical composition can, as explained above, be used for either the prevention or treatment of cancers and / or cell-stress-induced inflammation. The pharmaceutical composition can be used alone or in combination with additional therapies, to either support or even replace conventional treatment. In addition, a pharmaceutical composition according to the invention can also be used following conventional treatment for prevention against a recurrence of the cancer and / or cell-stress-induced inflammation conditions.

[0098] Presently, and as generally understood, the terms “cell-stress-induced inflammation” or “cell-stress-induced inflammation condition” refer to inflammatory responses triggered by cellular stress. This type of stress can occur due to a variety of factors including oxidative stress, heat shock, DNA damage, or other cellular injuries. Such stress leads to the activation of various cell signaling pathways, which promote the production of inflammatory cytokines and other mediators.

[0099] The p53 protein, as discussed above, is a critical tumor suppressor involved in various cellular processes including cell cycle regulation, apoptosis (programmed cell death), and DNA repair. It plays a significant role in maintaining cellular integrity in response to stress. When cellular stress occurs - such as DNA damage, hypoxia, or oncogenic stress - p53 becomes activated to prevent the propagation of damaged cells, which can lead to inflammation. Some exemplary conditions where cell stress- induced inflammation involves p53 are cancer, neurodegenerative diseases, ischemiareperfusion injury, chronic inflammatory diseases and atherosclerosis.

[0100] The link between dysfunctional p53 and inflammation is significant, particularly because p53 is supposed to act as a safeguard, preventing the proliferation of cells that have sustained damage. When this function is lost or impaired - e.g., due to expression of ASPP2K, which cannot regulate p53 - not only does it allow for the growth of potentially malignant cells, but it also affects the inflammatory pathways that are normally regulated by p53. This dysregulation can lead to a chronic inflammatory state, contributing to the progression of various diseases, as outlined above.

[0101] Also, it is noted that upon expression of ASPP2K (instead of ASPP2) the formation of proinflammatory factors, such as NF-KB (Nuclear Factor kappa-light-chain- enhancer of activated B cells; a transcription factor that plays a crucial role in regulating a wide range of cellular processes), is promoted. While ASPP2 binds NF-KB, thereby inhibiting its activation, ASPP2K lacks NF-KB binding sites, which can lead to the mentioned in- crease / activation of NF-KB, which in turn can lead to cancer development due to promoting cell proliferation and inhibiting apoptosis; also, since NF-kB is a major regulator of inflammation, an increase induces the expression of inflammatory mediators. As such, activation of NF-kB is associated with various inflammatory diseases, including rheumatoid arthritis, inflammatory bowel disease, and asthma; also, NF-kB-driven inflammation creates a tumor-promoting environment by producing cytokines and growth factors that support tumor growth and survival.

[0102] Similarly, wild-type ASPP2 binds to Bcl2 (b-cell lymphoma 2; an anti- apoptotic protein), thereby inhibiting the protein’s anti-apoptotic effects and promoting the death of, e.g., cancer cells, and promoting apoptosis in response to cellular stress or DNA damage. The Bcl2 binding sites are lost in the ASPP2K isoform leaving Bcl2 unbound to mediate anti-apoptotic functions.

[0103] With the invention as disclosed herein, the above issues can be addressed by shifting the reading-frame towards the wild-type isoform ASPPS2, which then can bind and restore NF-KB, resp. Bcl2 signaling.

[0104] More particularly, and preferably, and according to one aspect, a pharmaceutical composition according to the invention can be used to resensitize ASPP2K- positive cancer cells, i.e. , cells in which apoptosis induction via ASPP2 and p53 is disrupted, to apoptosis-inducing stimuli. These cells can be stimulated to undergo apoptosis applying the antisense oligonucleotide of the invention, and / or by means of tumor therapeutics such as, inter alia, chemotherapy and radiation therapy.

[0105] Accordingly, and according to an aspect of the invention, the pharmaceutical composition is for use with an apoptosis-inducing therapeutic agent and / or an apoptosis-inducing therapy.

[0106] Specifically, in a preferred embodiment, the antisense oligonucleotide is used in the pharmaceutical composition together with a pharmaceutically acceptable carrier, which can be a nanoparticle, preferably a lipid nanoparticle, on or in which the antisense oligonucleotide is adsorbed.

[0107] As such, also according to the invention, a modified exon-including antisense oligonucleotide can be used, either as such, or in a pharmaceutical composition. The antisense oligonucleotide of the invention can be linked to one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the resulting modified antisense oligonucleotide. In one embodiment such modified antisense oligonucleotides are prepared by covalently attaching conjugate groups to functional groups such as hydroxyl or amino groups. Conjugate groups of the invention include intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of the antisense oligonucleotides. Typical conjugate groups include cholesterol moieties and lipid moieties. Additional conjugate groups include carbohydrates, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes. Groups that enhance the pharmacodynamic properties, in the context of this invention, include groups that impart to the antisense oligonucleotide properties such as improved uptake, enhanced resistance todegradation, and / or enhance hybridization with RNA. Groups that enhance the pharmacokinetic properties, in the context of this invention, include groups impart to the antisense oligonucleotide properties such as improved uptake, distribution, metabolism or excretion.

[0108] Exon-including antisense oligonucleotides used as such or in the pharmaceutical compositions of the present invention can also be modified to have one or more stabilizing groups that are generally attached to one or both termini of the antisense oligonucleotide, e.g. cap structures. By "cap structure" or "terminal cap moiety" is meant chemical modifications, which have been incorporated at either terminus of an antisense oligonucleotide. These terminal modifications protect the antisense compounds having terminal nucleic acid molecules from exonuclease degradation, and can help in delivery and / or localization within a cell. The cap can be present at the 5'-terminus (5'-cap), or at the 3'-terminus (3'-cap), or can be present on both termini. For double-stranded antisense compounds, the cap may be present at either or both termini of either strand. Cap structures are well known in the art and include, for example, inverted deoxy abasic caps.

[0109] In some embodiments, an exon-including antisense oligonucleotide as such, or as present in a pharmaceutical composition, can be administered to a subject via, e.g., an oral route of administration, or intravenously, subcutaneously, intramuscular, etc. The subject may be a mammal, preferably a human. In certain embodiments, the subject may be in need of modulation of the level or expression of the ASPPSK variant.

[0110] In a preferred embodiment, in the pharmaceutical composition of the invention including at least one, or at least two or more apoptosis-inducing therapeutic agent (s), the at least one apoptosis-inducing therapeutic agent is a chemotherapeutic agent, preferably an anticancer drug.

[0111] In another preferred embodiment, when applying the pharmaceutical composition of the invention with at least one apoptosis-inducing therapy, the at least one apoptosis-inducing therapy is a radiotherapy or a targeted cancer therapy.

[0112] Combining a pharmaceutical composition with apoptosis-inducing therapies such as radiotherapy or targeted cancer therapy can leverage the natural process of programmed cell death (apoptosis) to effectively target and destroy cancer cells. Apart from the synergy, and enhancement, by combining therapies, it is also possible to overcome the resistance mechanisms, e.g., if a tumor becomes resistant to a targeted therapy alone. Also, with the combination it can be possible to reduce dosages of otherwise more harming therapies.

[0113] Consequently, the present invention also provides a method for treating a patient, wherein the method comprises the following steps: a) administering an exon-in- cluding antisense oligonucleotide or pharmaceutical composition according to the invention, and b) repeating steps a) if necessary.

[0114] The patient in need of the therapy may be identified by selecting the patient in view of an ASPP2K-marker. The advantage of such a method is that one of the molecular causes of cancer, viz. ASPP2 insufficiency and the thus reduced induction of apoptosis, can be counteracted in a specific manner.

[0115] The effects of antisense compounds have on the level of blocking ASPP2K expression and increasing ASPP2 wild-type expression, can first be tested in vitro in a variety of cell types. Cell types used for such analyses are available from com- merical vendors (e.g. American Type Culture Collection, Manassus, VA, Zen-Bio, Inc., Research Triangle Park, NC; Clonetics Corporation, Walkersville, MD) and cells are cultured according to the vendor's instructions using commercially available reagents (e.g. Invitro- gen Life Technologies, Carlsbad, CA).

[0116] In another aspect, the present invention also relates to splice-switching AONs, which induce exon skipping and, as a consequence, ASPP2K overexpression. The exon-skipping antisense oligonucleotides (AONs) of the invention modulate the splicing of ASPP2 pre-mRNA, leading to the exclusion (skipping) of certain exons during the formation of mature ASPP2 mRNA.

[0117] Accordingly, one aspect of the invention is directed to an antisense oligonucleotide comprising a targeting sequence with at least 90% sequence complementarity to a target nucleic acid sequence within a region of a pre-mRNA of human ASPP2, wherein the region of pre-mRNA of human ASPP2 is transcribed from a region the region -200 bases to +200 bases of exon 17 of human ASPP2, wherein the antisense oligonucleotide is exon-skipping.

[0118] In other words, the invention also concerns an antisense oligonucleotide comprising a targeting sequence with at least 90% sequence complementarity to a target nucleic acid sequence within a region of a pre-mRNA of human ASPP2, wherein the region has the SEQ ID No. 16, wherein the antisense oligonucleotide is exon-17-skipping.

[0119] According to an embodiment of this aspect of the invention, the nucleic acid molecule has between 10 to 50 nucleotides in length, preferably between 15 and 30, preferably between 19 and 24, complementary to a nucleotide sequence of the ASPP2 pre-mRNA, said nucleic acid being able to exclude exon 17 of ASPP2 pre-mRNA, wherein said nucleic acid molecule comprises a nucleotide sequence complementary to a sequence comprised in the region defined by positions - 200 bases to + 200 bases of exon 17 of the ASPP2 gene, preferably wherein the region has the SEQ ID No. 16 .

[0120] According to one aspect of the invention, the exon-skipping (or spliceshifting) AON of the invention targets the natural acceptor site of exon 17 until the first nt of exon 17 itself. In an embodiment of this aspect of the invention, the exon-skipping AON comprises or has a sequence as defined in SEQ ID no. 15.

[0121] Various modifications of the invention, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. Each reference (including, but not limited to, journal articles, patent application publications, GENBANK® accession numbers, and the like) cited in the present application is incorporated herein by reference in its entirety.BRIEF DESCRIPTION OF THE FIGURES

[0122] In the figures:Fig. 1 shows the -200 to +200 bp nucleic acid sequence region of exon 17 (ref to isoform 2) of human ASPP2, with the sequence of exon 17 in bold letters, with the coding strand / upper strand in Fig. 1 having SEQ ID NO. 1 , and the non-coding strand / lower strand in Fig. 1 having SEQ ID NO. 2 (A); and the isolated sequence of the coding strand of the exon 17 nucleic acid sequence has SEQ ID No. 3 (B); and the sequence of the pre- mRNA -200 to +200 of exon 17 (SEQ ID NO. 16) (C):Fig. 2 shows a table displaying exemplary and specific antisense oligonucleotides according to the invention, detailing the target sequence in the -200 to +200 bp nucleic acid sequence region of exon 17 (ref. to isoform 2) of human ASPP2, the targeting sequence of the antisense oligonucleotide, and the GO percentage; chemical modifications: m_ modification: 2' O- methyl RNA; “m_*” modification: Phosphorothioated 2'-O-methyl RNA; _* modification: Phosphorothioated DNA bases; +_ modification: locked nucleic acids (LNA); r_ modification: RNA;Fig. 3 shows the results of qRT-PCR experiments for validation of the exon inclusion and exon skipping AON candidates. (A-H) isoform specific qRT- PCR quantifying ASPP2Kexpression from the exon inclusion AON candidates. (i) isoform specific qRT-PCT quantifying ASPP2K expression induced by the exon skipping AON. Analyses were performed in triplicates Statistical test: unpaired t-test. ****p < 0.0001 , ***p < 0.001 , **p < 0.01 , *p < 0.05; andFig. 4 shows the results of one-step isoform-specific RT-PCR experiments with selected antisense oligonucleotides according to the invention assessingtheir effectiveness. (A) Agarose gel electrophoresis of the RT-PCR products under the different antisense oligonucleotide (AON) candidates for GAPDH (192bp), ASPP2 WT (exon 17-specific primer sets, 149bp) and ASPP2K (fusion site-specific primer sets, 128bp). (B-C) Quantification of the ASPP2 WT (B), the ASPP2K (D) and the GAPDH (C) band intensity of the agarose gel for the different AON treatment conditions using Imaged. Intensity measurements were performed in triplicates. Statistical test: unpaired t-test. ****p < 0.0001 , ***p < 0.001 , **p < 0.01 , *p < 0.05.EXAMPLES

[0123] Starting from the hypothesis that cis-acting splicing motifs exist that can promote the exon 17 skipping and generate the alternative splicing of ASPP2K, the inventors analyzed the human TP53BP2 gene sequence from NCBI (http: / / www.ncbi.nlm.nih.gov / ; gene ID 7159. Specifically, the exon 17 was focused on along with the 200bp of its upstream and downstream introns which is the maximum distance the splicing elements can be found. The sequence was then analyzed using the Human Splicing Finder system (http: / / www.umd.be / HSF3 / ), a bioinformatics server that combines multiple algorithms and matrices in a pool for splicing signals in order to provide a wide range of information about signals contained in any human genome sequence.

[0124] The sequence analysis revealed that the area upstream and downstream of exon 17 is heavily loaded with non-natural acceptor and branchpoint sites on the 5’ end of the exon 17, but also inside the exon itself and the 3’ end (data not shown). Importantly, some of these signals scored higher than the natural site sequences in the HSF, an indicator of signal strength during splicing that could result in an alternative splicing event. Furthermore, the exonic splicing regulator (ESR) profile of the sequence was analyzed. Interestingly, it was found that there is a big accumulation of ESEs motifs upstream but also downstream of exon 17 (data not shown), and a big cluster of ESSs in the middle and downstream of the exon that could contribute to the exon skipping event.

[0125] Using the sequence analysis from HSF and considering specific criteria for splice-switching antisense oligonucleotide (AON) design (such as length (20-30nt), Tm (>48°C), CG% (40%-60%) and sequence accessibility on the pre-mRNA, which should involve a partially open structure, etc.), candidate target sequences were identified that could potentially induce exon inclusion (see table in Figure 2). As a proof of principle, a splice-switching AON was designed to induce exon-skipping and thus overexpression of ASPP2K. This segment targets the natural acceptor site of the exon up to the first nt of exon 17 itself. Scrambled splice-switching AONs were used as controls. For exon inclusion AONs, i.e. AONs according to the invention, the target regions selected were sites containing ISS, ESS sites and sites of non-natural splice elements (non-natural branch point, acceptor and donor signals). The RNA structure of exon 17 with its upstream and downstream 200bp introns was analysed for its secondary RNA structure using the mfold web server (http: / / unafold.rna. albany.edu / ?q=mfold)..

[0126] The splice switching AON candidates were then introduced into RD (rhabdomyosarcoma) cells using lipofectamine transfection and the effect on expression of ASPP2K and ASPP2 was measured by qRT-PCR (see Figure 4), 48 hrs after transfection.

[0127] As a result, the exon-skipping AON resulted in significant overexpression of ASPP2K, which was 40-fold higher compared to scrambled AON (Figure 3I). Simultaneously ASPP2 WT expression decreased by 50% (data not shown). In contrast, analysis of 5 candidate exon-inclusion AONs with predicted efficacy in silico (aiming to restore inclusion of exon 17 and thereby ASPP2 WT expression) revealed that 3 AONs indeed successfully restored ASPP2 WT expression- while reducing ASPP2K expression levels (see Figure 3B, D, E).

[0128] To further identify the splicing modification ability of the exon-including antisense oligonucleotides, a confirmational lipofectamine transfection of RD cells was performed with the exon-including AONs, 340-362; 342-362; 471-492; 340 / 471 (combi); 342 / 471 (combi). Importantly, the highest downregulation of ASPP2K was achieved when two AONs were combined (see Fig. 3G, H).

[0129] To analyze the splicing pattern, cells were collected 24hrs after transfection and isoform-specific one-step RT-PCR was performed. Primers were designed for GAPDH, ASPP2 WT (targeting exon 17), and ASPP2K (targeting the fusion site) (see the table below) and products were run in an agarose gel (Figure 4A).

[0130] The table below lists the sequences of the primers used for one-step RT-PCR:

[0131] The intensities of the bands were furthermore quantified., Together, three out of five candidates managed to induce an exon inclusion, resulting in reduction of ASPP2K mRNA expression. This observation shows that not all possible candidate AONs are capable to suppress ASPP2K underlining the significance of the location of the splice switching antisense oligonucleotides and the importance of their modification to increase affinity and effectivity.

[0132] It was also found that the combination of splice switching AONs led to higher efficacy to attenuate ASPP2K expression (Figure 4C) while expression of the ASPP2 wildtype (WT) isoform is increased (Figure 4B).

[0133] With the experiments and the results as presented above, the proof-of- concept and suitability of exon-including antisense to restore ASPP2 WT function and attenuate oncogenic ASPP2K expression is confirmed.

[0134] To summarize, the inventors of the present invention analyzed the splicing elements of ASPP2 down- and upstream of exon 17, which is being spliced out in ASPP2K, as well as the cis-acting elements of the splicing machinery. It was found that the area around exon 17 is heavily loaded with motifs and splicing signals that favor alternative exon 17-skipping.

[0135] Promising sequences within this area were identified as targets for splicing modification (exon-including) and RNAse H resistant antisense oligonucleotides (AON) were designed accordingly. As a control, an exon-skipping AON was developed to overly express ASPP2K. AS expected, ASPP2K mRNA increased using the exon-skipping control AON - while exon-including AONs were identified that managed to modulate the splicing machinery to decrease ASPP2K mRNA levels while restoring ASPP2 WT.

[0136] Targeting the ASPP2 locus with exon-including splice switching antisense oligonucleotides as presently claimed can result in restoration of exon 17 transcription and ASPP2K elimination. Thus, the exon-including AONs of the present invention can be used to restore the onco-suppressing functions of ASPP2, thereby representing useful therapeutic and clinical tools in the treatment and / or prevention of diseases or conditions related to ASPP2K expression.

Claims

CLAIMS1. An antisense oligonucleotide comprising a targeting sequence with at least 90% sequence complementarity to a target nucleic acid sequence within a region of a pre-mRNA of human ASPP2, wherein the region of pre-mRNA of human ASPP2 is transcribed from a region the region -200 bases to +200 bases of exon 17 of human ASPP2, wherein the antisense oligonucleotide is exon-including.

2. The antisense oligonucleotide according to claim 1 , for use in the treatment and / or prevention of conditions and / or diseases related to ASPP2K-variant expression, in particular in which the induction of apoptosis is disturbed, reduced or inhibited.

3. The antisense oligonucleotide according to claim 2, wherein the disease or condition is cancer or cell-stress-induced inflammation.

4. The antisense oligonucleotide according to claim 3, wherein the cancer is selected from hematological neoplasms, solid tumors, precancerous conditions, and in particular acute or chronic myeloid or acute lymphoblastic leukemia.

5. The antisense oligonucleotide according to any of the preceding claims, wherein the region -200 bases to +200 bases of exon 17 of a human ASPP2 is transcribed from SEQ ID No. 1.

6. The antisense oligonucleotide according to any one of the preceding claims, wherein the antisense oligonucleotide is single stranded or double stranded, preferably a modified RNA or DNA oligonucleotide.

7. The antisense oligonucleotide according to any one of the preceding claims, wherein the targeting sequence is fully complementary to the target nucleic acid sequence.

8. The antisense oligonucleotide according to any one of the preceding claims, wherein the antisense oligonucleotide comprises modified nucleotides selected from O-methyl RNA nucleotides, phosphorothioate 2'0-methyl RNA nucleotides, phosphorothioate DNA nucleotides, locked-nucleic-acid nucleotides and natural RNA nucleotides.

9. The antisense oligonucleotide according to any one of the preceding claims, wherein the targeting sequence is a 15 to 40 nucleosides in length.

10. The antisense oligonucleotide according to any one of the preceding claims, wherein, in the targeting sequence, the proportion of guanine and cytosine nucleic bases is, either naturally of artificially modified, at least 40% and at most 60% of all nucleic bases.

11. The antisense oligonucleotide according to any one of the preceding claims, wherein the antisense oligonucleotide has a sequence that is selected from the group consisting of SEQ ID No. 11 (“340-362"), SEQ ID No. 12 (“342-364"), and SEQ ID No. 14 (“471-492").

12. Pharmaceutical composition comprising or consisting of a) at least one exon-including antisense oligonucleotide according to any one of the preceding claims; and b) a pharmaceutically acceptable carrier.

13. The pharmaceutical composition according to claim 12, for use with an apoptosisinducing therapeutic agent and / or an apoptosis-inducing therapy and / or with an anticancer agent and / or an anticancer therapy.

14. The pharmaceutical composition according to claim 12 or 13, wherein the pharmaceutically acceptable carrier is a nanoparticle, preferably a lipid nanoparticle, on or in which the antisense oligonucleotide is adsorbed.

15. The pharmaceutical composition according to claim 13, wherein the at least one apoptosis-inducing therapeutic agent or anticancer agent is a chemotherapeutic agent, preferably an anticancer drug.

16. The pharmaceutical composition according to claim 13, wherein the at least one apoptosis-inducing therapy or anticancer therapy is a radiotherapy or a targeted cancer therapy.

17. An antisense oligonucleotide comprising a targeting sequence with at least 90% sequence complementarity to a target nucleic acid sequence within a region of a pre-mRNA of human ASPP2, wherein the region of pre-mRNA of human ASPP2 is transcribed from a region the region -200 bases to +200 bases of exon 17 of human ASPP2, wherein the antisense oligonucleotide is exon-skipping.

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