WO2020148400A1

WO2020148400A1 - Antisense oligonucleotides for use in the treatment of crpc

Info

Publication number: WO2020148400A1
Application number: PCT/EP2020/051051
Authority: WO
Inventors: Jacobus Antonius Schalken; Gerardus Wilhelmus Christiaan Theodoor Verhaegh; John Pieter Michiel SEDELAAR; Maria Victoria LUNA VELEZ
Original assignee: Stichting Katholieke Universiteit
Priority date: 2019-01-16
Filing date: 2020-01-16
Publication date: 2020-07-23

Abstract

The present invention relates to the field of medicine. In particular, it relates to novel antisense oligonucleotides that may be used in the treatment, prevention and/or delay of castration-resistant prostate cancer.

Description

Antisense oligonucleotides for use in the treatment of CRPC

Field of the invention

The invention relates to the fields of medicine and immunology. In particular, it relates to novel antisense oligonucleotides that may be used in the treatment, prevention and/or delay of an AR- associated condition.

Background of the invention

Despite the clinical remission achieved by androgen deprivation therapy, advanced prostate cancer eventually progresses into recurrent or castration-resistant prostate cancer (CRPC)¹. CRPC is a lethal disease with no curative treatment available and with a median survival of 1 -2 years ²’³. Current therapeutics for CRPC with anti-androgen medication are directed to impair signaling from the full-length androgen receptor (AR-FL) by using an AR antagonist, or by depleting adrenal and intratumoral androgens. However, approximately 20 to 40% of patients have no response to current therapeutic agents and among patients who initially respond, nearly all eventually acquire resistance. Expression of AR variants (AR-Vs) is one of the main mechanisms responsible for the therapeutic failure. AR antagonists exert their antitumor activity through interactions with the ligandbinding domain (LBD) of the AR, which is absent in most AR-Vs.

Although many AR-Vs have been described, AR-splice variant 7 (AR-V7) is the most commonly and abundantly expressed variant in human CRPC tissues ⁴·⁵. AR-V7 originates from alternative splicing of the AR pre-mRNA. and is correlated with a bad prognosis and a high probability of disease recurrence ^4_7. AR-V7 lacks the ligand-binding domain (LBD) and is constitutively active, i.e. it can promote androgen-independent cell proliferation in vitro ⁸ and tumor growth in vivo under castrate androgen levels ⁵. Due to the lack of the LBD, the AR-V activity is insensitive to the AR antagonists: bicalutamide and enzalutamide, agents currently used as prostate cancer therapeutics

8-11

Thus it is clear that there remains a need for novel therapies for the treatment of CRCP that target AR-Vs.

Summary of the invention

In a first aspect, the invention provides for an antisense oligonucleotide for the downregulation of AR-V7 mRNA by redirecting splicing or promoting mRNA degradation that binds to and/or is complementary to a polynucleotide with the nucleotide sequence a shown in SEQ ID NO 4. Preferably the antisense oligonucleotide binds to or is complementary to a polynucleotide with SEQ ID NO:5 or SEQ ID NO:6. More preferably, the antisense oligonucleotide binds to or is complementary to a polynucleotide with SEQ ID NO: 8, 9, 11 , 12, 14, or 15. Most preferably, the antisense oligonucleotide binds to or is complementary to a polynucleotide selected from the group consisting of SEQ ID NO:7, 10 and 13.

In a second aspect, the invention provides for a set of antisense oligonucleotide for the downregulation of AR-V7 mRNA by redirecting splicing or promoting mRNA degradation comprising at least two antisense oligonucleotides as defined herein. Preferably, the set comprises or consists of SEQ ID NO: 16 and SEQ ID NO: 17, SEQ ID NO: 16 and SEQ ID NO: 18, or SEQ ID NO: 17 and SEQ ID NO: 18.

In a third aspect, the invention provides for a viral vector expressing an antisense oligonucleotide for the downregulation of AR-V7 mRNA by redirecting splicing as defined herein when placed under conditions conducive to expression of the molecule.

In a third aspect, the invention provides for a pharmaceutical composition comprising an antisense oligonucleotide for redirecting splicing as defined herein or a viral vector as defined herein and a pharmaceutically acceptable excipient.

In a fourth aspect, the invention provides for an antisense oligonucleotide for the downregulation of AR-V7 mRNA by redirecting splicing or promoting mRNA degradation as defined herein or a viral vector as defined herein and a pharmaceutically acceptable excipient.

In a fifth aspect, the invention provides for an antisense oligonucleotide for the downregulation of AR-V7 mRNA by redirecting splicing or promoting mRNA degradation for use as a medicament, preferably for use as a medicament for treating an AR (Androgen Receptor) related disease or a condition requiring redirecting splicing of the (pre)mRNA of AR or promoting AR-V7 mRNA degradation. Detailed Description of the invention

Castration-resistant prostate cancer (CRPC) can be caused by the elevated expression of androgen-receptor splice variants (AR-Vs). The most commonly and abundantly expressed AR-V in human CRPC tissue is AR-V7. AR-V7 originates from alternative splicing of the AR pre-mRNA. A typical splicing process requires the coordinated action of splicing factors and cis-acting regulatory elements. Intron 3 of the AR contains two splicing signals known as intronic and exonic splicing enhancers (ISE and ESE, respectively). Recognition of these cis elements by the splicing machinery results in the inclusion of a cryptic exon 3 (CE3) into the mRNA. This cryptic exon includes a premature stop codon leading to the synthesis of AR-V7 ¹².

By definition, antisense oligonucleotides (AONs) are substantially complementary (antisense) to their target, allowing them to bind to the corresponding pre-mRNA molecule, thereby, without wishing to be being bound by theory, preventing the binding of proteins essential for splicing. Usually, this lack of binding results in the skipping of the targeted exon, however AONs can also block (aberrant) splicing events by base-pairing with cryptic splice sites in the pre-mRNA in the nucleus

The present inventors have demonstrated that molecules inducing the downregulation of the AR- V7 mRNA be used to re- sensitize tumor cells to current androgen deprivation therapy. The inventors have developed antisense oligonucleotides capable of recognizing ISE and ESE of intron 3 of AR with antisense oligonucleotides preventing splicing and inclusion of CE3, leading to the expression of a full-length AR mRNA (AR-FL) and consequent downregulation of AR-V7. Additionally, the inventors developed a GapmeR antisense oligonucleotide that downregulates AR- V7 mRNA by promoting its degradation by RNAse H.

Accordingly, in a first aspect, the invention provides an antisense oligonucleotide for the downregulation of AR-V7 mRNA by redirecting splicing or promoting mRNA degradation that binds to and/or is complementary to a polynucleotide with the nucleotide sequence a shown in SEQ ID NO:4, wherein preferably the antisense oligonucleotide binds to or is complementary to a polynucleotide with SEQ ID NO:5 or SEQ ID NO:6, more preferably the antisense oligonucleotide binds to or is complementary to a polynucleotide with SEQ ID NO: 8, 9, 11 , 12, 14, or 15, most preferably the antisense oligonucleotide binds to or is complementary to a polynucleotide selected from the group consisting of SEQ ID NO: 7, 10 and 13 .

The terms "antisense oligonucleotide" and “AON” are used interchangeably herein and are understood to refer to an oligonucleotide molecule comprising a nucleotide sequence which is substantially complementary to a target nucleotide sequence in a pre-mRNA molecule, hnRNA (heterogenous nuclear RNA) or mRNA molecule. The degree of complementarity (or substantial complementarity) of the antisense sequence is preferably such that a molecule comprising the antisense sequence can form a stable hybrid with the target nucleotide sequence in the RNA molecule under physiological conditions. Binding of an AON to its target can easily be assessed by the person skilled in the art using techniques that are known in the field such as the gel mobility shift assay as described in EP1619249.

The term "complementary" used in the context of the invention indicates that some mismatches in the antisense sequence are allowed as long as the functionality, i.e. redirecting splicing or promoting mRNA degradation is achieved. Preferably, the complementarity is from 90% to 100%. In general this allows for 1 or 2 mismatches in an AON of 20 nucleotides or 1 , 2, 3 or 4 mismatches in an AON of 40 nucleotides, or 1 , 2, 3, 4, 5 or 6 mismatches in an AON of 60 nucleotides, etc. Optionally, said AON may further be tested by transfection into prostate cancer cells of patients. The complementary regions are preferably designed such that, when combined, they are specific for the intron or exon in the pre-mRNA or mRNA. Such specificity may be created with various lengths of complementary regions, as this depends on the actual sequences in other (pre-)mRNA molecules in the system. The risk that the AON will also be able to hybridize to one or more other (pre-)mRNA molecules decreases with increasing size of the AON. It is clear that AONs comprising mismatches in the region of complementarity but that retain the capacity to hybridize and/or bind to the targeted region(s) in the (pre-)mRNA, can be used in the invention. However, preferably at least the complementary parts do not comprise such mismatches as AONs lacking mismatches in the complementary part typically have a higher efficiency and a higher specificity than AONs having such mismatches in one or more complementary regions. It is thought, that higher hybridization strengths, (i.e. increasing number of interactions with the opposing strand) are favorable in increasing the efficiency of the process of interfering with the splicing or mRNA degradation machinery of the system.

The terms “modulate splicing” and “redirect splicing” are used herein interchangeably and encompass AON-based splice modulation therapy for the treatment of an AR related disease or a condition requiring redirecting splicing of the AR. The term“redirecting splicing” is herein defined as redirecting the AR pre-mRNA splicing to yield the original AR transcript and downregulation of the AR-V7 transcript.

In a preferred embodiment, the AON for the downregulation of AR-V7 mRNA by redirecting splicing binds to and/or is complementary to a polynucleotide with the nucleotide sequence a shown in SEQ ID NO:4, wherein preferably the antisense oligonucleotide binds to or is complementary to a polynucleotide with SEQ ID NO: 5, more preferably the antisense oligonucleotide binds to or is complementary to a polynucleotide with SEQ ID NO: 8, 9, 1 1 , or 12, most preferably the antisense oligonucleotide binds to or is complementary to a polynucleotide selected from the group consisting of SEQ ID NO: 7 and 10.

The AON according to the invention preferably does not contain a stretch of CpG, more preferably does not contain any CpG. The presence of a CpG or a stretch of CpG in an oligonucleotide is usually associated with an increased immunogenicity of said oligonucleotide (Dorn and Kippenberger, 2008). This increased immunogenicity is undesired since it may induce damage of the tissue to be treated, i.e. the eye. Immunogenicity may be assessed in an animal model by assessing the presence of CD4+ and/or CD8+ cells and/or inflammatory mononucleocyte infiltration. Immunogenicity may also be assessed in blood of an animal or of a human being treated with an AON according to the invention by detecting the presence of a neutralizing antibody and/or an antibody recognizing said AON using a standard immunoassay known to the skilled person. An inflammatory reaction, type l-like interferon production, IL-12 production and/or an increase in immunogenicity may be assessed by detecting the presence or an increasing amount of a neutralizing antibody or an antibody recognizing said AON using a standard immunoassay. The AON according to the invention furthermore preferably has acceptable RNA binding kinetics and/or thermodynamic properties. The RNA binding kinetics and/or thermodynamic properties are at least in part determined by the melting temperature of an oligonucleotide (Tm; calculated with the oligonucleotide properties calculator (www. unc. edu/-cail/biotool/oligo/index) for single stranded RNA using the basic Tm and the nearest neighbor model), and/orthe free energy of the AON-target intron/exon complex (using RNA structure version 4.5). If a Tm is too high, the AON is expected to be less specific. An acceptable Tm and free energy depend on the sequence of the AON. Therefore, it is difficult to give preferred ranges for each of these parameters. An acceptable Tm may be ranged between 35 and 70 °C and an acceptable free energy may be ranged between 15 and 45 kcal/mol. In all embodiments, the nucleotide in the antisense oligonucleotide according to the invention may be, wherein a nucleotide in the antisense oligonucleotide may be an RNA residue, a DNA residue, an RNA/DNA residue, or a nucleotide analogue or equivalent. Preferably, the nucleotide in the antisense oligonucleotide may be an RNA/DNA residue. More preferably, the antisense oligonucleotide is a GapmeR.

“GapmeR" or "gap oligomer", as used herein, refers to a chimeric oligomer having a central portion (a "gap") flanked by 3' and 5' "wings", wherein the gap has a modification that is different as compared to each of the wings. Such modifications may include nucleobase, monomeric linkage, and sugar modifications as well as the absence of a modification (such as unmodified RNA or DNA). Accordingly, a gapmer may be as simple as RNA wings separated by a DNA gap. In an embodiment, the GapmeR comprises an 2'-0 alkyl phosphorothioate modified nucleotide, such as 2'-0-methyl modified ribose (RNA), 2'-0-ethyl modified ribose, 2'-0-propyl modified ribose, and/or substituted derivatives of these modifications such as halogenated derivatives. Preferably the GapmeR comprises a 2'-0-methyl modified ribose.

In a preferred embodiment, the AON for the downregulation of AR-V7 mRNA by promoting mRNA degradation is a GapmeR. Preferably the GapmeR for promoting mRNA degradation binds to or is complementary to a polynucleotide with SEQ ID NO: 6, more preferably the GapmeR binds to or is complementary to a polynucleotide selected from the group consisting of 14 and 15, of most preferably the antisense oligonucleotide binds to or is complementary to SEQ ID NO: 13.

In a preferred embodiment the AON for the downregulation of AR-V7 mRNA by promoting mRNA degradation comprises or consists of SEQ ID NO: 18.

In some cases, the nucleotide linkages in the wings may be different than the nucleotide linkages in the gap. In certain embodiments, each wing comprises nucleotides with high affinity modifications and the gap comprises nucleotides that do not comprise that modification.

Alternatively, the nucleotides in the gap and the nucleotides in the wings may have high affinity modifications, but the high affinity modifications in the gap are different than the high affinity modifications in each of the wings. The modifications in the wings may confer resistance to cleavage by endogenous nucleases, including RNaseH, while the modifications in the gap may be substrates for RNase H. The modifications in the wings may confer resistance to cleavage by endogenous nucleases, including RNaseH, while the modifications in the gap maybe substrates for RNase H. The modifications in the wings may be the same or different from one another. The nucleotides in the gap may be unmodified and nucleotides in the wings may be modified. In one embodiment the GapmeR of the invention is modified to comprise a phosphorothioate backbone and a 2'-0-methyl modified ribose.

A GapmeR has a wing-gap-wing ratio, which may be represented numerically (wing#-gap#-wing#). The GapmeR maybe symmetrical for example 7-12-7, 7-11-7, 7-10-7, 7-9-7, 7-8-7, 7-7-7, 7-6-7, 7- 5-7, 7-4-7, 7-3-7, 6-12-6, 6-11-6, 6-10-6, 6-9-6, 6-8-6, 6-7-6, 6-6-6, 6-5-6, 6- 4-6, 6-3-6, 6-2-6, 5- 12-5, 5-11-5, 5-10-5, 5-9-5, 5-8-5, 5-7-5, 5-6-5, 5-5-5, 5- 4-5, 5-3-5, 4-12-4, 411-4, 4-10-4, 4-9-4, 4- 8-4, 4-7- 4, 4-6-4, 4-5-4, 4-4-4, 4-3-4,3-12-3, 3-11-3, 3-10-3, 3-9-3, 3-8-3, 3-7-3, 3-6-3, 3-5-3, or 3- 4-3. Preferably, the GapmeR has a 5-10-5 gap-wing ratio. A preferred AON for redirecting splicing according to the invention, has a length of from about 8 to about 40 nucleotides, preferably from about 10 to about 40 nucleotides, more preferably from about 14 to about 30 nucleotides, more preferably from about 16 to about 24 nucleotides, such as 16, 17, 18, 19, 20, 21 , 22, 23 or 24 nucleotides. Preferably, an AON according to the invention has a length of at least 8, 9, 10, 1 1 , 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.

A preferred AON for the downregulation of AR-V7 mRNA by redirecting splicing or promoting mRNA degradation according to the invention comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 16, 17 and 18.

It is preferred that an AON for redirecting splicing according to the invention comprises one or more residues that are modified to increase nuclease resistance, and/or to increase the affinity of the antisense oligonucleotide for the target sequence. Therefore, in a preferred embodiment, the AON comprises at least one nucleotide analogue or equivalent, wherein a nucleotide analogue or equivalent is defined as a residue having a modified base, and/or a modified backbone, and/or a non-natural internucleoside linkage, or a combination of these modifications.

In a preferred embodiment, the nucleotide analogue or equivalent comprises a modified backbone. Examples of such backbones are provided by morpholino backbones, carbamate backbones, siloxane backbones, sulfide, sulfoxide and sulfone backbones, formacetyl and thioformacetyl backbones, methyleneformacetyl backbones, riboacetyl backbones, alkene containing backbones, sulfamate, sulfonate and sulfonamide backbones, methyleneimino and methylenehydrazino backbones, and amide backbones. Phosphorodiamidate morpholino oligomers are modified backbone oligonucleotides that have previously been investigated as antisense agents.

Morpholino oligonucleotides have an uncharged backbone in which the deoxyribose sugar of DNA is replaced by a six membered ring and the phosphodiester linkage is replaced by a phosphorodiamidate linkage. Morpholino oligonucleotides are resistant to enzymatic degradation and appear to function as antisense agents by arresting translation or interfering with pre-mRNA splicing rather than by activating RNase H. Morpholino oligonucleotides have been successfully delivered to tissue culture cells by methods that physically disrupt the cell membrane, and one study comparing several of these methods found that scrape loading was the most efficient method of delivery; however, because the morpholino backbone is uncharged, cationic lipids are not effective mediators of morpholino oligonucleotide uptake in cells. A recent report, demonstrated triplex formation by a morpholino oligonucleotide and, because of the non-ionic backbone, these studies showed that the morpholino oligonucleotide was capable of triplex formation in the absence of magnesium.

It is further preferred that the linkage between the residues in a backbone do not include a phosphorus atom, such as a linkage that is formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages.

A preferred nucleotide analogue or equivalent comprises a Peptide Nucleic Acid (PNA), having a modified polyamide backbone (Nielsen et al., 1991). PNA-based molecules are true mimics of DNA molecules in terms of base-pair recognition. The backbone of the PNA is composed of N-(2- aminoethyl)-glycine units linked by peptide bonds, wherein the nucleobases are linked to the backbone by methylene carbonyl bonds. An alternative backbone comprises a one-carbon extended pyrrolidine PNA monomer (Govindaraju and Kumar, 2005). Since the backbone of a PNA molecule contains no charged phosphate groups, PNA-RNA hybrids are usually more stable than RNA-RNA or RNA-DNA hybrids, respectively (Egholm et al., 1993). A further preferred backbone comprises a morpholino nucleotide analog or equivalent, in which the ribose or deoxyribose sugar is replaced by a 6-membered morpholino ring. A most preferred nucleotide analog or equivalent comprises a phosphorodiamidate morpholino oligomer (PMO), in which the ribose or deoxyribose sugar is replaced by a 6-membered morpholino ring, and the anionic phosphodiester linkage between adjacent morpholino rings is replaced by a non-ionic phosphorodiamidate linkage.

In yet a further embodiment, a nucleotide analogue or equivalent according to the invention comprises a substitution of one of the non-bridging oxygens in the phosphodiester linkage. This modification slightly destabilizes base-pairing but adds significant resistance to nuclease degradation. A preferred nucleotide analogue or equivalent comprises phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphotriester, aminoalkylphosphotriester, H- phosphonate, methyl and other alkyl phosphonate including 3'-alkylene phosphonate, 5'-alkylene phosphonate and chiral phosphonate, phosphinate, phosphoramidate including 3'-amino phosphoramidate and aminoalkylphosphoramidate, thionophosphoramidate, thionoalkylphosphonate, thionoalkylphosphotriester, selenophosphate or boranophosphate.

A further preferred nucleotide analogue or equivalent according to the invention comprises one or more sugar moieties that are mono- or disubstituted at the 2', 3' and/or 5' position such as a -OH; - F; substituted or unsubstituted, linear or branched lower (CI-C10) alkyl, alkenyl, alkynyl, alkaryl, allyl, or aralkyl, that may be interrupted by one or more heteroatoms; 0-, S-, or N-alkyl; 0-, S-, or N-alkenyl; 0-, S-or N-alkynyl; 0-, S-, or N- allyl; O-alkyl-O-alkyl, -methoxy, -aminopropoxy; methoxyethoxy; dimethylaminooxyethoxy; and -dimethylaminoethoxyethoxy. The sugar moiety can be a pyranose or derivative thereof, or a deoxypyranose or derivative thereof, preferably ribose or derivative thereof, or deoxyribose or derivative of. A preferred derivatized sugar moiety comprises a Locked Nucleic Acid (LNA), in which the 2'-carbon atom is linked to the 3' or 4' carbon atom of the sugar ring thereby forming a bicyclic sugar moiety. A preferred LNA comprises 2'-0, 4'-C- ethylene-bridged nucleic acid (Morita et al., 2001). These substitutions render the nucleotide analogue or equivalent RNase H and nuclease resistant and increase the affinity for the target RNA. In another embodiment, a nucleotide analogue or equivalent according to the invention comprises one or more base modifications or substitutions. Modified bases comprise synthetic and natural bases such as inosine, xanthine, hypoxanthine and other -aza, deaza, -hydroxy, -halo, -thio, thiol, -alkyl, -alkenyl, -alkynyl, thioalkyl derivatives of pyrimidine and purine bases that are or will be known in the art.

It is understood by a skilled person that it is not necessary for all positions in an AON to be modified uniformly. In addition, more than one of the aforementioned analogues or equivalents may be incorporated in a single AON or even at a single position within an AON. In certain embodiments, an AON according to the invention has at least two different types of analogues or equivalents. Accordingly, in a preferred embodiment an antisense oligonucleotide for redirecting splicing according to the invention, comprises a 2'-0 alkyl phosphorothioate antisense oligonucleotide, such as 2'-0-methyl modified ribose (RNA), 2'-0-ethyl modified ribose, 2'-0-propyl modified ribose, and/or substituted derivatives of these modifications such as halogenated derivatives. Preferably, the AON according to the invention comprises a 2'-0-methyl modified ribose.

In an embodiment, the antisense oligonucleotide for the downregulation of AR-V7 mRNA by redirecting splicing or promoting mRNA degradation according the invention, comprises a 2'-0- methyl modified ribose (RNA) and a phosphorothiorate backbone.

In an embodiment, an AON for the downregulation of AR-V7 mRNA by redirecting splicing according to the invention, comprises or consists of SEQ ID NO: 16 and comprises a 2'-0-methyl modified ribose (RNA) and a phosphorothiorate backbone.

In an embodiment, an AON for the downregulation of AR-V7 mRNA by redirecting splicing according to the invention, comprises or consists of SEQ ID NO: 17 and comprises a 2'-0-methyl modified ribose (RNA) and a phosphorothiorate backbone.

In an embodiment, an AON for for the downregulation of AR-V7 mRNA by promoting mRNA degradation according to the invention, comprises or consists of SEQ ID NO: 18 and comprises a 2'-0-methyl modified ribose (RNA) and a phosphorothiorate backbone.

It will also be understood by a skilled person that different antisense oligonucleotides can be combined for redirecting splicing of AR and promoting AR-V7 mRNA degradation. Accordingly, the invention provides for a set of antisense oligonucleotide for redirecting splicing or promoting AR-V7 mRNA degradation comprising at least two antisense oligonucleotides as defined herein. Preferably, the set comprises or consists of:

- antisense oligonucleotide comprising or consisting of a sequence as represented by SEQ ID NO:

16 and 17;

- antisense oligonucleotide comprising or consisting of a sequence as represented by SEQ ID NO: 16 and 18 ; or

- antisense oligonucleotide comprising or consisting of a sequence as represented by SEQ ID NO: 17 and 18.

An AON for the downregulation of AR-V7 mRNA by redirecting splicing according to the invention may be indirectly administrated using suitable means known in the art. It may for example be provided to an individual or a cell, tissue or organ of said individual as such, as a so-called‘naked’ AON. It may also be administered in the form of an expression vector wherein the expression vector encodes an RNA transcript comprising the sequence of said AON according to the invention. The expression vector is preferably introduced into a cell, tissue, organ or individual via a gene delivery vehicle. In a preferred embodiment, there is provided a viral-based expression vector comprising an expression cassette or a transcription cassette that drives expression or transcription of an AON for redirecting splicing according to the invention. Accordingly, the invention provides for a viral vector expressing antisense oligonucleotide for redirecting splicing according to the invention when placed under conditions conducive to expression of the antisense oligonucleotide.

A cell can be provided with an AON for redirecting splicing according to the invention by plasmid- derived antisense oligonucleotide expression or viral expression provided by adenovirus- or adeno- associated virus-based vectors. Expression may be driven by an RNA polymerase II promoter (Pol II) such as a U7 RNA promoter or an RNA polymerase III (Pol III) promoter, such as a U6 RNA promoter. A preferred delivery vehicle is a viral vector such as an adeno-associated virus vector (AAV), or a retroviral vector such as a lentivirus vector and the like. Also, plasmids, artificial chromosomes, plasmids usable for targeted homologous recombination and integration in the human genome of cells may be suitably applied for delivery of an AON for redirecting splicing according to the invention. Preferred for the invention are those vectors wherein transcription is driven from Poll II promoters, and/or wherein transcripts are in the form fusions with U1 or U7 transcripts, which yield good results for delivering small transcripts. It is within the skill of the artisan to design suitable transcripts. Preferred are Pollll driven transcripts, preferably, in the form of a fusion transcript with an U1 or U7 transcript. Such fusions may be generated as previously described (Gorman et al., 1998).

A preferred expression system for an AON for the downregulation of AR-V7 mRNA by redirecting splicing according to the invention is an adenovirus associated virus (AAV)-based vector. Single chain and double chain AAV-based vectors have been developed that can be used for prolonged expression of antisense nucleotide sequences for highly efficient redirection of splicing. A preferred AAV-based vector, for instance, comprises an expression cassette that is driven by an RNA polymerase Ill-promoter (Pol III) or an RNA polymerase II promoter (Pol II). A preferred RNA promoter is, for example, a Pol III U6 RNA promoter, or a Pol II U7 RNA promoter.

The invention accordingly provides for a viral-based vector, comprising a Pol II or a Pol III promoter driven expression cassette for expression of an AON for redirecting splicing according to the invention.

An AAV vector according to the invention is a recombinant AAV vector and refers to an AAV vector comprising part of an AAV genome comprising an encoded AON for redirecting splicing according to the invention encapsidated in a protein shell of capsid protein derived from an AAV serotype as depicted elsewhere herein. Part of an AAV genome may contain the inverted terminal repeats (ITR) derived from an adeno-associated virus serotype, such as AAV1 , AAV2, AAV3, AAV4, AAV5, AAV8, AAV9 and others. A protein shell comprised of capsid protein may be derived from an AAV serotype such as AAV1 , 2, 3, 4, 5, 8, 9 and others. A protein shell may also be named a capsid protein shell. AAV vector may have one or preferably all wild type AAV genes deleted, but may still comprise functional ITR nucleic acid sequences. Functional ITR sequences are necessary for the replication, rescue and packaging of AAV virions. The ITR sequences may be wild type sequences or may have at least 80%, 85%, 90%, 95, or 100% sequence identity with wild type sequences or may be altered by for example in insertion, mutation, deletion or substitution of nucleotides, as long as they remain functional. In this context, functionality refers to the ability to direct packaging of the genome into the capsid shell and then allow for expression in the host cell to be infected or target cell. In the context of the invention a capsid protein shell may be of a different serotype than the AAV vector genome ITR. An AAV vector according to present the invention may thus be composed of a capsid protein shell, i.e. the icosahedral capsid, which comprises capsid proteins (VP1 , VP2, and/or VP3) of one AAV serotype, e.g. AAV serotype 2, whereas the ITRs sequences contained in that AAV5 vector may be any of the AAV serotypes described above, including an AAV2 vector. An “AAV2 vector” thus comprises a capsid protein shell of AAV serotype 2, while e.g. an“AAV5 vector” comprises a capsid protein shell of AAV serotype 5, whereby either may encapsidate any AAV vector genome ITR according to the invention.

Preferably, a recombinant AAV vector according to the invention comprises a capsid protein shell of AAV serotype 2, 5, 8 or AAV serotype 9 wherein the AAV genome or ITRs present in said AAV vector are derived from AAV serotype 2, 5, 8 or AAV serotype 9; such AAV vector is referred to as an AAV2/2, AAV 2/5, AAV2/8, AAV2/9, AAV5/2, AAV5/5, AAV5/8, AAV 5/9, AAV8/2, AAV 8/5, AAV8/8, AAV8/9, AAV9/2, AAV9/5, AAV9/8, or an AAV9/9 vector.

More preferably, a recombinant AAV vector according to the invention comprises a capsid protein shell of AAV serotype 2 and the AAV genome or ITRs present in said vector are derived from AAV serotype 5; such vector is referred to as an AAV 2/5 vector.

More preferably, a recombinant AAV vector according to the invention comprises a capsid protein shell of AAV serotype 2 and the AAV genome or ITRs present in said vector are derived from AAV serotype 8; such vector is referred to as an AAV 2/8 vector.

More preferably, a recombinant AAV vector according to the invention comprises a capsid protein shell of AAV serotype 2 and the AAV genome or ITRs present in said vector are derived from AAV serotype 9; such vector is referred to as an AAV 2/9 vector.

More preferably, a recombinant AAV vector according to the invention comprises a capsid protein shell of AAV serotype 2 and the AAV genome or ITRs present in said vector are derived from AAV serotype 2; such vector is referred to as an AAV 2/2 vector.

A nucleic acid molecule encoding an AON for redirecting splicing according to the invention represented by a nucleic acid sequence of choice is preferably inserted between the AAV genome or ITR sequences as identified above, for example an expression construct comprising an expression regulatory element operably linked to a coding sequence and a 3’ termination sequence. “AAV helper functions” generally refers to the corresponding AAV functions required for AAV replication and packaging supplied to the AAV vector in trans. AAV helper functions complement the AAV functions which are missing in the AAV vector, but they lack AAV ITRs (which are provided by the AAV vector genome). AAV helper functions include the two major ORFs of AAV, namely the rep coding region and the cap coding region or functional substantially identical sequences thereof. Rep and Cap regions are well known in the art, see e.g. (Chiorini et al., 1999) or US 5,139,941 , incorporated herein by reference. The AAV helper functions can be supplied on an AAV helper construct, which may be a plasmid. Introduction of the helper construct into the host cell can occur e.g. by transformation, transfection, or transduction prior to or concurrently with the introduction of the AAV genome present in the AAV vector as identified herein. The AAV helper constructs according to the invention may thus be chosen such that they produce the desired combination of serotypes for the AAV vector’s capsid protein shell on the one hand and for the AAV genome present in said AAV vector replication and packaging on the other hand.

“AAV helper virus” provides additional functions required for AAV replication and packaging. Suitable AAV helper viruses include adenoviruses, herpes simplex viruses (such as HSV types 1 and 2) and vaccinia viruses. The additional functions provided by the helper virus can also be introduced into the host cell via vectors, as described in US 6,531 ,456 incorporated herein by reference.

Preferably, an AAV genome as present in a recombinant AAV vector according to the invention does not comprise any nucleotide sequences encoding viral proteins, such as the rep (replication) or cap (capsid) genes of AAV. An AAV genome may further comprise a marker or reporter gene, such as a gene for example encoding an antibiotic resistance gene, a fluorescent protein (e.g. gfp) or a gene encoding a chemically, enzymatically or otherwise detectable and/or selectable product (e.g. lacZ, aph, etc.) known in the art.

A preferred AAV vector according to the invention is an AAV vector, preferably an AAV2/5, AAV2/8, AAV2/9 or AAV2/2 vector, expressing an AON for redirecting splicing or carrying an AON for promoting mRNA degradation according to the invention that is an AON that comprises, or preferably consists of, a sequence that is:

complementary or substantially complementary to a nucleotide sequence consisting of SEQ ID NO 4, preferably the antisense oligonucleotide is complementary to a polynucleotide with SEQ ID NO:5 or SEQ ID NO:6,

more preferably complementary or substantially complementary to a polynucleotide with a nucleotide sequence selected from the group consisting of SEQ ID NO: 8, 7, 9, 10, 1 1 , 12, 14, 13 and 15. Even more preferably, the AON comprises or consists of a polynucleotide with a nucleotide sequence selected from the group consisting of SEQ ID NO: 16, 17 and 18 .

Improvements in means for providing an individual or a cell, tissue, organ of said individual with an AON for the downregulation of AR-V7 mRNA by redirecting splicing or promoting mRNA degradation according to the invention, are anticipated considering the progress that has already thus far been achieved. Such future improvements may of course be incorporated to achieve the mentioned effect on restructuring of mRNA using a method according to the invention. An AON for the downregulation of AR-V7 mRNA by redirecting splicing or promoting mRNA degradation according to the invention can be delivered as such as a‘naked’ AON to an individual, a cell, tissue or organ of said individual. When administering an AON for redirecting splicing or promoting mRNA degradation according to the invention, it is preferred that the AON is dissolved in a solution that is compatible with the delivery method.

Alternatively, a preferred delivery method for an AON for redirecting splicing or a plasmid for expression of such AON is a viral vector or are nanoparticles.

Alternatively, a plasmid can be provided by transfection using known transfection agents. For intravenous, subcutaneous, intramuscular, intrathecal and/or intraventricular administration it is preferred that the solution is a physiological salt solution. Particularly preferred in the invention is the use of an excipient or transfection agents that will aid in delivery of each of the constituents as defined herein to a cell and/or into a cell, preferably a prostate cancer cell. Preferred are excipients or transfection agents capable of forming complexes, nanoparticles, micelles, vesicles and/or liposomes that deliver each constituent as defined herein, complexed or trapped in a vesicle or liposome through a cell membrane. Many of these excipients are known in the art. Suitable excipients or transfection agentia comprise polyethylenimine (PEI; ExGen500 (MBI Fermentas)), LipofectAMINE™ 2000 (Invitrogen) or derivatives thereof, or similar cationic polymers, including polypropyleneimine or polyethylenimine copolymers (PECs) and derivatives, synthetic amphiphils (SAINT-18), lipofectinTM, DOTAP and/or viral capsid proteins that are capable of self-assembly into particles that can deliver each constituent as defined herein to a cell, preferably a prostate cancer cell. Such excipients have been shown to efficiently deliver an oligonucleotide such as AONs to a wide variety of cultured cells in vitro, including prostate cancer cells such as DuCaP/VCaP/22Rv1¹³’¹⁴ or non-prostate cancer cells such as MIA-PaCa-2 ²⁷. Their high transfection potential is combined with an excepted low to moderate toxicity in terms of overall cell survival of control cell line MIA-PaCa-2. The ease of structural modification can be used to allow further modifications and the analysis of their further (in vivo) nucleic acid transfer characteristics and toxicity.

Lipofectin represents an example of a liposomal transfection agent. It consists of two lipid components, a cationic lipid N-[1-(2,3 dioleoyloxy)propyl]-N, N, N- trimethylammonium chloride (DOTMA) (cp. DOTAP which is the methylsulfate salt) and a neutral lipid dioleoylphosphatidylethanolamine (DOPE). The neutral component mediates the intracellular release. Another group of delivery systems are polymeric nanoparticles.

Polycations such as diethylaminoethylaminoethyl (DEAE)-dextran, which are well known as DNA transfection reagent can be combined with butylcyanoacrylate (PBCA) and hexylcyanoacrylate (PHCA) to formulate cationic nanoparticles that can deliver each constituent as defined herein, preferably an AON according to the invention, across cell membranes into cells.

In addition to these common nanoparticle materials, the cationic peptide protamine offers an alternative approach to formulate an oligonucleotide with colloids. This colloidal nanoparticle system can form so called proticles, which can be prepared by a simple self-assembly process to package and mediate intracellular release of an oligonucleotide. The skilled person may select and adapt any of the above or other commercially available alternative excipients and delivery systems to package and deliver an AON for use in the current invention to deliver it for the prevention, treatment or delay of AR-related disease or condition. "Prevention, treatment or delay of an AR related disease or condition" is herein preferably defined as preventing, halting, ceasing the progression of, or reversing partial or complete resistance that is caused by alternative splicing or the AR gene resulting in AR-Vs, especially AR-V7.

In addition, an AON for the downregulation of AR-V7 mRNA by redirecting splicing or promoting degradation of mRNA according to the invention could be covalently or non-covalently linked to a targeting ligand specifically designed to facilitate the uptake into the cell, cytoplasm and/or its nucleus. Such ligand could comprise (i) a compound (including but not limited to peptide(-like) structures) recognizing cell, tissue or organ specific elements facilitating cellular uptake and/or (ii) a chemical compound able to facilitate the uptake in to cells and/or the intracellular release of an oligonucleotide from vesicles, e.g. endosomes or lysosomes.

Therefore, in a preferred embodiment, an AON for the downregulation of AR-V7 mRNA by redirecting splicing or promoting degradation of mRNA according to the invention is formulated in a composition or a medicament or a composition, which is provided with at least an excipient and/or a targeting ligand for delivery and/or a delivery device thereof to a cell and/or enhancing its intracellular delivery.

It is to be understood that if a composition comprises an additional constituent such as an adjunct compound as later defined herein, each constituent of the composition may not be suitably formulated in one single combination or composition or preparation. Depending on their identity and specific features, the skilled person will know which type of formulation is the most appropriate for each constituent as defined herein. In a preferred embodiment, the invention provides a composition or a preparation which is in the form of a kit of parts comprising an AON for the downregulation of AR-V7 mRNA by redirecting splicing or promoting degradation of mRNA according to the invention and a further adjunct compound as later defined herein.

If required and/or if desired, an AON for redirecting splicing according to the invention, a set of antisense oligonucleotides according to the invention, a GapmeR according to the invention, or a vector, preferably a viral vector, according to the invention, carrying naked AONs or expressing an AON for redirecting splicing according to the invention can be incorporated into a pharmaceutically active mixture by adding a pharmaceutically acceptable carrier.

Accordingly, the invention also provides for a composition, preferably a pharmaceutical composition comprising an antisense oligonucleotide for redirecting splicing according to the invention, a set of antisense oligonucleotides according to the invention, a GapmeR according to the invention, or a viral vector according to the invention and a pharmaceutically acceptable excipient Such composition may comprise a single AON for redirecting splicing or viral vector according to the invention, but may also comprise multiple, distinct AONs for redirecting splicing or promoting degradation of mRNA or viral vectors according to the invention. Such a pharmaceutical composition may comprise any pharmaceutically acceptable excipient, including a carrier, filler, preservative, adjuvant, solubilizer and/or diluent. Such pharmaceutically acceptable carrier, filler, preservative, adjuvant, solubilizer and/or diluent may for instance be found in Remington, 2000. Each feature of said composition has earlier been defined herein.

A preferred route of administration is through intratumoral, intravascular, intravenous, or subcutaneous administration.

A preferred AON for the downregulation of AR-V7 mRNA by redirecting splicing or promoting mRNA degradation according to the invention, is for the treatment of an AR related disease or condition of an individual. In all embodiments of the invention, the term "treatment" is understood to include the prevention and/or delay of the AR-related disease or condition requiring redirecting splicing of the pre-mRNA of AR or promoting degradation of AR-V7 mRNA. An individual, which may be treated using an AON for redirecting splicing or promoting mRNA degradation according to the invention may already have been diagnosed as having an AR- related disease or condition, such as castration resistant prostate cancer.

Alternatively, an individual which may be treated using an AON for redirecting splicing according to the invention may not have yet been diagnosed as having a AR-related disease or condition requiring redirecting splicing of the pre-mRNA of AR or promotion of AR-V7 degradation but may be an individual having an increased risk of developing a AR-related disease or condition, such as CRPC in the future given his or her genetic background. A preferred individual is a human being. In all embodiments of the invention, the AR-related disease or condition preferably is cancer, more preferably prostate cancer, even more preferably castration resistant prostate cancer.

Accordingly, the invention further provides for an antisense oligonucleotide for redirecting splicing according to the invention, a GapmeR according to the invention, a set of antisense oligonucleotides according to the invention, or a viral vector according to the invention, or a (pharmaceutical) composition according to the invention for use as a medicament, preferably as a medicament for the treatment of an AR -related disease or condition requiring redirecting splicing of the pre-mRNA of AR or promotion of degradation of AR-V7 and for use as a medicament for the prevention, treatment or delay of an AR-related disease or condition requiring redirecting splicing of the pre- mRNA of AR or promotion of degradation of AR-V7. Each feature of all medical use embodiment herein has earlier been defined herein and is preferably such feature as earlier defined herein.

The invention further provides for the use of an AON for redirecting splicing according to the invention, a GapmeR according to the invention, a set of antisense oligonucleotides according to the invention, a vector according to the invention or a (pharmaceutical) composition according to the invention for treating an AR-related disease or condition requiring redirected splicing of the pre- mRNA of AR or promotion of degradation of AR-V7. Each feature of all medical use embodiment herein has earlier been defined herein and is preferably such feature as earlier defined herein. The invention further provides for, a method of treatment of an AR-related disease or condition requiring redirecting splicing of redirected splicing of the pre-mRNA of AR or promoting degradation of AR-V7, comprising said method comprising contacting a cell of said individual with an AON for redirecting splicing or promoting degradation of mRNA according to the invention, a vector according to the invention or a (pharmaceutical) composition according to the invention. Each feature of all medical use embodiment herein has earlier been defined herein and is preferably such feature as earlier defined herein.

The invention further provides for the use of an AON for the downregulation of AR-V7 mRNA by redirecting splicing according to the invention, a GapmeR according to the invention, a set of antisense oligonucleotides according to the invention, a vector according to the invention or a (pharmaceutical) composition according to the invention for the preparation of a medicament for the treatment of an Androgen Receptor (AR)-related disease or condition requiring redirecting splicing of the pre(mRNA) of AR or degradation of AR-V7. Each feature of all medical use embodiment herein has earlier been defined herein and is preferably such feature as earlier defined herein. The invention further provides for an antisense oligonucleotide for redirecting splicing according to the invention, a GapmeR according to the invention, a set of antisense oligonucleotides according to the invention, the use according the invention or the method according to the invention, wherein the AR related disease or condition requiring redirecting splicing of the pre-mRNA of AR or promoting degradation of AR-V7 is cancer, preferably prostate cancer, more preferably castration- resistant prostate cancer (CRPC).

Treatment in a use or in a method according to the invention is preferably at least once, and preferably lasts at least one week, one month, several months, one year, 2, 3, 4, 5, 6 years or longer, such as life-long. Each AON for redirecting splicing according to the invention or equivalent thereof as defined herein for use according to the invention may be suitable for direct administration to a cell, tissue and/or an organ in vivo of individuals already affected or at risk of developing an AR related disease or condition requiring redirecting splicing of the pre-mRNA of AR or promoting degradation of AR-V7 mRNA, such as prostate cancer or cancer resistant prostate cancer, and may be administered directly in vivo, ex vivo or in vitro. The frequency of administration of an AON, composition, compound or adjunct compound according to the invention may depend on several parameters such as the severity of the disease, the age of the patient, the mutation of the patient, the number of AON for redirecting splicing or promoting AR-V7 degradation according to the invention (i.e. dose), the formulation of the AON, composition, compound or adjunct compound according to the invention, the route of administration and so forth. The frequency of administration may vary between daily, weekly, at least once in two weeks, or three weeks or four weeks or five weeks or a longer time period.

Dose ranges of an AON, composition, compound or adjunct compound according to the invention are preferably designed on the basis of rising dose studies in clinical trials (in vivo use) for which rigorous protocol requirements exist. An AON according to the invention may be used at a dose which is ranged from 0.01 and 20 mg/kg, preferably from 0.05 and 20 mg/kg. A suitable intravascularly, intravenously, or subcutaneously dose would be between 0.01 and 20 mg/kg.

In a preferred embodiment, a viral vector, preferably an AAV vector as described earlier herein, as delivery vehicle for an AON according to the invention, is administered in a dose ranging from 1x1⁰⁹ — 1x10¹⁷ virus particles per injection, more preferably from 1x10¹⁰— 1x10¹² virus particles per injection.

The ranges of concentration or dose of AONs as depicted above are preferred concentrations or doses for in vivo, in vitro or ex vivo uses. The skilled person will understand that depending on the AONs used, the target cell to be treated, the gene target and its expression levels, the medium used and the transfection and incubation conditions, the concentration or dose of AONs used may further vary and may need to be optimized any further.

An AON for redirecting splicing according to the invention, a GapmeR according to the invention, a set of antisense oligonucleotides according to the invention, or a viral vector according to the invention, or a composition according to the invention for use according to the invention may be administered to a cell, tissue and/or an organ in vivo of individuals already affected or at risk of developing a AR related disease or a condition requiring redirecting splicing of the pre-mRNA of AR or degradation of AR-V7 mRNA, and may be administered in vivo, ex vivo or in vitro. An AON for redirecting splicing or degradation of mRNA according to the invention, or a viral vector according to the invention, or a composition according to the invention may be directly or indirectly administered to a cell, tissue and/or an organ in vivo of an individual already affected by or at risk of developing a AR related disease or a condition requiring redirecting splicing of the pre-mRNA of AR -related disease or condition, and may be administered directly or indirectly in vivo, ex vivo or in vitro.

The invention further provides for a method for redirecting splicing of AR in a cell, said method comprising contacting the cell, preferably a prostate cancer cell, with an antisense oligonucleotide for redirecting splicing according to the invention, a GapmeR according to the invention, a set of antisense oligonucleotides according to the invention, the vector according to the invention or the pharmaceutical composition according to the invention The features of this aspect are preferably those defined earlier herein. Contacting the cell with an AON for redirecting splicing according to the invention, a GapmeR according to the invention, a set of antisense oligonucleotides according to the invention, or a viral vector according to the invention, or a composition according to the invention may be performed by any method known by the person skilled in the art. Use of the methods for delivery of AONs for redirecting splicing, viral vectors and compositions as described earlier herein is included. Contacting may be directly or indirectly and may be in vivo, ex vivo or in vitro.

Unless otherwise indicated each embodiment as described herein may be combined with another embodiment as described herein.

Definitions

In this document and in its claims, the verb "to comprise" and its conjugations is used in its non- limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".

The word "about" or "approximately" when used in association with a numerical value (e.g. about 10) preferably means that the value may be the given value (of 10) more or less 5% of the value. The sequence information as provided herein should not be so narrowly construed as to require inclusion of erroneously identified bases. The skilled person is capable of identifying such erroneously identified bases and knows how to correct for such errors. In case of sequence errors, the sequence of the polypeptide obtainable by expression of the gene present in SEQ ID NO: 1 containing the nucleic acid sequence coding for the polypeptide should prevail.

All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety. Description of the figures

Figure 1 : Design of antisense oligonucleotides. Schematic representation of the antisense oligonucleotides designed to prevent splicing of AR pre-mRNA into AR-V7 mRNA. AON-ISE is complementary to the intronic splicing enhancer (ISE) sites predicted by ACESCAN2, and the cryptic “GA” splice acceptor dinucleotide motif, predicted by NetGene2. AON-ESE is complementary to the region harbouring the ESEfinder-predicted exonic splicing enhancer (ESE) sites. Predicted splicing enhancer sites are in bold, and the predicted cryptic splice acceptor site is in italics. The corresponding genomic coordinates (Human Genome Assembly February 2019, HG19) are marked by vertical lines pointing at the 5’ and/or 3’ junctions of exon 3, CE3 and exon 4.

Figure 2: AON -mediated AR-V7 knockdown. (A) Schematic diagram of the AR minigene construct. Minimal regions containing AR exon 2, CE3, exon 4 and their flanking regions are cloned into a CMV-driven pEGFP-N3 expression vector. Vertical lines mark positions of each AR gene fragment on chromosome X (Human Genome Assembly February 2019, HG19). Primers for RT-qPCR are marked with headed arrows. (B) AR negative MIA PaCa-2 cells were transfected with 500 ng AR minigene vector or with empty vector and with 0.5 pM antisense oligonucleotides (AON-ISE and AON-ESE) or control sense oligos (SON-ISE and SON-ESE). Relative expression of AR-FL and AR-V7 from the AR minigene were measured by RT-qPCR analysis, four days after transfection. Unpaired t- test; **, p<0.01 ; ***, p<0.001 . AON-treated vs non-treated cells; #, p<0.05 and ##, p<0.01 . Bars represent the mean ± SD of three independent experiments. (C-E) Expression levels of AR-V7, AR-FL, AR-V1 and AR-V3 in DuCaP and VCaP cells (determined by RT-qPCR), four days after transfection with 0.2 mM AONs or control oligos. Expression levels were compared to non-transfected cells (NT). Below each graphs, western blot analysis of AR-V7 (anti-AR-V7), AR- FL (N20) and truncated AR-Vs (N20) protein levels are shown. Protein levels of b-actin (anti-b- actin) were used as protein loading control. Unpaired t-test; *, p<0.05; **, p<0.01 ; ***, p<0.001 . Bars represent the mean ±SD of three independent experiments.

Figure 3: Effect of AON-ISE on AR-V7 -targeted gene expression. (A) Microarray analysis showing AR, UBE2C and BUB1B gene expression profiles in normal prostate (NP, n=7), benign prostate hyperplasia (BPH, n=12), primary prostate cancer (PCa, n=49), castration-resistant prostate cancer (CRPC, n=22) and metastasis (n=7). Microarray values (2log scale are shown as the mean ± SD of each group is depicted. Unpaired t-test; **, p<0.01 ; ***, p<0.001). (B) Pearson correlation of AR- FL, AR-V7, UBE2C and BUB1B mRNA expression, obtained by RT-qPCR, in castration-resistant prostate cancer (n=20) specimens. Two-tailed P values and Pearson r values are depicted. NS; p>0.05 (C) Relative UBE2C and BUB1B mRNA expression in VCaP cells, determined 96 hours after treatment with 0.1 nM R1881 , or R1881 in combination with 2 pM Enzalutamide. Unpaired t- test; *, p<0.05; **, p<0.01 ; ***, p<0.001 . Bars represent the mean ± SD of three independent experiments. (D) Relative mRNA expression levels of AR-V7, UBE2C and BUB1B in VCaP cells following transfection with an AR-V7 expression vector. Unpaired t-test; *, p<0.05; ***, p<0.001). Bars represent the mean ± SD of three independent experiments. (E-F) Relative mRNA expression from AR-V7 (E), UBE2C and BUB1B (F) in DuCaP and VCaP cells, as determined 96 hours after treatment with increasing doses of AON-ISE, compared to non-transfected cells (NT). Unpaired t- test; *, p<0.05; **, p<0.01 ; ***, p<0.001 . Bars represent the mean ± SD of three independent experiments.

Figure 4: Effect of AON-ISE in cell viability and apoptosis. (A) Dose-dependent effect of AON- ISE666 mediated AR-V7 knockdown on cell viability of DuCaP and VCaP cells compared to SON- treated cells. AR-negative MIA PaCa-2 cells were used as a negative control. Unpaired t-test; *, p<0.05; **, p<0.01 ; ***, p<0.001 . Bars represent the mean ± SD of three independent experiments. (B) Induction of apoptosis, as determined by Caspase 3/7 induction, in DuCaP and 669 VCaP cells after treatment with different doses of AON-ISE. AR-negative MIA PaCa-2 cells were used as a negative control. Bars represent the mean ± SD of three independent experiments. (C) Western blot analysis of full-length and cleaved PARP-1 protein (anti-PARP) in DuCaP and VCaP cells, 96 hours after transfection with 0.2 pM AON-ISE or sense oligonucleotides. Protein levels of b-actin (anti-p-actin) was used as loading control. (D) Relative cell viability of DuCaP, VCaP and MIA PaCa- 2 cells after treatment with increasing doses of GapmeR-AR-V7, compared to GapmeR-Control- treated cells. Unpaired t-test; *, p<0.05; **, p<0.01 . Bars represent the mean ± SD of three independent experiments. (E) Relative cell viability of AON-ISE or SON-ISE-transfected (0.2 pM) DuCaP and VCaP cells grown in medium containing 0.1 nM R1881 , or R1881 in combination with 2 pM Enzalutamide. Unpaired t-test; *, p<0.05; **, p<0.01 ; ***, p<0.001 . Bars represent the mean ±SD of three independent experiments. Figure 5: Time follow up ofAON-ISE’s effect. (A) Expression levels of AR-V7 and UBE2C in DuCaP and VCaP cells, determined at different time points after treatment with 0.2 pM of AON-ISE, normalized to values from un-transfected cells. Unpaired t-test; *, p<0.05; **, p<0.01 ; ***, p<0.001 . Bars represent the mean ±SD of three independent experiments. (B) Relative Caspase 3/7 activity measured at different time points in DuCaP and VCaP cells treated with 0.2 pM of AON-ISE or SON- ISE. Unpaired t-test; **, p<0.01 ; ***, p<0.001 . Bars represent the mean ± SD of three independent experiments. (C) Increase in DuCaP and VCaP sub-G1 cell population at day 3 and day 4, following knockdown of AR-V7 by AON-ISE, as analysed by propidium iodide staining and flow cytometry. Graphs show gated percentages of cells corresponding to sub-G1 (light grey), G1 (medium gray), S (dark grey) and G2/M (medium grey) cell cycle phases.

Figure 6: AR mRNA expression and AR copy number in CRPC tissue and in cell line models. (A) Relative mRNA expression of AR-FL and AR-V7 in AR-positive prostate cancer cell lines 22Rv1 , DuCaP, LNCaP and VCaP, and AR-negative cell lines 5637, MIA PaCa-2 and PC3, as determined by real-time RT-PCR. AR expression levels were normalized to the expression of the HP1 BP3 housekeeping gene. (B) Expression levels of AR-FL and AR-V7 in castration-resistant prostate cancer (CRPC, n=20) specimens and in CRPC-derived cell lines 22Rv1 , DuCaP and VCaP as a reference. Bars represent the mean of each group. (C) AR and SPIN4 gene copy numbers were determined by real time PCR using genomic DNA from 22Rv1 , DuCaP, LNCaP and VCaP cells, and white blood cells from a healthy female. PCR values of the X-linked genes were normalized to PCR values of the autosomal GAPDH gene, and then normalized to the ratio found in female cells.

Figure 7: AR-FL and AR-V7 signaling. (A) Relative AR-V7, AR-FL and KLK3 mRNA expression in VCaP cells, determined 96 hours after treatment with 0.1 nM R1881 , or R1881 in combination with 2 pM Enzalutamide. Unpaired t-test; *, p < 0.05; **, p < 0.01 ; ***, p < 0.001 . Bars represent the mean ± SD of three independent experiments. (B) Relative mRNA expression levels of TMPRSS2- ERG in VCaP cells following treatment with increasing doses of AON-ISE, compared to non- transfected cells (NT). Unpaired t-test; *, p<0.05; **, p<0.01 ; ***, p<0.001 . Bars represent the mean ± SD of three independent experiments.

Figure 8: GapmeR-mediated knockdown of AR-V7. Relative expression of AR-V7 in DuCaP and VCaP cells after treatment with control GapmeR or increasing doses of AR-V7 GapmeR. AR-V7 expression levels were normalized to HP1BP3. Unpaired t-test; *, p < 0.05. Bars represent the mean ± SD of three independent experiments.

Figure 9: Effect of AON -!SE -mediated AR-V7 knockdown in 22Rv1 cells. (A) Dose- dependent effect of AON-ISE-mediated AR-V7 knockdown on cell viability of 22Rv1 cells compared to non- transfected cells (NT). Unpaired t-test; *, p<0.05; **, p<0.01 ; ***, p<0.001 . Bars represent the mean ± SD of three independent experiments. (B) Induction of apoptosis, as determined by Caspase 3/7 induction, in 22Rv1 cells after treatment with different doses of AON-ISE. Unpaired t-test; ***, p<0.001 . Bars represent the mean ± SD of three independent experiments. (C) Relative mRNA expression levels of AR-FL, AR-V7 and UBE2C in 22Rv1 cells following treatment with increasing dose of AON-ISE, compared to controls. Unpaired t-test; *, p<0.05. Bars represent the mean ± SD of three independent experiments.

Figure 10: Assessment of cell death in MIA PaCa-731 2. (A) Relative Caspase 3/7 activity measured at different time points in cells treated with 0.2 pM of AON-ISE or SON-ISE, and of non- transfected (NT) cells as a reference. Bars represent the mean ±SD of three independent experiments. (B) Cell cycle analysis of MIA PaCa-2 cells after treatment with AON-ISE and SON-ISE, assessed on day 0, 2, 3 and 4 after transfection. Ethanol-fixated and RNase-treated cells were stained with propidium and analyzed by flow cytometry. Percentages of cells in each phase of the cell cycle were determined using the Kaluza® Flow Analysis software, and are depicted in the graphs. (C) Cell doubling times were calculated by entering cell viability values, determined using CellTiterGLO, at different time points into the publically available Doubling Time calculator (http://www.doubling740time.com/compute.php). Unpaired t-test; **, p < 0.01 ; ***, p < 0.001 . Bars represent the mean ±SD of three independent measurements.

Description of the sequences

Table 1 : Sequences

Examples

Materials and Methods

Splicing signals prediction

A 4 kb sequence containing an AR intronic region known as cryptic exon 3 (CE3) and its flanking sequences (516 bp upstream and 2 418 bp downstream of CE3) was screened for the presence of intronic and exonic splice enhancer motifs. The publically available computer-based algorithms ACESCAN2 (http://genes.mit.edu/ acescan2/index.html) and ESEFinder (http://rulai.cshl.edu/cgi- bin/tools/ESE3/esefinder.cgi) were used to predict potential ISEs and ESEs, respectively. The cryptic splicing acceptor site was detected by screening the same sequence with the NetGene2 server (http://www.cbs.dtu.dk/services/NetGene2/).

AON design

Two RNA antisense oligonucleotides, a twenty-two nucleotides long AON-ISE and a nineteen nucleotides long AON-ESE, together with two control sense RNA oligonucleotides SON-ISE and SON-ESE were synthesized and modified with a phosphorothioate backbone and 2'-0-methyl groups at the sugar chain (Eurogentec, the Netherlands). Oligos were dissolved in nuclease-free water. A twenty nucleotides long GapmeR antisense oligonucleotides, GapmeR-AR-V7, was designed using SFold software 48 (http://sfold.wadsworth.org/cgi-bin/index.pl). The chimeric GapmeR (RNA5-DNA10-RNA5) was chemically modified and synthesized as described above for the RNA AONs. The GapmeR sequence described by Wheeler et al. (2012) ²⁸ was used as a control. Analytical ion exchange high-pressure liquid chromatography (HPLC) and Matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF MS) were chosen to assess the purity of all oligonucleotides, and a purity of more than 90% was considered as pure. Antisense oligonucleotide sequences are listed in table 2.

Table 2: AON’s

Cell culture

The castration-resistant prostate cancer-derived 22Rv1 (ATCC# CRL-2505), DuCaP and VCaP, the prostate cancer derived LNCaP (ATCC# CRL-1740), PC-3 (ATCC# CRL-1435) and the bladder carcinoma-derived

(ATCC# HTB-9) cell lines were maintained as monolayer cultures in Roswell Park Memorial Institute (RPMI)-1640 medium (Invitrogen), supplemented with 2 mM L-Glutamine and 10% Fetal Calf Serum (FCS; Sigma). Pancreatic carcinoma MIA-PaCa-2 cells (ATCC# CRL-1420) were grown in Dulbecco’s modified Eagle medium (DMEM) (Invitrogen) with 4.5 g/ml glucose and 1 mM pyruvate, supplemented with 2 mM L-Glutamine and 10% FCS and 2.5% of Horse serum (Invitrogen). For hormone-stimulation experiments, 0.1 nM of synthetic androgen R1881 (PerkinElmer) was added to the medium in combination with 2 pM Enzalutamide (Selleck Chemicals) or 0.2% DMSO (as a vehicle control). Results were reproduced in at least three independent experiments. All cultures were maintained in a humidified atmosphere at 37°C and 5% C02. Cell lines were authenticated in 2016 using the PowerPlex 21 system (Promega) by Eurofins Genomics (Germany)..

Construction of minigene and AR-V7 expression vector

The AR minigene was built according to sequence coordinates described by Liu et al. (2014)¹². Briefly, three PCR amplicons were generated using Phusion High-Fidelity DNA Polymerase (New England Biolabs) and joined together by SOEing PCR. Genomic DNA from the (normal) human genomic DNA was used as the template to amplify AR exon 3, CE3, exon 4 and their flanking regions. Exon 4 was amplified including a downstream 447-base-pair flanking region. CE3 was amplified including 364 base-pair upstream and 1 067-base-pair downstream flanking regions and AR exon 4 amplicon included 469 base pairs from the upstream flanking region. For SOEing PCR, all three fragments contain 20 base-pair overlapping sequences incorporated as overhangs in the forward and reverse primers. The assembled minigene was directionally cloned into the pEGFP- N3 vector (Clontech) between the Bglll and Notl sites (thereby removing the eGFP region). For cloning of the eukaryotic expression vector pCMV-AR-V7, CE3 was amplified from human genomic DNA. The fusion between an exon 3 and CE3 was achieved by SOEing PCR using Phusion High- Fidelity DNA Polymerase (New England Biolabs). The forward primers for the sewed amplicons were complemented with a 5’ GAGATG overhang and a Hindlll site, and the reverse primers with a 5’ GTTGTT following an Mfel restriction site. The insert was directionally cloned into the pEGFP- N3-derived CMV-driven expression vector backbone vector. Correct cloning was verified by Sanger DNA sequence analysis of PCR products, purified using Wizard PCR preps DNA purification system (Promega). Primer sequences for cloning and sequencing analysis are listed in Table 3.

Table 3: Primer sequences for cloning of the minigene and AR-V7 expression vector.

Transfection with antisense oligonucleotides

One day before transfection, 140,000 cells (DuCaP/VCaP) or 70,000 cells (22Rv1/MIA-PaCa-2) were seeded per well of a 24-well plate, in a total volume of 500 pi medium. After trypsinization cells were collected and seeded in charcoal-stripped serum (CSS)-containing medium to wash away traces of androgens previously reported to be present in FCS³⁰. Transfection mixtures were prepared by combining oligonucleotides (AON / SON or GapmeRs) in a desired concentration with X-tremeGENE™ 9 transfection reagent (Roche), both dissolved in Opti-MEM I Reduced serum-free medium (Invitrogen). A mix of transfection reagent alone, i.e. without oligonucleotide, was used as non-transfected control. Mixes were incubated at room temperature for 15 minutes before addition to the cells in a dropwise manner. For overexpression studies, 140,000 VCaP cells were seeded per well in 24-well plates. Twenty-four hours later cells were transfected with 250 ng of pCMV-AR- V7 expression vector or empty vector control. For minigene experiments, 70,000 MIA-PaCa-2 cells were seeded per well in 24-well plates and after 24 hours, cells were co-transfected with 500 ng of minigene or empty vector and 0.5 pM of the desired oligonucleotide. All experiments were performed at least three times. RNA isolation 379 and reverse transcription-PCR

Total RNA was isolated using TRIzol reagent (Invitrogen) according to manufacturer’s protocol. Concentration and purity of the RNA was determined on a Nanodrop-1000 spectrophotometer (Thermo Scientific). Subsequently, 2 pg of total RNA was treated with DNasel and used to 5 synthesize cDNA using random hexamer primers and Superscript II Reverse Transcriptase (Invitrogen). Real-time PCR (qPCR) analysis was performed using LightCycler 480 SYBR Green I Master Mix (Roche) and gene-specific primers (Table 4). Crossing-point (Cp) values were determined using the LightCycler 480 SW 1 .5 software (Roche). RNA not subjected to reverse transcriptase was used as a control for non-specific PCR amplification. Expression levels of the 10 human heterochromatin protein 1 binding protein 3 (HP1 BP3), the hypoxanthine phosphoribosyltransferase 1 (HPRT1) and the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes were used for normalization and relative gene expression levels were calculated according to the mathematical model for relative quantification in real-time PCR 51 . To determine TMPRSS2-ERG fusion transcript levels a forward primer directed to exon 1 of the TMPRSS2 15 transcript together with a reverse primer directed to exon 4 of the ERG transcript were used³¹.

Table 4: Primer sequences for (real-time) PCR analysis

Western blot analysis

One day before transfection, 1 200,000 (DuCaP/VCaP) cells were seeded in 10-cm dishes. When cells reached 70% confluency, oligonucleotide transfection was performed. Four days after transfection cells were harvested and washed. Cell pellets were lysed using Laemmli lysis buffer (1 mM CaCI2, 2% SDS, 60 mM Tris-Glycine pH 6.8) supplemented with 1 :50 b-mercaptoethanol (Merck). Lysates were homogenized by sheering them through a 0.5 x 25 mm syringe needle. Protein concentration was measured using the Odyssey CLx Imaging System (LI-COR) and Image Studio software (LI-COR), after staining with Coomassie brilliant blue (Merck) with serial dilutions of BSA as a standard. A total of 100 pg of protein was subjected to SDS-PAGE using 7.5% polyacrylamide gels. Proteins were electrotransferred onto PVDF membranes (Hybond 0.45 pm, Amersham Biosciences). Membranes were blocked for 1 hour in PBST/5% non-fat dry milk and incubated overnight with the primary antibody. The mouse monoclonal-antibody anti-AR-V7 (Precision Antibody, #AG10008), the rabbit polyclonal AR antibody N20 (Santa Cruz, SC-816), the rabbit monoclonal-antibody anti-PARP (Cell Signaling, #46D1 1) and the mouse monoclonal- antibody anti-p-actin (Sigma-Aldrich, clone AC-15) were used, diluted 1 :500, 1 :50 000, 1 :1 000 and

1 :5 000 in PBS-T/5% non-fat dry milk, respectively. The PO-conjugated Donkey-anti-Rabbit antibody (Amersham Biosciences, N4934) or Sheep-anti-Mouse antibody (Amersham Biosciences, NXA931) diluted 1 :50 000 in PBS-T were used as secondary antibodies. Protein bands were detected using ECL and Hyperfilm (Amersham Biosciences). Results were reproduced in two independent experiments.

Tissue collection and processing

CRPC tissue (n=20) was obtained by transurethral resection of prostate tumor tissue (TURP). TURP specimens were snap frozen in liquid nitrogen. Regions of CRPC tumors (n=20) with high percentage of epithelial tumor cells (>50%) were selected for cryo-sectioning. The use of patient materials was approved by the local ethics committee of the Radboud university medical center (CMO Arnhem-Nijmegen).

Microarray analysis

Microarray gene expression analysis on normal prostate, prostate cancer and CRPC tissue samples were performed and described previously by Leyten GH et al. (2015) ³².

Cell viability assay

To assess cell viability, 10,000 (22Rv1), 20,000 cells (DuCaP/VCaP) or 500 cells (MIA-PaCa-2) were cultured in 96-well culture plates. Transfection with oligonucleotides was done 24 hours after seeding. Four days after transfection, cell viability was measured using 3-(4,5-dimethylthiazol-2-yl)- 2,5-dephenyltetrazolium bromide (MTT, 1 mg/ml) assays. Alternatively, cell viability was measured using CellTiter-Glo luminescence assays (Promega), following the manufacturer’s instructions. Absorbance (at 490 nm) and luminescence were measured using a 428 Victor3 multilabel reader (PerkinElmer). Medium only was used as background control. To calculate the relative cell viability, cell viability values for each condition were normalized to the average of the cell viability values for control oligo-transfected cells. Each experiment was performed in triplicate and repeated at least three times. Apoptosis assay

In parallel to the cell viability assays, cells were seeded into 96-well plates for assessment of Caspase-3/7 activity using the Apo-ONE Homogenous Caspase-3/7 Assay (Promega), following manufacturer’s instructions. After 4 hours of incubation, luminescence was measured on a Victor3 multilabel reader (Perkin Elmer). The luminescence signal from medium alone was used as background. Caspase-3/7 activity was normalized to values in control oligo-transfected cells. Each experiment was performed in triplicate and repeated at least three times.

Cell cycle analysis

One day before transfection, 280,000 cells (DuCaP/VCaP) were seeded per well of a 12-well plate. The next day, cells were transfected with oligonucleotides as described above. Samples were harvested at different time points after transfection. Briefly, cells were harvested and washed with 0.9% NaCI. Cell pellets were resuspended in HBSS buffer (Invitrogen) and cells were fixed with ice- cold ethanol (58%). Fixated cells were centrifuged, resuspended in PBS and treated with RNase A (100 pg/ml, Sigma) for 40 min at 37°C. Subsequently, cells were stained with propidium iodide (40 pg/ml, Sigma) for 15 min in the dark. The samples were analyzed on a FC500 Flow Cytometer (Beckman-Coulter) and histograms were created and analyzed using Kaluza® Flow Analysis software (Beckman-Coulter). Results were reproduced in two independent experiments.

Statistical analysis

The data are presented as means ± SD from at least three independent experiments. Two- tailed paired and unpaired t-tests were performed using GraphPad Prism (GraphPad Software, Inc). Pearson correlation coefficients were used to determine the relationships between relative gene expression profiles, considering a 95% confidence interval. A p-value of <0.05 was considered statistically significant and p<0.05 is represented by one star (*), p<0.01 is represented by two stars (**) and p<0.001 is represented by three stars (***).

Results

Identification of cis-acting splicing enhancer elements in AR cryptic exon 3

For the identification of cis-acting splicing enhancer elements within the so-called cryptic exon 3 (CE3) sequence^{4 5} and its flanking regions, we used the publically available computer-based algorithms ACESCAN2¹⁵ and ESEFinder^{16 17} to predict potential intronic and exonic splicing enhancer sites, respectively. Four ISE sites were identified in the flanking region upstream of CE3 and its cryptic splice acceptor (SA) site. This SA site was detected by screening the same sequence with the NetGene2 server¹⁸ Two ESE sites were found close to the 3’ end of the CE3 sequence. One antisense oligonucleotide (AON), named AON-ISE, was designed such that it encompasses all four ISE motifs as well as the detected cryptic SA site (Fig. 1). A second AON, designated AON- ESE, was designed encompassing both ESE motifs in CE3 (Fig. 1). Both AONs were generated with a phosphorothioate backbone¹⁹ and 2'-0-methyl group modifications at the sugar chain²⁰’²¹ to make them resistant to RNAse activity.

AON-mediated suppression of AR-V7 mRNA synthesis and expression

Next, we evaluated the splicing inhibitory potential of the AONs in vitro. An AR minigene was created with CE3 and its flanking regions inserted in between exon 3 and exon 4 and flanking regions of the human AR gene (Fig. 2A). The AR minigene was transiently transfected into AR- negative MIA88 PaCa-2 cells (Fig. 6A), and both an AR-FL (exon 3-exon 4) and an AR-V7 (exon 3-CE3) transcript were expressed, suggesting that canonical and alternative splicing occurs in the minigene-encoded AR transcript (Fig. 2B). Of note, a natural preference for canonical splicing was apparent as levels of the AR-FL transcript were almost 2-fold higher than those of AR-V7 transcript. Minigene-transfected MIA PaCa-2 cells were subsequently treated with either AON-ISE or AON- ESE. Both splicing-directed AONs displayed a significant reduction of AR-V7 transcript expression but did not affect the expression levels of AR-FL (Fig. 2B). The specificity of both AONs was assessed by transfecting control oligonucleotides containing 96 the AON sequence in the sense orientation (SONs). Neither of the sense oligonucleotides, SON-ISE or SON-ESE, affected the levels of either AR minigene-encoded transcript, whilst expression levels were comparable to non- treated minigene-expressing cells (Fig. 2B). We further tested the AONs ability to knockdown AR- V7 in the CRPC-derived DuCaP and VCaP cell line models. Both cell lines express AR-FL and AR- V7 at levels comparable to those from CRPC specimens (Fig 6A and B). Upon addition of either AON, a strong decrease in AR-V7 mRNA expression was noted in both cell lines (Fig. 2C). Western blot analysis of whole cell extracts using an AR-V7-specific antibody directed against CE3-encoded amino acids showed a reduction of AR-V7 protein levels upon treatment with the AONs (Fig. 2C). Treatment with control SONs didn’t affect AR-V7 mRNA or protein levels. AR-FL mRNA as well as protein levels remained unchanged upon treatment with AONs (Fig. 2D). Because CE3 lies in close proximity to other intronic regions that can serve as cryptic exons to generate other AR variants, such as AR-V1 or AR-V3, we assessed the expression levels of these variants. AR-V1 mRNA levels were not affected in neither cell line. However, AR-V3 expression was significantly reduced in VCaP upon addition of AON-ISE, albeit to a lesser extent than that of AR-V7 expression. Staining with an AR N-terminus-specific antibody (N20) detected two protein bands of about 75 kDa. The upper band had the same size as the translated product in AR-V7 -transfected HeLa cells (data not shown), suggesting it corresponds to endogenous AR-V7 protein levels (67 kDa). This band was weakened after treatment with AON-ISE, similarly to band detected with AR-V7-specific antibody. Interestingly, the lower molecular size band (~66 kDa) was also reduced in VCaP cells, presumably corresponding to AR-V3 (Fig. 2E). Altogether, these results showed that AONs complementary to the splice enhancer motifs in and around CE3 can efficiently prevent AR-V7 mRNA synthesis in vitro.

AON-mediated knockdown of AR-V7 results in downregulation 121 of AR-V7-target genes AR-Vs have been described to have an overlapping but distinct transcriptional output than AR-FL ⁶’²². Amongst the genes described to be regulated by AR-Vs, specifically by AR-V7, are the cell cycle regulatory genes UBE2C and BUB1B ⁸·¹¹. Microarray analysis of prostate (cancer) specimens showed that, similarly to AR expression, UBE2C and BUB1B are significantly upregulated in CRPC tissue compared to benign tissues and androgen-sensitive primary prostate cancer and metastatic tissues (Fig. 3A). To be able to discriminate between AR-FL and AR-V7 expression, a qPCR validation was performed using CRPC samples from an independent cohort. Both AR-FL and AR- V7 expression positively correlated with the expression of UBE2C, but only AR-FL correlated with BUB1B expression (Fig. 3B).

We next assessed the dependency of UBE2C and BUB1B expression on AR-FL-mediated transactivation. Treatment of VCaP cells with androgens induced expression of AR-target gene KLK3 whereas treatment with Enzalutamide, a new-generation (full-length) AR antagonist, inhibited it ( Fig. 7A). Androgen stimulation also resulted in a marked induction of UBE2C and BUB1B which was reverted with the addition of Enzalutamide (Fig. 3C). Forced expression of AR-V7 in VCaP cells resulted in a significant upregulation of UBE2C and a weak, but observable increase of BUB1 B expression (Fig. 3D). These last results were obtained from cells grown in androgen-depleted medium, and hence this expression profile was considered a consequence of AR-V7 activity, exclusively. From these results it is clear that UBE2C is part of both AR-FL and AR-V7 transcriptional program and, therefore, it can be used to monitor the efficiency of AON-induced AR- V7 knockdown.

Cells were transfected with various concentrations of AON-ISE ranging from 0.02 pM to 0.5 pM. A significant AR-V7 knockdown was achieved with doses above 0.02 pM, with a clear dose- dependent decrease of AR-V7 mRNA levels in both cell lines (Fig. 3E). The dose-dependent AON- ISE-mediated knockdown of AR-V7 resulted in a dose-dependent suppression of UBE2C. Although the maximum

level of AR-V7 splicing inhibition was achieved at 0.5 pM AON-ISE, a dose of 0.35 pM suppressed UBE2C the most. Treatment with AON-ISE at a dose of 0.2 pM and 0.35 pM doses resulted in a significant downregulation of BUB1B in VCaP cells, but the AON did not affect BUB1B expression in DuCaP cells (Fig. 3F). AON-ISE treatment resulted in a specific AR-V7 knockdown and subsequently downregulation of the AR-V7 -target gene, UBE2C. Intra-chromosomal translocation of the transmembrane protease serine 2 ( TMPRSS2 ) gene to the ETS family member ERG is the most prevalent fusion in prostate cancer ²³ and the fusion gene is expressed in the VCaP cell line. AR-V7, as well as AR-FL, have been described to mediate transcriptional activation of TMPRSS2 ^{4 12 24} interestingly, under castrated conditions, AON-ISE treatment of VCaP cells resulted in downregulation of TMPRSS2-ERG mRNA levels (Fig. 7B), suggesting the involvement of AR-V7 in the transcriptional regulation of this fusion gene.

Effects of AON-ISE-mediated AR-V7 knockdown on cell proliferation and apoptosis

Prostate cancer cells rely on androgens for proliferation and survival, via activation of AR-FL and its targeted genes. One of the functional consequences of AR-V7 protein expression is its capacity to maintain proliferation of tumor cells in the absence of androgens. Thus, we evaluated the ability of AON-ISE to inhibit androgen-independent cell proliferation. To eliminate any contribution of AR- FL, cells were grown in androgen-depleted medium. A dose-dependent effect on cell viability was observed in DuCaP and VCaP upon treatment with AON-ISE but not with control SONs (Fig. 4A). To exclude that the effect of AON-ISE on cell viability is AR-independent, we assessed the effect of the AON on cell viability of AR-negative MIA PaCa-2 cells. Treatment with three different concentrations of AON-ISE had no effect on MIA PaCa-2 cell viability (Fig. 4A). The reduction of cell viability was found to be a result of the induction of apoptosis, which was marked by an increase in Caspase-3/7 activity (Fig. 4B) and, consequently, a cleavage of the Poly (ADP-ribose) polymerase 1 (PARP-1) protein ^{25 26} (Fig. 4C).

A GapmeR antisense oligonucleotide was designed to bind complementary to AR-V7 mRNA, inducing its degradation by RNAse H ( Fig. 8). GapmeR treatment caused a reduction in cell viability of both DuCaP and VCaP cells similarly to the AON-ISE treatment, without affecting MIA PaCa-2 cell viability (Fig. 4D). This validated the association of AR-V7 knockdown to the decrease in cell viability.

Reactivation of full-length AR signaling was expected to revert the cell survival inhibitory effect of the AR-V7 -targeting AONs. Upon stimulation of DuCaP or VCaP cells with the synthetic androgen R1881 AR-FL activity became re-activated (Fig. 7A) and cell viability was not significantly affected by AON-ISE compared to control SON-ISE-treated cells. Addition of Enzalutamide to the medium re-sensitized cells to AON-ISE treatment (Fig. 4E), demonstrating that AON-ISE is able to inhibit AR-V7-mediated and androgen-independent induction of cell proliferation.

Lastly, to validate the effects of AON-ISE in a cell line with a different genetic background, the CRPC-derived 22Rv1 cell line was used. 22Rv1 cells express AR-V7 at a similar level than DuCaP and VCaP cells, and the ratio of AR -V7-to- AR-FL mRNA levels in 22Rv1 cells was higher than in these two cell lines, making it an ideal model to study AR-V7 activity (Fig. 6A and B). AON-ISE treatment reduced cell viability of 22Rv1 cells by induction of apoptosis at all doses tested (Fig. 9A and B). In addition, AR-V7 and UBE2C but not AR-FL mRNA levels were significantly downregulated upon treatment with AON-ISE (Fig. 9C).

Effect of AON-ISE treatment over time

To assess whether the effect on cell viability matches the downregulation of UBE2C, apoptosis and gene expression were assessed in time following AON-ISE treatment. A robust downregulation of AR-V7 was observed in both DuCaP and VCaP cells at all time points up to 8 days after a single administration of the AON. The highest knockdown efficiency was observed between day 2 and day 4 hours after transfection with a slow decrease in knockdown thereafter. Consistently, expression levels of UBE2C followed a similar trend (Fig. 5A).

Caspase-3/7 induction was observed from day 2 onwards in both CRPC cell lines after treatment with AON-ISE (Fig. 5B). Cell cycle profiling of these cells showed an increase in the sub-G1 cell population in AON-ISE-treated cells at day 3 after transfection (Fig. 5C). While the number of apoptotic cells in VCaP remained stable over time, an increase was noted in DuCaP cell cultures (Fig. 5C). Results in MIA PaCa-2 cells showed no significant difference in the induction of Caspase activity or the number of (sub-G1) apoptotic cells between AON and SON treated cells (Fig. 10).

References

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Claims

1 An antisense oligonucleotide for the downregulation of AR-V7 mRNA by redirecting splicing or promoting mRNA degradation that binds to and/or is complementary to a polynucleotide with the nucleotide sequence a shown in SEQ ID NO 4 , wherein preferably the antisense oligonucleotide binds to or is complementary to a polynucleotide with SEQ ID NO:5 or SEQ ID NO:6, more preferably the antisense oligonucleotide binds to or is complementary to a polynucleotide with SEQ ID NO: 8, 9, 11 , 12, 14, or 15, most preferably the antisense oligonucleotide binds to or is complementary to a polynucleotide selected from the group consisting of SEQ ID NO:7, 10 and 13.

2. An antisense oligonucleotide for the downregulation of AR-V7 mRNA by redirecting splicing or promoting mRNA degradation according to claim 1 , wherein a nucleotide in the antisense oligonucleotide may be an RNA residue, a DNA residue, an RNA/DNA residue, or a nucleotide analogue or equivalent, preferably wherein the nucleotide in the antisense oligonucleotide may be an RNA/DNA residue.

3. An antisense oligonucleotide for the downregulation of AR-V7 mRNA by redirecting splicing or promoting mRNA degradation according to claim 1 or 2, wherein the antisense oligonucleotide has a length of from about 8 to about 40 nucleotides, preferably from about 10 to about 40 nucleotides, more preferably from about 14 to about 30 nucleotides, more preferably from about 16 to about 24 nucleotides, such as 16, 17, 18, 19, 20, 21 , 22, 23 or 24 nucleotides.

4. An antisense oligonucleotide for the downregulation of AR-V7 mRNA by redirecting splicing or promoting mRNA degradation according to any one of the proceeding claims, wherein said antisense oligonucleotide comprises or consists of a sequence selected from the group consisting of SEQ ID NO 16, 17 and 18.

5. An antisense oligonucleotide for the downregulation of AR-V7 mRNA by redirecting splicing or promoting mRNA degradation according any one of the preceding claims, comprising a 2'-0 alkyl phosphorothioate antisense oligonucleotide, such as 2'-0-methyl modified ribose (RNA), 2'-0-ethyl modified ribose, 2'-0-propyl modified ribose, and/or substituted derivatives of these modifications such as halogenated derivatives, preferably comprising a 2'-0-methyl modified ribose.

6. An antisense oligonucleotide for the downregulation of AR-V7 mRNA by redirecting splicing or promoting mRNA degradation according to claim 5, wherein the antisense oligonucleotide comprises a 2'-0-methyl modified ribose (RNA) and a phosphorothiorate backbone.

7. A set of antisense oligonucleotide for the downregulation of AR-V7 mRNA by redirecting splicing or promoting mRNA degradation comprising at least two antisense oligonucleotides as defined in claims 1-6, preferably wherein the set comprises or consists of: - SEQ ID NO: 16 and SEQ ID NO: 17;

- SEQ ID NO: 16 and SEQ ID NO: 18; or

- SEQ ID NO: 17 and SEQ ID NO: 18.

8. A viral vector expressing an antisense oligonucleotide for the downregulation of AR-V7 mRNA by redirecting splicing as defined in any one of claims 1 -4 when placed under conditions conducive to expression of the antisense oligonucleotide.

9. A pharmaceutical composition comprising an antisense oligonucleotide for redirecting splicing or promoting mRNA degradation according to any one of claims 1 -4 or a viral vector according to claim 8 and further comprising a pharmaceutically acceptable excipient.

10. A pharmaceutical composition according to claim 8, wherein the pharmaceutical composition is for intratumoral, intravascular, intravenous, or subcutaneous administration, preferably intravenous administration.

1 1 . The antisense oligonucleotide for the downregulation of AR-V7 mRNA by redirecting splicing or promoting mRNA degradation according to any one of claims 1 -4, the set according to claim 7, the vector according to claim 8 or the pharmaceutical composition according to any one of claims 8-10 for use as a medicament, preferably for use as a medicament for treating an AR (Androgen

Receptor) related disease or a condition requiring redirecting splicing of the (pre)mRNA of AR or promoting AR-V7 mRNA degradation.

12. Use of the antisense oligonucleotide for the downregulation of AR-V7 mRNA by redirecting splicing or promoting mRNA degradation according to claims 1 -4, the set according to claim 7, the vector according to claim 8 or the pharmaceutical composition according to any one of claims 8-10 for treating an AR related disease or a condition requiring redirecting splicing of the (pre)mRNA of AR or promoting AR-V7 mRNA degradation.

13. A method for downregulating of AR-V7 mRNA by redirecting splicing of AR or promoting AR-

V7 mRNA degradation in a cell, the method comprising contacting the cell with an antisense oligonucleotide for the downregulation of AR-V7 mRNA by redirecting splicing or promoting mRNA degradation as defined in claims 1 -4, the set according to claim 7, the vector according to claim 8 or the pharmaceutical composition according to any one of claims 8-10.

14. The antisense oligonucleotide for the downregulation of AR-V7 mRNA by redirecting splicing or promoting mRNA degradation for use according to claim 1 1 , the use according to claim 12 or the method according to claim 13, wherein the AR related disease or a condition is cancer, preferably prostate cancer, more preferably castration-resistant prostate cancer (CRPC).