EP4658783A1

EP4658783A1 - Splice-switching antisense oligonucleotide (sso) modulating the expression of a tumor specific protein derived from te-exon junction (jet) transcript

Info

Publication number: EP4658783A1
Application number: EP24703345.9A
Authority: EP
Inventors: Marianne BURBAGE; Sebastian Amigorena; Blandine BAUDON
Original assignee: Centre National de la Recherche Scientifique CNRS; Institut National de la Sante et de la Recherche Medicale INSERM; Institut Curie
Current assignee: Centre National de la Recherche Scientifique CNRS; Institut National de la Sante et de la Recherche Medicale INSERM; Institut Curie
Priority date: 2023-02-02
Filing date: 2024-02-02
Publication date: 2025-12-10
Also published as: WO2024161014A1

Abstract

The present disclosure relates to a splice-switching antisense oligonucleotide (SSO) that modulates the expression of a tumor protein derived from TE-exon junction (JET) transcript in a tumor cell and its use in the treatment of a cancer in a subject in need thereof.

Description

SPLICE-SWITCHING ANTISENSE OLIGONUCLEOTIDE (SSO) MODULATING THE EXPRESSION OF A TUMOR SPECIFIC PROTEIN DERIVED FROM TE- EXON JUNCTION (JET) TRANSCRIPT

TECHNICAL FIELD OF THE DISCLOSURE

BACKGROUND

Harnessing the immune system to generate effective responses against tumors is a central goal of cancer immunotherapy. Part of the effective immune response involves T lymphocytes specific for tumor antigens. T cell activation requires their interaction with antigen-presenting cells (APCs), commonly dendritic cells (DCs), expressing TCR-cognate peptides presented in the context of a major histocompatibility molecule (MHC) and costimulation signals. Subsequently, activated T cells can recognize peptide-MHC complexes presented by all cell types, even malignant cells. Neoplasms often contain infiltrating T lymphocytes reactive with tumor cells.

However, the efficiency of immune responses against tumors is severely dampened by various immunosuppressive strategies developed by tumors; e.g., tumor cells express receptors that provide inhibitory signals to infiltrating T cells, or they secrete inhibitory cytokines. The development of checkpoint blockade therapy has provided means to bypass some of these mechanisms, leading to more efficient killing of cancer cells. The promising results yielded by this approach have opened up new avenues for the development of T cellbased immunotherapy. Checkpoint inhibitors are, however, effective in a minority of patients and only in limited types of cancer.

A major goal in immunotherapy is to increase the proportion of responding patients and extend the cancer indications. Vaccination, administration of anti-tumor antibodies, or administration of immune cells specific for tumor antigens have all been proposed to increase the anti-tumor immune response, and can be administered alone, with other therapies such as chemotherapy or radiation, or as a combination therapy with checkpoint blockers. The selection of antigens able to trigger anti-tumor immunity without targeting healthy tissues has been a long-standing challenge. The search for tumor neoantigens has mostly been focused on mutated sequences appearing as in cancer cells. These antigens are often unique to each patient. Tumor antigens (the ones preferentially expressed in tumor cells) are, however, self-antigens that represent poor targets for vaccination (probably due to central tolerance). Identifying shared true neoantigens (absent from tissues) is a major challenge for the field.

The inventors have previously shown that splicing events between coding exons and transposable element represent a source of recurrent, immunogenic tumor specific proteins in tumor cells (WO2021/043804, WO2022/189620, WO2022/189626 and

WO2022/189639).

However, in order to have an effective immune response against tumor cells expressing these tumor specific antigens, there remains a need to develop a new strategy for inducing the expression of these tumor specific antigens in tumor cells to enhance the anti-tumor immune response.

Strategies to inhibit the expression of oncogenes and the carcinogenic splice variants of essential genes at the mRNA level have been developed in the past few decades. Splicemodulating antisense oligonucleotides have been used for suppressing the genes involved in the progression of cancer (Dean N.M., Bennett C.F. Oncogene. 2003;22:9087-9096; Mercatante D.R., Mohler J.L., Kole R. J. Biol. Chem. 2002;277:49374-49382) or for blocking specific splice junctions to create novel isoforms that may serve as neoantigens (W02020/157760). However, this strategy was never developed to modulate splicing events between coding exons and transposable element (TE).

SUMMARY

In the present application, the inventors showed for the first time that splice-switching antisense oligonucleotide can efficiently be used to modulate splicing events between coding exons and transposable element (TE) and modulate the expression of tumor-specific protein derived from TE-exonic sequence splicing junction (JET) transcript.

The present disclosure relates to a Splice-switching antisense oligonucleotide (SSO) that modulates splicing events between coding exons and transposable element (TE) and modulates the expression of a tumor specific protein derived from an exon-transposable element splicing junction (JET) transcript resulting from the splice junction between a TE and exonic sequence, wherein said SSO comprises a complementary nucleic acid sequence, preferably of 18 to 30 nucleotides in length to a splice modulator site (e.g., splice silencer site) localized within a pre-mRNA sequence encoding said JET transcript.

In a particular embodiment, said splice modulator site (e.g. splice silencer site) is localized within a TE sequence or a sequence adjacent to TE or exonic sequence of said pre-mRNA JET transcript, in particular within a sequence of up to 200 nucleotides adjacent to said TE or exonic sequence. In a particular embodiment, the SSO comprises a chemically modified nucleic acid sequence, preferably a 2’-O-methyl (2’0me) modified phosphorothioate oligonucleotide. In another particular embodiment, said JET transcript includes an openreading frame coding for a non-canonical peptide or protein isoform.

In a particular embodiment, the present disclosure relates to a Splice-switching antisense oligonucleotide (SSO) that induces the expression of a tumor specific antigen derived from an exon-transposable element splicing junction (JET) transcript resulting from the splice junction between a TE and exonic sequence, wherein said SSO comprises a complementary nucleic acid sequence to a splice silencer site localized within a pre-mRNA sequence encoding said JET transcript, preferably wherein said TE and exonic sequences are selected from the pairs consisting of: SEQ ID NO: 1 and 2; 3 and 4, 5 and 6, 7 and 8, 9 and 10, 11 and 12, 13 and 14, 15 and 16, 17 and 18, and 24 and 25, more preferably SEQ ID NO: 7 and 8, SEQ ID NO: 15 and 16 or SEQ ID NO: 24 and 25. In a particular embodiment, said complementary nucleic acid sequence is of 18 to 30 nucleotides in length. In another particular embodiment, said SSO comprises a chemically modified nucleic acid sequence, preferably a 2’-O-methyl (2’0Me) modified phosphorothioate oligonuleotide. In a particular embodiment, the TE is located at the 5’-end of the JET transcript and the exonic sequence is located at the 3’-end of the JET transcript or the TE is located at the 3’-end of the JET transcript and the exonic sequence is located at the 5’-end of the JET transcript.

In a particular embodiment, said JET transcript results from the splice junction between a TE sequence and exonic sequence of SEQ ID NO: 7 and 8 and preferably said SSO is SEQ ID NO: 21. In another particular embodiment, said JET transcript results from the splice junction between a TE sequence and exonic sequences of SEQ ID NO: 15 and 16 and preferably said SSO is SEQ ID NO: 22 or 23. In another particular embodiment, said JET transcript results from the splice junction between a TE sequence and exonic sequences of SEQ ID NO: 24 and 25 and preferably said SSO is SEQ ID NO: 27. In another embodiment, the present disclosure relates to a Splice-switching antisense oligonucleotide (SSO) that induces the expression of a tumor suppressor protein derived from an exon-transposable element splicing junction (JET) transcript resulting from the splice junction between a TE and exonic sequence, wherein said SSO comprises a complementary nucleic acid sequence to a splice silencer site localized within a pre-mRNA sequence encoding said JET transcript, preferably wherein said TE and exonic sequences are selected from the pairs consisting of: SEQ ID NO: 28 and 29. In a particular embodiment, said complementary nucleic acid sequence is of 18 to 30 nucleotides in length. In another particular embodiment, said SSO comprises a chemically modified nucleic acid sequence, preferably a 2’-O-methyl (2’0Me) modified phosphorothioate oligonuleotide. In a particular embodiment, the TE is located at the 5’-end of the JET transcript and the exonic sequence is located at the 3’-end of the JET transcript or the TE is located at the 3’-end of the JET transcript and the exonic sequence is located at the 5’-end of the JET transcript.

In a particular embodiment, said JET transcript results from the splice junction between a TE sequence and exonic sequence of SEQ ID NO: 28 and 29 and preferably said SSO is SEQ ID NO: 31 or 32.

In another embodiment, the present disclosure relates to a Splice-switching antisense oligonucleotide (SSO) that inhibits the expression of a tumor suppressor protein derived from an exon-transposable element splicing junction (JET) transcript resulting from the splice junction between a TE and exonic sequence, wherein said SSO comprises a complementary nucleic acid sequence to a splice enhancer site localized within a pre-mRNA sequence encoding said JET transcript, preferably wherein said TE and exonic sequences are selected from the pairs consisting of: SEQ ID NO: 28 and 29. In a particular embodiment, said complementary nucleic acid sequence is of 18 to 30 nucleotides in length. In another particular embodiment, said SSO comprises a chemically modified nucleic acid sequence, preferably a 2’-O-methyl (2’0Me) modified phosphorothioate oligonuleotide. In a particular embodiment, the TE is located at the 5’-end of the JET transcript and the exonic sequence is located at the 3’-end of the JET transcript or the TE is located at the 3’-end of the JET transcript and the exonic sequence is located at the 5’-end of the JET transcript.

In a particular embodiment, said JET transcript results from the splice junction between a TE sequence and exonic sequence of SEQ ID NO: 28 and 29 and preferably said SSO is SEQ ID NO: 33. The present disclosure also relates to a pharmaceutical composition comprising the SSO as described above and a pharmaceutical acceptable excipient.

In another aspect, the present disclosure relates to the SSO or pharmaceutical composition as described above for modulating expression of a tumor specific protein derived from an exon-transposable element splicing junction (JET) transcript in a tumor cell of a subject in need thereof suffering from a cancer, preferably lung cancer.

In another aspect, the present disclosure relates to the SSO or pharmaceutical composition as described above for inducing expression of a tumor specific antigen derived from an exon- transposable element splicing junction (JET) transcript in a tumor cell of a subject in need thereof suffering from a cancer, preferably lung cancer.

In another aspect, the present disclosure relates to the SSO or pharmaceutical composition as described above for modulating the expression of a tumor suppressor protein derived from an exon-transposable element splicing junction (JET) transcript in a tumor cell of a subject in need thereof suffering from a cancer, preferably lung cancer.

In a particular embodiment, said SSO is administered in combination with an immunotherapy, preferably selected from the group consisting of: immune inhibitory checkpoint inhibitors, T-cell transfer therapy, monoclonal antibodies and immune system activators.

The present disclosure also relates to a method for identifying a SSO that modulates the expression of a tumor specific antigen derived from TE-exon junction (JET) transcript comprising the steps of: a) identifying a putative splice modulator site localized within a pre-mRNA encoding JET transcript, preferably said JET transcript results from the splice junction between a TE sequence and exonic sequence selected from the pairs consisting of: SEQ ID NO: 1 and 2; 3 and 4, 5 and 6, 7 and 8, 9 and 10, 11 and 12, 13 and 14, 15 and 16, 17 and 18, 24 and 25 and, 28 and 28, more preferably SEQ ID NO: 7 and 8, SEQ ID NO: 15 and 16, SEQ ID NO: 24 and 25 or SEQ ID NO: 28 and 29, b) introducing a SSO comprising a complementary sequence to said splice modulator site localized within a pre-mRNA encoding JET transcript into a cell, c) determining the expression level of the JET transcript in said cell, d) selecting said SSO when the expression level of the JET transcript in said cell is higher or lower than a control value.

The present disclosure also relates to a kit for modulating the expression of a tumor specific protein derived from JET transcript comprising a SSO comprising a complementary sequence to said splice modulator site localized within a pre-mRNA encoding JET transcript into a cell, preferably wherein said JET transcript results from the splice junction between a TE sequence and exonic sequences of SEQ ID NO: 7 and 8, 15 and 16, 24 and 25, and 28 and 28, more preferably said SSO comprises or consists of a sequence selected from the group consisting of: SEQ ID NO: 21, 22, 23, 27, 31 to 33.

LEGEND FIGURES

Figure 1. SSO 2242 HD(-49-69) increases 2242 JET expression. TaqMan qPCR results for JET 2242 transcript (A) and normal junction between exon 12 and 13 (B) after SSO transfection at 500nM in A459 cell line. Control of the experiment is Scramble transfection in A459 cell line.

Figure 2. Determination of the optimal concentration of SSO 2242 HD(-49-69). TaqMan qPCR results for 2242 JET and normal junction expression in cells treated with different concentrations of SSO (A). A549 human lung cells viability percentage in cells treated with different concentrations of SSO (B). Control of the experiment is Scramble transfection in A459 cell line.

Figure 3. Determination of the duration SSO 2242 HD(-49-69) effect. qPCR results for 2242 JET (A) and normal junction (B) expression for different incubation times (24h, 48h and 72h). Control of the experiment is Scramble transfection in cell line.

Figure 4. SSO 3175 HA(-44-69) and 3175HD(-20-45) both increase 3175 JET expression in A549 cells line. TaqMan qPCR results for JET 3175 and normal junction between after SSO transfection at lOOnM in A549 cell line. Control of the experiment is Scramble transfection A549 cell line.

Figure 5. SSO 3175 HA(-44-69) and 3175HD(-20-45) both increase 3175 JET expression in H1650 cell line. TaqMan qPCR results for JET 3175 and normal junction between after SSO transfection at lOOnM in H1650 cell line. Control of the experiment is Scramble transfection Hl 650 cell line. Figure 6. SSO 3175 HA(-44-69) increases 3175 JET expression in H1650 cell line until at least 5 days. TaqMan qPCR results for JET 3175 and normal junction. Control of the experiment is Scramble transfection H1650 cell line. SSO transfection was done at day 1 followed by the same transfection at day 3 (both at 50nM in Hl 650 cell line). Lysis buffer (RLT buffer) was added at day 5.

Figure 7. 148 JET expression is increased by SSO 148_HA(-94-74). TaqMan qPCR results for JET 148 transcript after SSO transfection at lOOnM in H1650 cell line. Control of the experiment is Scramble transfection in H1650Luc cell line.

Figure 8: PTEN JET expression is modulated by SSO PTEN_HA(+56+78), PTEN_HD(+ 13+38) and PTEN_HD(+53+78). TaqMan qPCR results for JET PTEN transcript (A) and normal junction between exon 5 and 6 (B) after SSO transfection at 50nM in Hl 650 cell line. Control of the experiment is Scramble transfection in H1650Luc cell line.

DETAILED DISCLOSURE

SSO targeting a tumor-specific protein derived from a JET transcript

Transposable elements represent between 40 and 50 % of mammalian genomes and originates from sequences capable of transposing, i.e. replicating and inserting themselves in a distal part of the genome. De novo insertions, resulting from full TE transposition cycles, can have dramatic consequences on cell physiology, but they are rare and often private events. Most TE-derived sequences in humans are partially degenerated and have lost the capacity to perform a full transposition cycle. Yet, they retain the potential to regulate gene expression in several ways: they can act as promoter and initiate transcription or provide alternative translation start sites or can also contribute alternative splice. In response to such widespread impact on gene expression and genome stability, mammalian cells have evolved multiple epigenetic mechanisms, including DNA methylation and various histone modifications, to repress TE expression and transposition. Such mechanisms can be compromised in tumor cells, leading a partial release of TE transcriptional control.

The inventors have previously shown that splicing events between exons and TEs can generate tumor specific proteins such as tumor specific antigens for presentation on major histocompatibility complex (MHC), tumor suppressor proteins, or onco-proteins.

The inventors in the present application showed for the first time that splicing switching oligonucleotides (SSO) targeting a splice modulator site within pre-mRNA encoding TE- exon junction modulates the expression of a tumor protein derived from TE-exonic splicing junction (JET) transcript in a tumor cell. Treatment of subject with SSO will effectively modulate expression of tumor protein derived from JET transcript in the subject tumor cell.

In particular, the inventors in the present application showed for the first time that splicing switching oligonucleotides (SSO) targeting a splice inhibitor site within pre-mRNA encoding TE-exon junction induces the expression of tumor specific antigen derived from TE-exonic splicing junction (JET) transcript in a tumor cell. Treatment of subject with SSO will effectively induce expression of tumor-specific antigen derived from JET transcript in the subject tumor cell that will be targeted by immunotherapy.

The present disclosure relates to a SSO that modulates expression of a tumor specific protein, preferably induces tumor specific antigen derived from JET transcript into a cell (e.g. tumor cell).

A “tumor protein” or “tumor specific protein” is a protein expressed specifically in a tumor cell. In particular, said tumor protein expression is higher in a tumor cell as compared to a non-tumoral cell.

The tumor protein derived from TE-exonic splicing junction (JET) transcript can be a tumor specific antigen or a protein encoded by an oncogene or by a tumor suppressor gene.

A tumor specific antigen is a peptide that arises from somatic alterations recognized as nonself by immune system and is presented by antigen-presenting cells (APC), such as dendritic cells (DC) and tumor cells themselves. Cross-presentation plays an important role as the APC is able to translocate exogenous antigens from the phagosome into the cytosol for proteolytic cleavage into the major histocompatibility complex (MHC) epitopes by the proteasome.

According to the present disclosure, the alteration at the origin of tumor specific antigen corresponds to the splice junctions between an Exon and a transposable element (TE) and the transcription of a fusion mRNA sequence that comprises a transposable element (TE) sequence and an exonic sequence. This may arise from somatic (i.e. : specifically in the tumor clone) transposition. It may also arise not from de novo transposition but from tumor specific transcriptional de-repression such that a TE and nearby gene are co-transcribed.

In a particular embodiment, the tumor specific antigens are major histocompatibility complex I or II -bound peptides derived from a fusion transcript comprising a Transposable element (TE) and an exonic sequence resulting from the splice junctions between Exons and transposable element (TE) (JET).

Typically, the tumor specific antigen binds MHC class I or class II with a binding affinity Kd of less than about 10'⁴, 10'⁵, 10'⁶, 10'⁷, 10'⁸ or 10'⁹ M. Affinity of the tumor specific antigen for MHC alleles can be determined in silico using appropriate software such as netMHCpan.

The tumor specific antigen derived from JET transcript binds to human leukocyte antigen (HLA) and can be recognized by T cells. Said tumor specific antigen can thus be capable of raising a specific T-cell response, in particular cytotoxic T-cells response and/or a specific helper T-cell response initiating an anti-cancer immune response.

A tumor specific antigen according to the present disclosure may be completely absent from normal healthy samples (i.e., not expressed in normal healthy samples) and thus be specific to tumor samples. Alternatively, it may be expressed at low levels in normal cells and differentially expressed in tumor samples as compared to normal (healthy) samples.

In a particular embodiment, said tumor specific antigen comprises at least 8, 9, 10, 11, or 12 amino acids, derived from a fusion transcript comprising a transposable element (TE) and exonic sequence. The peptide may be 8-9, 8-10, 8-11, 12-25, 13-25, 12- 20, or 13-20 amino acids in length.

Although the transcript overlaps a junction between a TE sequence and an exonic sequence, it is understood that the tumor specific antigens itself may not comprise the junction.

Tumor protein derived from a JET transcript could also provide a novel protein isoform that comprises non-exonic amino acid sequence (e.g., TE sequence), derived from variant transcripts that are identified as comprising non-exonic sequence and that are furthermore recurrent and translated. These variant transcripts are expressed and can generate protein isoforms that are stable in cells.

The tumor protein such as tumor suppressor protein or oncoprotein derived from JET transcript may have the same function as the canonical gene or in most cases a different function compared to canonical isoforms resulting in cancer. Splicing events of a protooncogene or tumor suppressor could also affect a regulatory mechanism of the protooncogene or tumor suppressor gene expression and lead to higher or lower gene expression. Oncogenes (also named cancer genes) are genes encoding protein (oncoprotein) whose action positively promotes cell proliferation or growth. The normal non mutant versions are known as proto-oncogenes. The mutant versions are excessively or inappropriately active leading to tumor growth. Oncogenes can be identified in the Cancer Gene Marker Database (CGMD) (Pradeepkiran, J., Sainath, S., Kramthi Kumar, K. et al. CGMD:. Sci Rep 5, 12035 (2015) “An integrated database of cancer genes and markers”). Oncogenes (ONCs) can also be downloaded from Network of Cancer Genes database (NCG 5.0) (A’ O, Dall'Olio GM, Mourikis TP, Ciccarelli FD, Nucleic Acids Res. 2016 Jan 4; 44(Dl):D992-9; “NCG 5.0: updates of a manually curated repository of cancer genes and associated properties from cancer mutational screenings”). Non-limitative examples of oncogenes include: L-MYC, LYL-1, LYT-10, LYT-10/Cal, MAS, MDM-2, MLL, MOS, MTG8/AML1, MYB, MYH11/CBFB, NEU, N-MYC, OST, PAX-5, PBX1/E2A, PIM-1, PRAD-1, RAF, RAR/PML, RAS-H, RAS-K, RAS-N, REL/NRG, RET, RH0M1, RH0M2, ROS, SKI, SIS, SET/CAN, SRC, TALI, TAL2, TAN-1, TIAM1, TSC2,and TRK.

Insertion of TE sequence in oncogenes can alter their oncogenic activity. Insertion of TE sequence, in oncogene active domains could therefore result in constitutive activity of the oncogenes, similar to driver mutations. These fusions giving chimeric oncogenes could thus represent a new family of oncogenic proteins. If this is the case, targeting the activity of these oncogene derived from JET transcript with SSO could represent a potential therapeutic approach for cancer where these chimeric oncogenes are expressed.

In a particular embodiment, the SSO according to the present disclosure can modulate the splice event, and modulate, preferably inhibit the expression of an oncoprotein derived from a JET transcript resulting from the splice junction between a TE and exonic sequence.

Tumor suppressor genes (also named anti-oncogenes) represent the opposite side of cell growth control, normally acting to inhibit cell proliferation and tumor development. Thus tumor suppressor genes are genes that normally suppress cell division or growth. Loss of TSG function promotes uncontrolled cell division and tumor growth. Rb, a tumor suppressor gene that was identified by the genetic analysis of retinoblastoma an encoding a transcriptional regulatory protein, served as the prototype for the identification of additional tumor suppressor genes that contribute to the development of many different human cancers. Tumor suppressor genes are notably described in “Cooper GM. The Cell: A Molecular Ap^proach. 2nd edition. Sunderland (MA): Sinauer Associates; 2000. Tumor Suppressor Genes”. Tumor-suppressor genes (TSGs) can also be downloaded from Tumor Suppressor Gene database (TSGene 2.0) (see for reference Zhao M, Kim P, Mitra R, Zhao J, Zhao Z; Nucleic Acids Res. 2016 Jan 4; 44(D1):D1O23-31; “TSGene 2.0: an updated literature-based knowledgebase for tumor suppressor genes”). In this context, non-limitative examples of tumor suppressor genes include: APC, BRCA1, BRCA2, DPC4, INK4, MADR2, NF1, NF2, p53, PTC, PTEN, Rb, RBI, VHL, WT1, BUB1, BUBR1, TGF-PRII, Axin, DPC4, p300, PPARy, pl6, Bcl2, SWVSNF, DPC4, CD95, PTEN (Phosphatidylinositol 3,4,5- trisphosphate 3-phosphatase and dual-specificity protein phosphatase), and hSNF5, preferably PTEN. In a particular embodiment of the present disclosure, the tumor protein derived from TE-exonic splicing junction (JET) transcript is a tumor suppressor protein, preferably PTEN.

PTEN (Phosphatase and TENsin homolog) tumor suppressor gene (based on lipid phosphatase activity) (UniProtKB: P60484, last update on Jan 24, 2024) is often mutated in malignant tumor with high incidence in the general population. See Eng, “ PTEN: one gene, many syndromes,” Hum Mutat, 22: 183-981 (2003); Trotman et al, “Ubiquitination regulates PTEN nuclear import and tumor suppression,” Cell 128: 141-56 (2007); Shen et al., “Essential role for nuclear PTEN in maintaining chromosomal integrity,” Cell, 128 : 157— 70 (2007). PTEN negatively regulates the PI3K/AKT pathway which controls cell growth, proliferation, survival and metabolism. Mutation or dysregulation of PTEN expression is commonly observed in a broad spectrum of cancers.

Insertion of TE sequence in tumor suppressors could inactivate their suppressor functions, leading typically to a loss of function (for example through introduction of stop codons, changes in ORF or disruptive amino acid stretches), thereby contributing to the oncogenic process. In this particular embodiment, the SSO according to the present disclosure can modulate the splice event, and modulates, preferably inhibits the expression of a tumor suppressor protein derived from a JET transcript resulting from the splice junction between a TE and exonic sequence.

In another aspect, insertion of TE sequence in tumor suppressors could also result in constitutive activity of the tumor suppressor protein. These fusions giving chimeric tumor suppressor protein could thus represent a new family of tumor suppressor protein. If this is the case, enhancing the activity of these tumor suppressor protein derived from JET transcript with SSO could represent a potential therapeutic approach for cancer. Oncogenes, tumor suppressor genes or “double agent” genes (with both oncogenic and tumor-suppressor functions) can be systematically identified through database search and text mining. Indeed, information on oncogenes or tumor suppressor genes can typically be found in Ensembl database (but see also Shen L, Shi Q, Wang W. Double agents: genes with both oncogenic and tumor-suppressor functions. Oncogenesis. 2018;7(3):25. Published 2018 Mar 13). Double agent genes may be identified as genes overlapped between the two above mentioned databases (see also Shen et al., Oncogenesis 2018 above).

Transposable element (TE) -exon splicing junction transcript

Tumor specific protein (e.g., tumor specific antigen) according to the present disclosure are derived from a fusion transcript comprising a Transposable element (TE) and an exonic sequence resulting from the splice junctions between Exons and transposable element (TE) (JET).

In the present application, the terms “fusion transcript”, “TE-exon transcript” or “Junction Exon-TE (JET) transcript” or “Transposable element (TE) -exon splicing junction transcript” are defined as a transcript that aligns in part with an exon sequence and in part with a transposable element (TE) sequence and has a normalized number of read greater than 2.10'⁶. The normalized number of reads is defined as the number of reads that cover the fusion divided by the library size of the sample.

A “messenger RNA (mRNA)” or “transcript” is a single-stranded RNA molecule that corresponds to the genetic sequence of a gene and is read by the ribosome in the process of producing a protein. mRNA is created during the process of transcription, where the enzyme RNA polymerase converts genes into primary transcript mRNA (also known as pre-mRNA). This pre-mRNA usually still contains introns, regions that will not go on to code for the final amino acid sequence. These are removed in the process of RNA splicing, leaving only exons, regions that will encode the protein. This exon sequence constitutes mature mRNA also named herein “transcript” or “mRNA”. Mature mRNA is then read by the ribosome, and, utilizing amino acids carried by transfer RNA (tRNA), the ribosome creates the peptide sequence a process called translation.

RNA splicing is an essential process wherein precursor messenger RNA (pre-mRNA) is reshaped into mature mRNA. In alternative splicing, exons of any pre-mRNA get rearranged to form mRNA variants and subsequently protein isoforms, which are distinct both by structure and function. The process of splicing is catalyzed by the RNA-protein complex known as the spliceosome. During splicing, introns are removed, and exons are joined together.

“transposable elements” (TEs) are DNA sequences that can move from one location on the genome to another. TE element can be divided in class I (retrotransposons, including those containing LTRs, long interspersed nuclear elements (LINEs), short interspersed nuclear elements SINEs and SINE-VNTR-Alu (SVA)) that replicate with an RNA intermediate coupled with reverse transcription and class II (DNA transposons) that replicates with DNA intermediate. This includes both autonomous and non-autonomous elements from both classes. According to the present disclosure the TE sequences can be for example selected from TE of class I, such as retrotransposons including Endogenous RetroVirus (ERVs), Long interspersed nuclear elements (LINEs) and short interspersed nuclear element (SINEs), SINE-VNTR-Alu (SVA) and mammalian long terminal repeat transposon (MaLR), and TE of class II, such as DNA transposons endogenously part of the genome.

Retrotransposons are by far more abundant, and their characteristics are similar to retroviruses, such as HIV. Retrotransposons function via reverse transcription of an RNA intermediate replicative mechanism. They are commonly grouped into three main orders: retrotransposons with long terminal repeats (LTRs) flanking the retroelement main body, which encode reverse transcriptase, similar to retroviruses; retroposons with long interspersed nuclear elements (LINEs, LINE- Is, or Lis), which encode reverse transcriptase but lack LTRs, and are transcribed by RNA polymerase II; and retrotransposons with short interspersed nuclear elements (SINEs) that do not encode reverse transcriptase and are transcribed by RNA polymerase III.

DNA transposons have a transposition mechanism that does not involve an RNA intermediate. The transpositions are catalyzed by several transposase enzymes. LTRs include endogenous retroviruses (ERVs), while non-LTR TEs subdivide into long- interspersed (LINEs) and short interspersed elements (SINEs), nonautonomous transposons mobilized by the LINE integration machinery. These lineages are composed of phylogenetically related families, further branching out into multiple subfamilies, each originating from one precursor copy. With time, the accumulation of mutations introduced divergence in the consensus sequence within members of each subfamily. For review on TE retrotransposon, see Richardson, Sandra R et al. “The Influence of LINE-1 and SINE Retrotransposons on Mammalian Genomes.” Microbiology spectrum vol. 3,2 (2015):

MDNA3-0061-2014.

A typical LI element is approximately 6,000 base pairs (bp) long and consists of two nonoverlapping open reading frames (ORF) which are flanked by untranslated regions (UTR) and target site duplications. LINE-1 retrotransposons have been amplifying in mammalian genomes for greater than 160 million years. In humans, the vast majority of LINE- 1 sequences have amplified since the divergence of the ancestral mouse and human lineages approximately 65- 75 million years ago. Sequence comparisons between individual genomic LINE-1 sequences and a consensus sequence derived from modern, active LINE- Is can be used to estimate the age of genomic LINE-ls (Khan H, Smit A, Boissinot S; Genome Res. 2006 Jan; 16(l):78-87). LI subfamilies typically categorize into old (L1M, AluJ), intermediate (LIP, L1PB, AluS), young (L1HS, LIPA, AluY) and related (HAL, FAM) subfamilies. In humans, the only autonomously active family is the long-interspersed element- 1 (LINE-1 or LI), however a few LI copies are still retrotransposition competent, all of them belonging to the youngest human- specific L1HS subfamily.

SVA elements comprise an evolutionarily young, non-autonomous retrotransposon family that arose in primate lineages approximately 25 million years ago (Hancks DC, Kazazian HH Jr, Semin Cancer Biol. 2010 Aug; 20(4):234-45). A typical SVA element is approximately 2,000 bp and has a composite structure that consists of: 1) a hexameric CCCTCT repeat; 2) an inverted Alu-like element repeat; 3) a set of GC-rich variable nucleotide tandem repeats (VNTRs); 4) a SINE-R sequence that shares homology with HERVK-10, an inactive LTR retrotransposon; and 5) a canonical cleavage polyadenylation specificity factor (CPSF) binding site that is followed by a poly (A) tract. The youngest SVA subfamilies include SVA-D, SVA-E, SVA-F, and SVA-F 1 subfamilies.

According to the present disclosure an “exon” or “exonic sequence” is any part of a gene that will encode a part of the final mature RNA produced by that gene after introns have been removed by RNA splicing. The term exon refers to both the DNA sequence within a gene and to the corresponding sequence in RNA transcripts. In RNA splicing, introns are removed and exons are covalently joined to one another as part of generating the mature messenger RNA. An exonic sequence as per the present application comprises at least a portion of one or more exon. Typically, the exonic sequence comprises at least a portion of one or 2 exons. The untranslated sequences in 3’ end and in 5’ end (3’UTR and 5’UTR) present in mature RNA after splicing are exonic sequences but are non-coding sequences because these sequences are located upstream of the start codon for the translation (5’UTR) or downstream of the stop codon ending the translation (3’UTR).

According to the present disclosure, in JET transcripts, the TE can be donor (in 5’ position) or acceptor (in 3’ acceptor) and correspondingly the exon can be acceptor or donor. TE-exon splicing results in the incorporation of parts of the “non-coding” genome into the coding genome, thereby exposing non-coding genomic sequences to the translation machinery. These JET transcripts include an ORF (open reading frame).

When the TE is acceptor, the ORF of the fusion transcript is canonical (i.e. the same as the canonical transcript), whereas when the TE is the donor the ORF can be canonical (generally ORF1) or can be shifted by 1 or 2 nucleotides (typically ORFs 2 and 3 respectively).

The fusion transcripts include not only the fused TE and exon sequences but can also further include exon(s), upstream the fusion breakpoint (between the exon and the TE) if the exon is donor or downstream the fusion breakpoint if the TE is donor, corresponding to the various transcript isoforms.

A reading frame is a way of dividing the sequence of nucleotides in a nucleic acid (DNA or RNA) molecule into a set of consecutive, non-overlapping triplets.

An open reading frame (ORF) is the part of a reading frame that has the ability to be translated into a peptide. An ORF is a continuous stretch of codons that contain a start codon (for example AUG) at a transcription starting site (TSS) and a stop codon (for example UAA, UAG or UGA). An ATG codon within the ORF (not necessarily the first) may indicate where translation starts. The transcription termination site is located after the ORF, beyond the translation stop codon. In eukaryotic genes with multiple exons, ORFs span intron/exon regions, which may be spliced together after transcription of the ORF to yield the final mRNA for protein translation.

A “canonical ORF” as herein intended is a protein coding sequence with specified reading frame within a mRNA sequence which is described or annotated in databases such as for example Ensembl genome/transcriptome/proteome database collection (typically HG19). Typically, a canonical ORF is the same as one of the exons in normal healthy cells. A “non-canonical ORF” as herein intended is a protein coding sequence with specified reading frame within a mRNA sequence which is not described (i.e. unannotated) in genome databases such as for example in Ensembl genome/transcriptome/proteome database. Typically, a non-canonical ORF means thus that the reading frame is shifted compared to the usual reading frame of exons in normal healthy cells. In some embodiments however, a non- canonical can be described in genome databases (such as Ensembl database), but the mRNA sequence represents minor species in normal cells. By minor species it is typically intended less that 5 % , notably less than 2 %, or preferentially less than 1 % species in normal cells.

A “reference genome, or “representative genome” is a digital nucleic acid sequence data base, assembled by scientists as a representative example of species set of genes. As they are often assembled from the sequencing of DNA from a number of donors, reference genomes do not accurately represent the set of genes of any single individual (animal or person). Instead a reference provides a haploid mosaic of different DNA sequences from each donor. According to the present disclosure, the reference genome is GRCh37.pl3 (genome reference consortium, Ref Seq CGF_000001405.25, June 28, 2013).

In a particular embodiment, said tumor specific protein (e.g., tumor specific antigen) is derived from a JET transcript, wherein the TE can be donor (in 5’ position) or acceptor (in 3’ acceptor), preferably wherein the TE is a donor.

In a particular embodiment, the tumor specific protein (e.g., tumor specific antigen) is derived from a JET transcript wherein the TE is located at the 5 ’end of the JET transcript and the exonic sequence is located at the 3 ’-end of the JET transcript.

In another particular embodiment, the tumor specific protein (e.g., tumor specific antigen) is derived from a JET transcript wherein the exonic sequence is located at the 5’end of the JET transcript and the TE is located at the 3 ’-end of the JET transcript.

In a particular embodiment, the JET transcript according to the present disclosure includes an open-reading frame coding for a non-canonical peptide or protein isoform.

In a particular embodiment, said JET transcript is selected from the fusion transcript as disclosed in Tables 9; 11, 13, 17 and 19, and 10, 12, 14, 16, 18 and 20 of WO2022/189626 application incorporated herein by reference providing the chromosome reference (column 1 in Tables 9 and 10, column 2 in Tables 12-20), start position (column 2 in table 9 and 10, and column 3 in Tables 12-20) and breakpoint position (column 3 in Table 9 and 10, and column 4 in Tables 12-20) of the donor sequence (exon or TE) and the chromosome reference (column 4 in Tables 9 and 10, column 7 in Tables 12-20), breakpoint position (column 5 in table 9 and 10, and column 8 in Tables 12-20) and end position (column 6 in Table 9 and 10, and column 9 in Tables 12-20) of acceptor sequence (TE or exon respectively) (GRCh37.pl3 (genome reference consortium, Ref Seq CGF_000001405.25) such that each fusion transcript sequence can be unambiguously retrieved and Tables 9; 11, 13 and 17, and 10, 12, 14, 16 and 18 of WO2022/189639 application incorporated herein by reference providing the chromosome reference (column 2 in Tables 9-18), start position (column 3 in table 9-18), and breakpoint positions (column 4 in Tables 9-18) of the donor sequence (exon or TE) and the chromosome reference (column 7 in Tables 9-18), breakpoint position (column 8 in table 9-18), and end position (column 9 in Tables 9-18of acceptor sequence (TE or exon respectively) (GRCh37.pl3 (genome reference consortium, Ref Seq CGF 000001405.25) such that each fusion transcript sequence can be unambiguously retrieved.

In a more particular embodiment, the fusion transcript results from the splice junction between an Exon and transposable element (TE) sequence, wherein said TE and exonic sequences are selected from the pairs consisting of: SEQ ID NO: 1 and 2; 3 and 4, 5 and 6, 7 and 8, 9 and 10, 11 and 12, 13 and 14, 15 and 16, 17 and 18, 24 and 25, SEQ ID NO: 28 and 29, preferably SEQ ID NO: 7 and 8, SEQ ID NO: 15 and 16, SEQ ID NO: 24 and 25 and SEQ ID NO: 28 and 29 .

Table 1 below herein provides the fusion Identify Number (column 1), coordinates to the donor (exon or TE) and acceptor sequences (TE or exon respectively) (GRCh37.pl 3, genome reference consortium, Ref Seq CGF 000001405.25) such that each fusion transcript sequence can be unambiguously retrieved.

Table 1: Fusion Identify Number (column 1), coordinates of the donor (exon or TE) and acceptor sequences (TE or exon respectively) (GRCh37.pl3, genome reference consortium, Ref Seq CGF 000001405.25).

The sequences of the donor and acceptor sequences of the preferred JET transcripts are presented in the Table 2 below.

Table 2: Donor and acceptor sequences of JET transcripts.

According to the present disclosure, the JET transcripts can be predicted from the bioinformatic tumor transcriptome database TCGA (The Cancer Genome Atlas) as described in WO2021/043804, WO2022/189620, WO2022/189626 and WO2022/189639. Typically, JET transcripts can be carried out by mapping mRNA sequences from cancer sample against a corresponding reference genome or transcriptome (such as the human reference genome Hgl9 ENSEMBL (RNA sequences, GRCh37) with an adapted software, such as for example: Spliced Transcripts Alignment to a Reference (i.e.: STAR - see Dobin, Alexander et al. “STAR: ultrafast universal RNA-seq aligner.” Bioinformatics (Oxford, England) vol. 29,1 (2013): 15-21), TopHat2 (Kim, Daehwan et al. “TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions.” Genome biology vol. 14,4 R36. 25 Apr. 2013, doi: 10.1186/gb- 2013-14-4-r36) or HIS AT (Kim, Daehwan et al. “HIS AT: a fast spliced aligner with low memory requirements.” Nature methods vol. 12,4 (2015): 357-60. doi: 10.1038/nmeth.3317). STAR is a standalone software that uses sequential maximum mappable seed search followed by seed clustering and stitching to align RNA-seq reads. It can typically detect canonical junctions, non-canonical splices, and fusion/chimeric transcripts. Typically, detection of the junctions can be performed as detailed in WO2021/043804, WO2022/189620, WO2022/189626 and WO2022/189639 based on the definitions from ENSEMBL and RepeatMasker databases respectively, downloaded from the UCSC Genome Browser. Thus, in some embodiments, the normal and abnormal junctions are determined in silico using dedicated databases, such as for example Ensembl and Repeatmasker databases, and the JET transcripts having junctions between a TE and an exonic sequence are extracted in silico.

More particularly, RNAseq reads from a sample (or cell) of interest are aligned to a reference genome (such as typically the hgl9 genome) using typically STAR two- pass mode27 to identify un-annotated junctions. As previously indicated JETs are identified as a junction between an exon (most particularly a coding DNA sequence - CDS- exon) and a TE (or repeated element, RE). As per the present disclosure TE (or RE) can be identified (i.e. filtered) according to the definition of commonly used databases in the field such as ENSEMBL (GRCh37) and RepeatMasker.

Splice-switching antisense oligonucleotide

The inventors in the present application showed for the first time that splice-switching antisense oligonucleotide (SSO) can be used to modulate (i.e., induce or inhibit) expression of a tumor protein (e.g., tumor antigen) derived from a JET transcript in a tumor cell.

Splice-switching oligonucleotides (SSOs) are antisense oligonucleotides that base-pair with a pre-mRNA and disrupt the normal splicing repertoire of the transcript by blocking the RNA-RNA base-pairing or protein-RNA binding interactions that occur between components of the splicing machinery.

The term “antisense oligonucleotide” or “antisense nucleic acid” as used herein refers to a single-stranded oligonucleotide having a nucleobase sequence that is complementary to a corresponding segment of a target nucleic acid, e.g., a target genomic sequence, pre-mRNA, or mRNA molecule.

The term “complementarity” refers to the capacity of base pairing between the nucleobases of a first nucleic acid strand and the nucleobases of a second nucleic acid strand, mediated by hydrogen binding (e.g., Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen 1 bonding) between corresponding nucleobases. For example, in DNA, adenine (A) is complementary to thymine (T); and guanosine (G) is complementary to cytosine (C). For example, in RNA, adenine (A) is complementary to uracil (U); and guanosine (G) is complementary to cytosine (C). In certain embodiments, complementary nucleobase means a nucleobase of an antisense oligonucleotide that is capable of base pairing with a nucleobase of its target nucleic acid. For example, if a nucleobase at a certain position of an antisense oligonucleotide is capable of hydrogen bonding with a nucleobase at a certain position of a target nucleic acid, then the position of hydrogen bonding between the oligonucleotide and the target nucleic acid is considered to be complementary at that nucleobase pair. Nucleobases comprising certain modifications may maintain the ability to pair with a counterpart nucleobase and thus, are still capable of nucleobase complementarity.

In a particular embodiment, said SSO comprises an oligonucleotide chemically modified. Chemical modification can be introduced at the backbone, nucleobase and/or sugar moiety.

In one embodiment, the SSO is modified by the substitution of at least one nucleotide with a modified nucleotide, such that in vivo stability is enhanced as compared to a corresponding unmodified oligonucleotide. In a related embodiment, the modified nucleotide is a sugar- modified nucleotide. In another embodiment, the modified nucleotide is a nucleobase- modified nucleotide.

In a particular embodiment, chemically modification is made to the phosphodiester backbone of said oligonucleotide to provide stability against nuclease degradation. For example, the non-bridging oxygen atom of the phosphate group is replaced with carbon (methyl phosphonate, phosphotriester), sulphur (phosphorothioate), nitrogen (phosphoroamidate) or boron (boranophosphate), preferably the SSO is phosphorothioate oligonucleotide.

In another particular embodiment, the modified SSO is a sugar-modified oligonucleotide to increase the binding affinity of oligonucleotides, protects oligonucleotide from nuclease degradation and increase its specificity. In particular, the modified nucleotide is a 2'-deoxy ribonucleotide. In certain embodiments, the 2'-deoxy ribonucleotide is 2'-deoxy adenosine or 2'-deoxy guanosine. In another embodiment, the modified nucleotide is a 2'-O-methyl (e.g., 2'-O-methylcytidine, 2'-O-methylpseudouridine, 2'-O-methylguanosine, 2'-O- methyluridine, 2'-O-methyladenosine, 2'-O-methyl)ribonucleotide, 2’-O-methoxyethyl ribonucleotide, locked nucleic acid (LNA), 2'-fluoro, 2'-amino, 2'-thio modified ribonucleotide, hexitol nucleic acid (HNA), cyclohexenyl nucleic acid (CeNA), altriol nucleic acid (ANA), 2’-O, 4’-C-ethylene bridged nucleic acid (ENA) or morpholino nucleic acid (MNA), preferably the SSO is a 2’-O-methyl oligonucleotide.

In a particular embodiment, said SSO is a 2’-O-methyl RNA phosphorothioate.

Usually, according to the present disclosure, SSO comprises a complementary sequence from 12 to 30 nucleobases in length. Those skilled in the art appreciate that when affinityincreasing chemical modifications are used, the SSO can be shorter and still retain specificity. Those skilled in the art will further appreciate that an upper limit on the size of the SSO is imposed by the need to maintain specific recognition of the target sequence, and to avoid secondary structure forming self-hybridization of the SSO and by the limitations of gaining cell entry. These limitations imply that an SSO of increasing length (above and beyond a certain length which will depend on the affinity of the SSO) will be more frequently found to be less specific, inactive or poorly active.

The SSOs according to the present disclosure may be made through the well-known technique of solid phase synthesis. Any other means for such synthesis known in the art may additionally or alternatively be used. It is well known to use similar techniques to prepare oligonucleotides such as the phosphorothioates and alkylated derivatives.

According to the present disclosure, the Splice-switching antisense oligonucleotides comprises a complementary sequence to a splice modulator (i.e., silencer or activator) site localized within a pre-mRNA encoding JET transcript resulting from the splice junction between a TE and exonic sequence as described above.

In a particular embodiment, said SSO comprises a complementary sequence to said splice modulator (i.e., silencer or activator) site that is localized within TE sequence or a nucleotide sequence (i.e., intronic sequence) adjacent to TE or exonic sequence of said pre-mRNA JET transcript, in particular within a sequence of up to 200 nucleotides, preferably between 40 and 200 nucleotides adjacent to said TE or exonic sequence.

In particular, said splice modulator (i.e., silencer or activator) site is localized within the TE sequence or the intronic sequence adjacent to TE or exonic sequence, preferably within a sequence of up to 200 nucleotides, preferably between 40 and 200 nucleotides adjacent to the TE or exonic sequence. In more particular embodiment, said splice modulator (i.e., silencer or activator) site is localized within the TE sequence or the intronic sequence adjacent to donor or acceptor splice site, preferably within a sequence of up to 200 nucleotides, preferably between 40 and 200 nucleotides adjacent donor or acceptor splice site.

According to the present disclosure, the SSO modulates expression of a tumor specific protein derived from an exon-transposable element junction (JET) transcript in a tumor cell by targeting a splicing modulator site localized within a pre-mRNA encoding JET transcript and thus modulating the level of splice events between TE and exonic sequence.

According to the present disclosure, “splice modulator sites”, “splicing modulator sites” or “splice modulators” are binding sites for cis-regulatory elements that are RNA-binding protein able to reduce or increase the probability that a nearby site will used as a splice junction.

According to the present disclosure, “splice silencer sites”, “splicing silencer sites” or “splice silencers” are binding sites for cis-regulatory elements that are RNA-binding protein able to reduce the probability that a nearby site will used as a splice junction. Splice silencer sites can be intronic splice silencer sites when located in the intron itself, or exonic splice silencer sites when located in a neighboring exon. The majority of splicing repressors are heterogeneous nuclear ribonucleotide (hnRNPs) such as hnRNPAl and Polypyrimidine Tract Binding Protein 1 (PTBP1, aka hnRNPI).

Splicing enhancers, splice enhancer site or splicing activator sites are sites to which splicing activator proteins bind, increasing the probability that a nearby site will be used as a splice junction. These also may occur in the intron (intronic splicing enhancers, ISE) or exon (exonic splicing enhancers, ESE). Most of the activator proteins that bind to ISEs and ESEs are members of the SR protein family. Such proteins contain RNA recognition motifs and arginine and serine-rich (RS) domains.

The localization of putative splicing silencers sites within a pre-mRNA encoding JET transcript can be predicted by any methods well-known in the art, for example by a tool named SpliceAid (http://www.introni.it/splicing.html) (Piva F. et al. Hum Mutat. 2012 Jan;33(l):81-5). SpliceAid is a database of experimentally assessed target RNA sequences in humans. In a particular embodiment of the present disclosure, the SSO induces expression of a tumor specific antigen peptide derived from an exon-transposable element junction (JET) transcript in a tumor cell by targeting and repressing a splicing silencer site localized within a pre- mRNA encoding JET transcript and thus enhancing the level of splice events between TE and exonic sequence.

In a particular embodiment, said SSO comprises a complementary sequence to said splice silencer site localized within a pre-mRNA encoding JET transcript resulting from the splice junction between a TE and exonic sequence, wherein said TE and exonic sequences are selected from the pairs consisting of: SEQ ID NO: 1 and 2; 3 and 4, 5 and 6, 7 and 8, 9 and 10, 11 and 12, 13 and 14, 15 and 16, 17 and 18, and SEQ ID NO: 24 and 25 more preferably

SEQ ID NO: 7 and 8, SEQ ID NO: 15 and 16 or SEQ ID NO: 24 and 25

Examples of two part of pre-mRNA JET sequences comprising splicing silencer sites are represented in the Table 3 below.

Tab e 3: part of pre-mRNA encoding JET sequences resulting from the splice junction between a TE and exonic sequences of SEQ ID NO: 7 and 8 (JET2242), a TE and exonic sequences of SEQ ID NO: 15 and 16 (JET3175) and a TE and exonic sequences of SEQ ID NO: 24 and 25 (JET148). Sequences with capital letters corresponds to the donor sequence (mRNA sequence of SEQ ID NO: 7 for JET 2242, mRNA sequence of SEQ ID NO: 15 for JET 3175 and mRNA sequence of SEQ ID NO: 24 for JET 148 (TE sequence)). Potential splicing silencer sites are in bold. SSO target sequence are underlined.

Examples of four SSO targeting splicing silencer sites comprised within a pre-mRNA encoding JET transcript resulting from the splice junction between a TE and exonic sequence of SEQ ID NO: 7 and 8 (JET 2242), between a TE and exonic sequence of SEQ ID NO: 15 and 16 (JET 3175) and between a TE and exonic sequence of SEQ ID NO: 24 and 25 (JET148) are represented in the Table 4 below.

Tab e 4: SSO sequences targeting corresponding pre-mRNA JET sequences.

In a particular embodiment, said SSO comprises a sequence complementary to a splicing silencer site localized within a pre-mRNA encoding 2242 JET transcript resulting from the split junction between a TE and exonic sequence SEQ ID NO: 7 and 8, said complementary sequence comprises or consists of SEQ ID NO: 21.

In another particular embodiment, said SSO comprises a sequence complementary to a splicing silencer site localized within a pre-mRNAs encoding 3175 JET transcript resulting from the split junction between a TE and exonic sequence of SEQ ID NO: 15 and 16, said complementary sequence comprises or consists of SEQ ID NO: 22 or 23.

In another particular embodiment, said SSO comprises a sequence complementary to a splicing silencer site localized within a pre-mRNAs encoding 148 JET transcript resulting from the split junction between a TE and exonic sequence of SEQ ID NO: 24 and 25, said complementary sequence comprises or consists of SEQ ID NO: 27. According to the present disclosure, the SSO as described above induces expression of a tumor specific antigen derived from a JET transcript, preferably in a cell, more preferably in a tumor cell.

As used, herein, by “SSO inducing expression of a tumor specific antigen derived from a JET transcript” is meant that said SSO is capable to induce a higher expression level of tumor specific antigen or JET transcript in a cell (e.g. tumor cell) in comparison to a control value.

The expression of an antigen specific antigen derived from JET transcript is higher in a cell (e.g. tumor cell) when the expression level of said antigen specific antigen or JET transcript in a cell treated with said SSO is at least 1.5-fold higher, or 2, 3, 4, 5-fold higher or even more than in a control value. The expression level of antigen specific antigen or JET transcript which may be at the protein or RNA level may be determined by any suitable methods known by skilled persons.

For example, the nucleic acid contained in the sample (e.g., tumor cells treated with SSO) is first extracted according to standard methods, for example using lytic enzymes or chemical solutions or extracted by nucleic-acid-binding resins following the manufacturer's instructions. The extracted mRNA is then detected by hybridization (e.g., Northern blot analysis) and/or amplification (e.g., RT-PCR) by using primer pairs and probes specific to said genes as described in the examples of the present disclosure. Quantitative or semi- quantitative RT-PCR is preferred. In another particular embodiment, the mRNA expression level is measured by RNA seq method.

The expression level of tumor specific antigen protein may also be determined by any suitable methods known by skilled persons. The quantity of the protein may be measured, for example, by semi-quantitative Western blots, enzyme-labelled and mediated immunoassays, such as ELISAs, biotin/avidin type assays, radioimmunoassay, immunoelectrophoresis, mass spectrometry, or immunoprecipitation or by protein or antibody arrays.

The expression level is then compared to a control value. As used herein, the term "control value " may refer to the expression level of JET transcript or tumor specific antigen in an untreated cell (i. e. no transfected with SSO) or a cell transfected with a non-specific oligonucleotide, also named scramble oligonucleotide. In a particular embodiment, the scramble oligonucleotide has the same nucleotide sequence composition as the input SSO sequence and has no match with any mRNA of the selected organism database. Tumor specific antigen or JET transcript expression level in said cell is higher when the expression level of the JET transcript in said cell is at least 1.5-fold higher, or 2, 3, 4, 5-fold higher or even more than in a control value.

Cells according to the present disclosure are eukaryotic cells, preferably mammalian cells, more preferably human cells. In a particular embodiment, said cells are tumor cells, more preferably human tumor cells. Cells can be immortalized cell lines derived from tumor cells that keep dividing and growing over time, such A459-cell line or Hl 650 cell line, or cells collected from any tissues or organs affected by a cancer or tumor. The tumor may be a solid or a non-solid tumor, for example from breast cancer, lung cancer and/or melanoma.

For example, the SSO according to the present disclosure can be identified by the method comprising the steps of a) introducing a SSO comprising a complementary sequence to a pre-mRNA encoding JET transcript into a cell, preferably said JET transcript results from the splice junction between a TE sequence and exonic sequence selected from the pairs consisting of SEQ ID NO: 1 and 2; 3 and 4, 5 and 6, 7 and 8, 9 and 10, 11 and 12, 13 and 14, 15 and 16, 17 and 18, and 24 and 25 more preferably SEQ ID NO: 7 and 8, SEQ ID NO: 15 and 16 or SEQ ID NO: 24 and 25, b) determining the expression level of the JET transcript in said cell, c) selecting said SSO when the expression level of the JET transcript in said cell is higher than a control value.

Thus, the present disclosure also relates to a method for identifying a SSO that induces expression of a tumor specific antigen derived from TE-exon junction (JET) transcript in a cell comprising the above-mentioned steps.

In a more particular embodiment, the present disclosure relates to a method for identifying a SSO that induces expression of a tumor specific antigen derived from TE-exon junction (JET) transcript in a cell comprising the steps of: a) identifying a putative splice silencer site localized within a pre-mRNA encoding JET transcript, preferably said JET transcript results from the splice junction between a TE sequence and exonic sequence selected from the pairs consisting of: SEQ ID NO: 1 and 2; 3 and 4, 5 and 6, 7 and 8, 9 and 10, 11 and 12, 13 and 14, 15 and 16, and 17 and 18, and 24 and 25, more preferably SEQ ID NO: 7 and 8, SEQ ID NO: 15 and 16 or SEQ ID NO: 24 and 25, b) introducing a SSO comprising a complementary sequence to said splice silencer site localized within a pre-mRNA encoding JET transcript into a cell, preferably within the TE sequence or a sequence of up to 200 nucleotides adjacent to said TE or exonic sequence, c) determining the expression level of the JET transcript in said cell, d) selecting said SSO when the expression level of the JET transcript in said cell is higher than a control value.

According to the present method, said SSO can be delivered to a cell using any-known techniques including but being not limited to calcium phosphate transfection, DEAE- Dextran transfection, electroporation, microinjection, biolistic, viral infection or liposome- mediated transfection.

The JET level expression may be determined by any suitable methods known by skilled persons as described above.

The expression level of JET transcript is compared to a control value. As used herein, the term “control value” may refer to the expression level of JET transcript in an untreated cell (i. e. no transfected with SSO) or a cell transfected with a non-specific oligonucleotide, also named scramble oligonucleotide. In a particular embodiment, the scramble oligonucleotide has the same nucleotide sequence composition as the input SSO sequence and has no match with any mRNA of the selected organism database.

The JET transcript expression level is higher in said cell when the expression level of the JET transcript is at least 1.5-fold higher, or 2, 3, 4, 5-fold higher or even more in said cell than in a control value.

Cells that can be used in the method according to the present disclosure are eukaryotic cells, preferably mammalian cells, more preferably human cells, preferably tumor cells, more preferably human tumor cells. Cells can be immortalized cell lines derived from tumor cells that keep dividing and growing over time, such A459-cell line or Hl 650 cell line, or cells collected from any tissues or organs affected by a cancer or tumor. The tumor may be a solid or a non-solid tumor, for example from breast cancer, lung cancer and/or melanoma. In another particular embodiment, the present disclosure relates to a method for identifying a SSO that induces expression of a tumor specific antigen derived from TE-exon junction (JET) transcript in a cell comprising the steps of: a) introducing at least one SSO comprising a complementary sequence to intronic sequence within a pre-mRNA encoding JET transcript into a cell, b) determining the expression level of the JET transcript in said cell, c) selecting said SSO when the expression level of the JET transcript in said cell is higher than a control value.

In another particular embodiment of the present disclosure, the SSO modulates the expression of a tumor protein such as a tumor suppressor protein or oncoprotein derived from an exon-transposable element junction (JET) transcript in a tumor cell by targeting and inhibiting a splicing modulator site localized within a pre-mRNA encoding JET transcript and thus modulating the level of splice events between TE and exonic sequence.

In a particular embodiment, said tumor protein is a tumor suppressor protein such as PTEN and SSO comprises a complementary sequence to a splice modulator site localized within a pre-mRNA encoding JET transcript resulting from the splice junction between a TE and exonic sequence, preferably wherein said TE and exonic sequences are selected from the pairs consisting of: SEQ ID NO: 28 and 29.

In a particular embodiment, the SSO induces the expression of a tumor suppressor protein such as PTEN derived from an exon-transposable element junction (JET) transcript in a tumor cell by targeting and inhibiting a splicing silencer site localized within a pre-mRNA encoding JET transcript and thus increasing the level of splice events between TE and exonic sequence and said SSO comprises a complementary sequence to said splice silencer site localized within a pre-mRNA encoding JET transcript resulting from the splice junction between a TE and exonic sequence, preferably wherein said TE and exonic sequences are selected from the pairs consisting of: SEQ ID NO: 28 and 29.

As used, herein, by “SSO inducing expression of a tumor specific protein such as tumor suppressor protein (e.g., PTEN) derived from a JET transcript” is meant that said SSO is capable to induce a higher expression level of tumor specific protein such as tumor suppressor protein (e.g., PTEN) or JET transcript in a cell (e.g. tumor cell) in comparison to a control value. The expression of a tumor specific protein such as a tumor suppressor protein (e.g., PTEN) derived from JET transcript is higher in a cell (e.g. tumor cell) when the expression level of said tumor specific protein or JET transcript in a cell treated with said SSO is at least 1.5- fold higher, or 2, 3, 4, 5-fold higher or even more than in a control value. The expression level of tumor specific protein or JET transcript which may be at the protein or RNA level may be determined by any suitable methods known by skilled persons.

The expression level of tumor specific protein such as tumor suppressor protein (e.g., PTEN) may also be determined by any suitable methods known by skilled persons. The quantity of the protein may be measured, for example, by semi-quantitative Western blots, enzyme- labelled and mediated immunoassays, such as ELISAs, biotin/avidin type assays, radioimmunoassay, immunoelectrophoresis, mass spectrometry, or immunoprecipitation or by protein or antibody arrays.

The expression level is then compared to a control value. As used herein, the term "control value " may refer to the expression level of JET transcript or tumor specific protein such as tumor suppressor protein (e.g., PTEN) in an untreated cell (i. e. no transfected with SSO) or a cell transfected with a non-specific oligonucleotide, also named scramble oligonucleotide. In a particular embodiment, the scramble oligonucleotide has the same nucleotide sequence composition as the input SSO sequence and has no match with any mRNA of the selected organism database.

In another particular embodiment, the SSO inhibits the expression of a tumor suppressor protein such as PTEN derived from an exon-transposable element junction (JET) transcript in a tumor cell by targeting and inhibiting a splicing activator site localized within a pre- mRNA encoding JET transcript and thus decreasing the level of splice events between TE and exonic sequence and said SSO comprises a complementary sequence to said splice enhancer site localized within a pre-mRNA encoding JET transcript resulting from the splice junction between a TE and exonic sequence, preferably wherein said TE and exonic sequences are selected from the pairs consisting of: SEQ ID NO: 28 and 29.

As used, herein, by “SSO inhibiting expression of a tumor specific protein such as tumor suppressor protein (e.g., PTEN) derived from a JET transcript” is meant that said SSO is capable to decrease the expression level of tumor specific protein such as tumor suppressor protein (e.g., PTEN) derived from or JET transcript in a cell (e.g. tumor cell) in comparison to a control value.

The expression of a tumor specific protein such as a tumor suppressor protein (e.g., PTEN) derived from JET transcript is lower in a cell (e.g. tumor cell) when the expression level of said tumor specific protein or JET transcript in a cell treated with said SSO is at least 1.5- fold lower, or 2, 3, 4, 5-fold lower or even less than in a control value. The expression level of tumor specific protein or JET transcript which may be at the protein or RNA level may be determined by any suitable methods known by skilled persons, as described above.

Examples of two part of pre-mRNA JET sequences comprising splicing modulator sites are represented in the Table 5 below.

Tab e 5: part of pre-mRNA encoding JET sequences resulting from the splice junction between a TE and exonic sequences of SEQ ID NO: 28 and 29 (JET PTEN). Sequences with capital letters corresponds to the acceptor sequence (mRNA sequence of SEQ ID NO: 29). Potential splicing silencer sites are in bold. Potential splicing enhancer sites are in italic. SSO target sequence are underlined.

Examples of three SSO targeting splicing activator or silencer sites comprised within a pre- mRNA encoding JET transcript resulting from the splice junction between a TE and exonic sequence of SEQ ID NO: 28 and 29 are represented in the Table 6 below.

Tab e 6: SSO sequences targeting corresponding pre-mRNA JET sequences.

In a particular embodiment, said SSO comprises a sequence complementary to a splicing silencer site localized within a pre-mRNA encoding PTEN JET transcript resulting from the split junction between a TE and exonic sequence SEQ ID NO: 28 and 29, said complementary sequence comprises or consists of SEQ ID NO: 31 or 32.

According to a particular embodiment, the SSO as described in the paragraph above induces the expression of a tumor specific protein, such as tumor suppressor protein (e.g., PTEN) derived from a JET transcript, preferably in a cell, more preferably in a tumor cell.

In another particular embodiment, said SSO comprises a sequence complementary to a splicing activator site localized within a pre-mRNAs encoding PTEN JET transcript resulting from the split junction between a TE and exonic sequence of SEQ ID NO: 28 and 29, said complementary sequence comprises or consists of SEQ ID NO: 33.

According to another particular embodiment, the SSO as described in the paragraph above inhibits the expression of a tumor specific protein, such as tumor suppressor protein (e.g., PTEN) derived from a JET transcript, preferably in a cell, more preferably in a tumor cell.

For example, the SSO according to the present disclosure can be identified by the method comprising the steps of: a) introducing a SSO comprising a complementary sequence to a pre-mRNA encoding JET transcript into a cell, preferably said JET transcript results from the splice junction between a TE sequence and exonic sequence consisting of the pairs: SEQ ID NO: 28 and 29, b) determining the expression level of the JET transcript in said cell, c) selecting said SSO when the expression level of the JET transcript in said cell is higher or lower than a control value.

Thus, the present disclosure also relates to a method for identifying a SSO that modulates (e.g., induces or inhibits) expression of a tumor protein such as a tumor suppressor protein (e.g. PTEN) derived from TE-exon junction (JET) transcript in a cell comprising the above- mentioned steps.

In a more particular embodiment, the present disclosure relates to a method for identifying a SSO that modulates (i.e., induces or inhibits) expression of a tumor specific protein such as a tumor suppressor protein (e.g. PTEN) derived from TE-exon junction (JET) transcript in a cell comprising the steps of: a) identifying a putative splice silencer site localized within a pre-mRNA encoding JET transcript, preferably said JET transcript results from the splice junction between a TE sequence and exonic sequence consisting of the pairs: SEQ ID NO: 28 and 29, b) introducing a SSO comprising a complementary sequence to said splice silencer site localized within a pre-mRNA encoding JET transcript into a cell, preferably within the TE sequence or a sequence of up to 200 nucleotides adjacent to said TE or exonic sequence, c) determining the expression level of the JET transcript in said cell, d) selecting said SSO when the expression level of the JET transcript in said cell is higher or lower than a control value.

The JET level expression may be determined by any suitable methods known by skilled persons as described above. The expression level of JET transcript is compared to a control value. As used herein, the term “control value” may refer to the expression level of JET transcript in an untreated cell (i. e. no transfected with SSO) or a cell transfected with a non-specific oligonucleotide, also named scramble oligonucleotide. In a particular embodiment, the scramble oligonucleotide has the same nucleotide sequence composition as the input SSO sequence and has no match with any mRNA of the selected organism database.

The JET transcript expression level is lower in said cell when the expression level of the JET transcript is at least 1.5-fold lower, or 2, 3, 4, 5-fold lower or even less in said cell than in a control value.

Cells that can be used in the method according to the present disclosure are eukaryotic cells, preferably mammalian cells, more preferably human cells, preferably tumor cells, more preferably human tumor cells. Cells can be immortalized cell lines derived from tumor cells that keep dividing and growing over time, such A459-cell line or Hl 650 cell line, or cells collected from any tissues or organs affected by a cancer or tumor. The tumor may be a solid or a non-solid tumor, for example from breast cancer, lung cancer and/or melanoma.

In another particular embodiment, the present disclosure relates to a method for identifying a SSO that modulates (i.e., induces or inhibits) expression of a tumor specific protein such as tumor suppressor protein (e.g., PTEN) derived from TE-exon junction (JET) transcript in a cell comprising the steps of: a) introducing at least one SSO comprising a complementary sequence to intronic sequence within a pre-mRNA encoding JET transcript into a cell, b) determining the expression level of the JET transcript in said cell, c) selecting said SSO when the expression level of the JET transcript in said cell is higher or lower than a control value.

Nucleic acid construct and expression vector

In one embodiment, the SSO according to the present disclosure is encoded by one or more nucleic acid constructs. The term “nucleic acid construct” as used herein refers to a man-made nucleic acid molecule resulting from the use of recombinant DNA technology. A nucleic acid construct is a nucleic acid molecule, either single- or double-stranded, which has been modified to contain segments of nucleic acids sequences, which are combined and juxtaposed in a manner, which would not otherwise exist in nature. A nucleic acid construct usually is a “vector”, i.e. a nucleic acid molecule which is used to deliver exogenously created DNA into a host cell.

The nucleic acid construct as described above may be contained in an expression vector. The vector may be an autonomously replicating vector, i.e., a vector that exists as an extra- chromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extra-chromosomal element, a mini-chromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one that, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated.

Examples of appropriate vectors include, but are not limited to, recombinant integrating or non- integrating viral vectors and vectors derived from recombinant bacteriophage DNA, plasmid DNA or cosmid DNA. Preferably, the vector is a recombinant integrating or nonintegrating viral vector. Examples of recombinant viral vectors include, but not limited to, vectors derived from herpes virus, retroviruses, lentivirus, vaccinia viruses, adenoviruses, adeno-associated viruses or bovine papilloma virus.

Pharmaceutical composition

The SSO according to the present disclosure is preferably used in the form of a pharmaceutical composition comprising a therapeutically effective amount of said product(s) as described above.

In the context of the disclosure, a therapeutically effective amount refers to a dose sufficient for reversing, alleviating or inhibiting the progress of the disorder or condition to which such term applies, or reversing, alleviating or inhibiting the progress of one or more symptoms of the disorder or condition to which such term applies.

The term “effective dose” or “effective dosage” is defined as an amount sufficient to achieve, or at least partially achieve, the desired effect. The effective dose is determined and adjusted depending on factors such as the composition used, the route of administration, the physical characteristics of the individual under consideration such as sex, age and weight, concurrent medication, and other factors, that those skilled in the medical arts will recognize.

In the various embodiments of the present disclosure, the pharmaceutical composition comprises a pharmaceutically acceptable carrier and/or vehicle.

A “pharmaceutically acceptable carrier” refers to a vehicle that does not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.

Preferably, the pharmaceutical composition contains vehicles, which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.

The pharmaceutical forms suitable injectable use include sterile aqueous solutions or suspensions. The solution or suspension may comprise additives which are compatible with viral vectors and do not prevent viral vector particle entry into target cells. In all cases, the form must be sterile and must be fluid to the extent that easy syringe ability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. An example of an appropriate solution is a buffer, such as phosphate buffered saline (PBS) or Ringer lactate.

A number of methods are conveniently used to deliver the nucleic acids (e.g. SSO, nucleic acid construct or expression vector encoding SSO) to the patient. For instance, the nucleic acid can be delivered directly, as “naked DNA or RNA”. The nucleic acids can also be delivered complexed to cationic compounds, such as cationic lipids. Delivery systems may optionally include cell-penetrating peptides, nanoparticulate encapsulation, virus like particles, liposomes, exosomes or any combination thereof. Dendrimers are a supermolecular delivery system which can be synthesized with various functional groups, making them a versatile non-viral particle delivery system. Polymers are also employed as delivery vehicles.

Therapeutic use

The present disclosure thus relates to a SSO as described above for treating a cancer disease in a subject in need thereof.

In particular, the composition comprising a SSO, nucleic acid construct, expression vector encoding it, or pharmaceutical composition as described above according to the present disclosure may be used as a medicament, in particular for use in modulating expression of tumor-specific protein derived from a TE-exon junction (JET) transcript in a subject in need thereof suffering from a cancer.

In particular, the composition comprising a SSO, nucleic acid construct, expression vector encoding it, or pharmaceutical composition as described above according to the present disclosure may be used as a medicament, in particular for use in inducing expression of tumor-specific antigen derived from a TE-exon junction (JET) transcript in a subject in need thereof suffering from a cancer.

In another particular embodiment, the composition comprising a SSO, nucleic acid construct, expression vector encoding it, or pharmaceutical composition as described above according to the present disclosure may be used as a medicament, in particular for use in inducing expression of tumor suppressor protein (e.g., PTEN) derived from a TE-exon junction (JET) transcript in a subject in need thereof suffering from a cancer.

In another particular embodiment, the composition comprising a SSO, nucleic acid construct, expression vector encoding it, or pharmaceutical composition as described above according to the present disclosure may be used as a medicament, in particular for use in inhibiting expression of tumor suppressor protein (e.g., PTEN) derived from a TE-exon junction (JET) transcript a in a subject in need thereof suffering from a cancer.

In another particular embodiment, the composition comprising a SSO, nucleic acid construct, expression vector encoding it, or pharmaceutical composition as described above according to the present disclosure may be used as a medicament, in particular for use in inhibiting expression of an oncoprotein derived from a TE-exon junction (JET) transcript a in a subject in need thereof suffering from a cancer. In the context of the disclosure, the term “treating” or “treatment”, as used herein, means reversing, alleviating or inhibiting the progress of the disease caused by a cancer or condition to which such term applies, or reversing, alleviating or inhibiting the progress of one or more symptoms of the disorder or condition to which such term applies.

The terms “cancer”, “tumor”, are used interchangeably herein to refer to cells that exhibit relatively abnormal, uncontrolled, and/or autonomous growth, so that they exhibit an aberrant growth phenotype characterized by a significant loss of control of cell proliferation.

The term “metastatic cancer”, “metastatic tumor or “metastasis” are used interchangeably herein to refer to a secondary tumor that is the result of the invasion of cancer cells through the bloodstream or lymphatic vessels to other sites and tissues in the body.

Cancers are classified by the type of cell that resembles the tumor and, therefore, the tissue presumed to be the origin of the tumor. These are the histology and the location, respectively.

According to the present application, cancer may affect any one of the following tissues or organs: breast; liver; kidney; heart, mediastinum, pleura; floor of mouth; lip; salivary glands; tongue; gums; oral cavity; palate; tonsil; larynx; trachea; bronchus, lung; pharynx, hypopharynx, oropharynx, nasopharynx; esophagus; digestive organs such as stomach, intrahepatic bile ducts, biliary tract, pancreas, small intestine, colon; rectum; urinary organs such as bladder, gallbladder, ureter; rectosigmoid junction; anus, anal canal; skin; bone; joints, articular cartilage of limbs; eye and adnexa; brain; peripheral nerves, autonomic nervous system; spinal cord, cranial nerves, meninges; and various parts of the central nervous system; connective, subcutaneous and other soft tissues; retroperitoneum, peritoneum; adrenal gland; thyroid gland; endocrine glands and related structures; female genital organs such as ovary, uterus, cervix uteri; corpus uteri, vagina, vulva; male genital organs such as penis, testis and prostate gland; hematopoietic and reticuloendothelial systems; blood; lymph nodes; thymus.

The term "cancer" according to the disclosure therefore comprises leukemias, seminomas, melanomas, teratomas, lymphomas, neuroblastomas, gliomas, rectal cancer, endometrial cancer, kidney cancer, adrenal cancer, thyroid cancer, blood cancer, skin cancer, cancer of the brain, cervical cancer, intestinal cancer, liver cancer, colon cancer, stomach cancer, intestine cancer, head and neck cancer, gastrointestinal cancer, lymph node cancer, esophagus cancer, colorectal cancer, pancreas cancer, ear, nose and throat (ENT) cancer, breast cancer, prostate cancer, cancer of the uterus, ovarian cancer and lung cancer and the metastases thereof. Examples thereof are lung carcinomas, mamma carcinomas, prostate carcinomas, colon carcinomas, renal cell carcinomas, cervical carcinomas, or metastases of the cancer types or tumors described above. The term cancer according to the present disclosure also comprises cancer metastases and relapse of cancer.

The disclosure also provides a method for inducing expression of tumor-specific antigen in a subject in need thereof suffering from a cancer comprising administering to a patient a therapeutically effective amount of the SSO, nucleic acid construct or expression vector encoding said SSO or pharmaceutical composition as described above.

By “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary to achieve the desired therapeutic result. The therapeutically effective amount of the product of the disclosure or pharmaceutical composition that comprises it may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the product or pharmaceutical composition to elicit a desired response in the individual. Dosage regimens may be adjusted to provide the optimum therapeutic response. A therapeutically effective amount is also typically one in which any toxic or detrimental effect of the product or pharmaceutical composition is outweighed by the therapeutically beneficial effects.

As used herein, the term “subject”, “patient” or “individual” denotes a mammal. Preferably, a patient, a subject or individual according to the disclosure is a human, in particular a human subject or patient, more preferably suffering from a cancer.

The product of the present disclosure is generally administered according to known procedures, at dosages and for periods of time effective to induce a therapeutic effect in the patient.

The administration can be systemic or local. Systemic administration is preferably parenteral such as subcutaneous (SC), intramuscular (IM), intravascular such as intravenous (IV) or intraarterial; intraperitoneal (IP); intradermal (ID), interstitial or else. The administration may be for example by injection or perfusion. In some particular embodiments, the administration is parenteral, preferably intravascular such as intravenous (IV) or intraarterial. The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques, which are within the skill of the art. Such techniques are explained fully in the literature. In a particular embodiment, said SSO is administered in combination with an immunotherapy to enhance anti-tumor immune response.

Immunotherapy is a type of cancer treatment that activates the immune system to fight disease such as cancer. Immunotherapy that can be used to treat cancer includes as nonlimiting examples: immune inhibitory checkpoint inhibitors which are drugs that block inhibitory immune checkpoint protein, T-cell transfer therapy, monoclonal antibodies or immune system activators.

As used herein the term “immune checkpoint protein” has its general meaning in the art and refers to a molecule that is expressed by T cells and NK cells and regulates the immune system. According to the present disclosure, immune checkpoint proteins are preferably inhibitory immune checkpoint proteins that dampen effector immune response. Inhibitory immune checkpoint molecules are recognized in the art to constitute immune checkpoint pathways similar to the CTLA-4 and PD-1 dependent pathways (see e.g., Pardoll, 2012. Nature Rev Cancer 12:252-264; Mellman et al., 2011. Nature 480:480- 489).

As used herein, the term “inhibitory immune checkpoint inhibitor” or “immune checkpoint inhibitor” has its general meaning in the art and refers to any compound inhibiting the function of an immune inhibitory checkpoint protein. Inhibition includes reduction of function and full blockade. In particular, the immune checkpoint inhibitor particularly suitable for enhancing the proliferation, migration, persistence and/or cytotoxic activity of T cells in the patient and in particular the tumor-infiltrating of CD8⁺ T cells of the patient.

In an embodiment, said immune checkpoint inhibitor of immune cells (T and B lymphocytes) is selected from the group consisting of anti-PD-Ll, anti-PD-1, anti-CTLA-4, anti-HVEM, anti-BTLA, anti-TIGIT, anti-TIM-1/3, anti-LAG-3, and anti-OX40 agonist, anti-CD40 agonist, CD40-L, TLR agonists, and B-cell receptor agonists, in particular selected from the group consisting of anti-PD-Ll, anti-PD-1 and anti-CTLA-4. In a particular embodiment of the disclosure, the immune checkpoint inhibitor is an anti-PD-Ll antibody.

In a particular embodiment, examples of immune checkpoint inhibitors are inhibitors that affect the PD-l/PDL-1 and CTLA-4 pathways and can be selected from the group consisting of: Ipilimumab, Nivolumab, Pembrolizumab, Atezolizumab, Avelumab and durvalumab. According to the present disclosure said immunotherapy can be T cell transfer therapy. As used herein, T-cell transfer therapy, also called adoptive cell therapy, adoptive immunotherapy, or immune cell therapy has its general meaning in the art and refers to a treatment that boosts the natural ability of T cells to fight cancer. In this treatment, immune cells are taken from the patient tumor. T-cell transfer therapy can be tumor infiltrating lymphocytes or CAR T-cell therapy.

According to the present disclosure, immunotherapy can be monoclonal antibodies that binds to specific targets on cancer cells such as anti-CD20, anti-HER2, anti-EGFR, anti- VEGF, anti-CD52 or anti-CD33 or immune system activators such as cytokines including as non-limiting examples: interferon alpha (IFN-a), interleukin-2 (IL-2), interleukin- 11 (IL- 11), interleukin-21 (IL-21), erythropoietin, granulocyte-macrophage colony-stimulating factor (GM-CSF) or granulocyte colony-stimulating factor (G-CSF).

Kit

In another aspect, the present disclosure also relates to a kit for use in modulating expression of a tumor specific protein derived from a JET transcript in a cell comprising a SSO comprising a complementary sequence to said splice modulator site localized within a pre- mRNA encoding said JET transcript resulting from the splice junction between a TE and exonic sequences, preferably wherein said TE and exonic sequences are selected from the pairs consisting of: SEQ ID NO: 1 and 2; 3 and 4, 5 and 6, 7 and 8, 9 and 10, 11 and 12, 13 and 14, 15 and 16, 17 and 18, 24 and 25 and, 28 and 29 preferably SEQ ID NO: 7 and 8, SEQ ID NO: 15 and 16, SEQ ID NO: 24 and 25 or SEQ ID NO: 28 and 29.

In particular embodiment, the present disclosure relates to a kit for use in inducing expression of a tumor specific antigen derived from a JET transcript in a cell comprising a SSO comprising a complementary sequence to said splice silencer site localized within a pre-mRNA encoding said JET transcript resulting from the splice junction between a TE and exonic sequences, preferably wherein said TE and exonic sequences are selected from the pairs consisting of: SEQ ID NO: 1 and 2; 3 and 4, 5 and 6, 7 and 8, 9 and 10, 11 and 12, 13 and 14, 15 and 16, 17 and 18, and 24 and 25, preferably SEQ ID NO: 7 and 8, SEQ ID NO: 15 and 16 or SEQ ID NO: 24 and 25.

In another particular embodiment, the present disclosure relates to a kit for use in modulating the expression of a tumor suppressor protein derived from a JET transcript in a cell comprising a SSO comprising a complementary sequence to a splice modulator site localized within a pre-mRNA encoding said JET transcript resulting from the splice junction between a TE and exonic sequences, preferably wherein said TE and exonic sequences are SEQ ID NO: 28 and 29.

In a particular embodiment, the present disclosure relates to a kit for use in inducing expression of a tumor specific antigen derived from 2242 JET transcript resulting from the splice junction between a TE sequence and exonic sequence of SEQ ID NO: 7 and 8 and preferably wherein said SSO comprises or consists of SEQ ID NO: 21.

In another particular embodiment, the present disclosure relates to a kit for use in inducing expression of a tumor specific antigen derived from 3175 JET transcript resulting from the splice junction between a TE sequence and exonic sequence of SEQ ID NO: 15 and 16 and preferably wherein said SSO comprises or consists of SEQ ID NO: 22 or 23.

In another particular embodiment, the present disclosure relates to a kit for use in inducing expression of a tumor specific antigen derived from 148 JET transcript resulting from the splice junction between a TE sequence and exonic sequence of SEQ ID NO: 24 and 25 and preferably wherein said SSO comprises or consists of SEQ ID NO: 27.

In another particular embodiment, the present disclosure relates to a kit for use in inducing expression of a tumor suppressor derived from PTEN JET transcript resulting from the splice junction between a TE sequence and exonic sequence of SEQ ID NO: 28 and 29 and preferably wherein said SSO comprises or consists of SEQ ID NO: 31 or 32.

In another particular embodiment, the present disclosure relates to a kit for use in inhibiting expression of a tumor suppressor derived from PTEN JET transcript resulting from the splice junction between a TE sequence and exonic sequence of SEQ ID NO: 28 and 29 and preferably wherein said SSO comprises or consists of SEQ ID NO: 33.

The kit may also comprise instructions indicating the methods for preparing and/or using the reagents to determine the expression level of said genes according to the methods of the disclosure.

In vitro method for inducing expression of tumor specific antigen derived from JET transcript

In a further aspect, the present disclosure also concerns the non-therapeutic use of the SSO as described above for modulating the expression of a tumor specific protein derived from a JET transcript in a cell, for example for research tool. In a particular embodiment, the present disclosure also concerns the non-therapeutic use of the SSO as described above for inducing expression of a tumor specific antigen derived from a JET transcript in a cell, for example for research tool, in particular in an antigen-presenting cell for example to induce the expression of a neoantigen peptide to prime tumor specific T cells or amplify neoantigen-specific TILs before transplanting cells into a patient.

The present disclosure relates to an in vitro method for modulating the expression of a tumor specific protein derived from a JET transcript in a cell, preferably a tumor cell, comprising the step of introducing a SSO as described above comprising a sequence complementary to a splicing modulator site within a pre-mRNA encoding said JET transcript into said cell, preferably within a TE sequence or a nucleotide sequence adjacent to TE and exonic sequences, preferably within a sequence of up to 200 nucleotides, preferably between 40 and 200 nucleotides adjacent to said TE or exonic sequence.

Preferably, the present disclosure relates to an in vitro method for inducing expression of a tumor specific antigen derived from a JET transcript in a cell, preferably a tumor cell, comprising the step of introducing a SSO as described above comprising a sequence complementary to a splicing silencer site within a pre-mRNA encoding said JET transcript into said cell, preferably within a TE sequence or a nucleotide sequence adjacent to TE and exonic sequences, preferably within a sequence of up to 200 nucleotides, preferably between 40 and 200 nucleotides adjacent to said TE or exonic sequence.

In a particular embodiment, said JET transcript results from the splice junction between a TE and exonic sequences, preferably wherein said TE and exonic sequences are selected from the pairs consisting of: SEQ ID NO: 1 and 2; 3 and 4, 5 and 6, 7 and 8, 9 and 10, 11 and 12, 13 and 14, 15 and 16, 17 and 18, 24 and 25, and, 28 and 29, preferably SEQ ID NO: 7 and 8, SEQ ID NO: 15 and 16, SEQ ID NO: 24 and 25 or SEQ ID NO: 28 and 29.

Said method involves introducing said SSO into a cell. Said SSO as described above may be synthesized in situ in the cell as a result of the introduction of nucleic acid construct, preferably expression vector encoding said SSO as described above into the cell. Alternatively, said SSO may be produced outside the cell and then introduced thereto.

Said SSO, nucleic acid construct or expression vector can be introduced into cell by any methods known in the art and include, as non-limiting examples, stable transformation methods in which the nucleic acid construct or expression vector is integrated into the cell genome, transient transformation methods in which the SSO, nucleic acid construct or expression vector is not integrated into the genome of the cell and virus-mediated methods. For example, transient transformation methods include for example microinjection, electroporation or particle bombardment.

In a particular embodiment, said SSO can be delivered to a cell using any-known techniques including but being not limited to calcium phosphate transfection, DEAE-Dextran transfection, electroporation, microinjection, biolistic, viral infection or liposome-mediated transfection.

Cells that can be used in the method according to the present disclosure are eukaryotic cells, preferably mammalian cells, more preferably human cells. In a particular embodiment, said cells are tumor cells, more preferably human tumor cells. Cells can be immortalized cell lines derived from tumor cells that keep dividing and growing over time, such A459-cell line or H1650 cell line, or cells collected from any tissues or organs affected by a cancer or tumor in a patient. The tumor may be a solid or a non-solid tumor, for example from breast cancer, lung cancer and/or melanoma.

The invention will now be exemplified with the following examples, which are not limitative with reference to the attached figures.

EXAMPLES

1. Material and methods

1.1 SSO design

Considering that JETs are splice variants, it is interesting to have the possibility to increase the expression of a specific JET to generate tumor-specific antigen presented by MHC in tumor cells. To modulate splicing events of pre-mRNA encoding JET transcript, the inventors used Splice Switching Oligonucleotides (SSO). SSO sequences are antisense oligonucleotides composed of 20-25 nucleotides of modified RNA. Commonly used modifications are a phosphorothioate backbone (PS) and a 2’-O-methyl (2’0Me) group to protect against RNAase H degradation and increase the stability and affinity for the targeted sequence.

The SSO are designed to have a length between 20-25 nucleotides and to not have off-target in the genome. Bsgenome R library is used to evaluate SSO off-targets. The evaluation is made in the whole human genome (“Bsgenome. Hsapiens.UCSC.hgl9”). The vmatchPattern() function allows to research the presence of a sequence in the genome. The max. mismatch argument is used to check the redundancy of a given sequence with 0 mismatch and with 1 mismatch. Sequences that are not redundant even with one mismatch are kept.

SSO sequences are also designed to avoid the formation of dimers and secondary structure. The online OligoEvaluator™ of Sigma-Aldrich® is used to predict the secondary structure and dimer formation. A weak secondary structure is acceptable.

1.2 SSO nomenclature

According to the existing nomenclature (Mann CJ. Et al. J Gene Med. 2002 Nov- Dec;4(6):644-54), SSO is named by indicating JET transcript reference, the species, and the localization from an acceptor (A) or a donor (D) splice site. The donor site corresponds to the GT motif after an exon or a TE. For the case of JET, the breakpoint in the TE is one splice site and the beginning or the end of the TE correspond to the other site.

For example, ‘’3175_HD(-20-45)” means JET 3175, in human (H), localized from 20 to 45 nucleotides before ( - ) the donor site (D). The TE of 3175 JET is donor, meaning that the donor splice site of the TE corresponds to the breakpoint and the acceptor splice site is the beginning of the TE.

1.3 Sites to target with SSO

To modify JET expression there are two ways of designing SSO.

SSO could be designed to create a walk along the intronic sequence containing the TE that participate to the JET. SSO is also designed considering the binding sites of enhancer/repressor proteins. A tool named SpliceAid (http://www.introni.it/splicing.html) is used to determine splicing silencer site on which repressor protein is known to bind SpliceAid is a database of experimentally assessed target RNA sequences in humans. This tool is useful to estimate the localization of possible regulation sites. Analysis of available CLIP-seq data from the studied cell line can also be used to design SSO targeting co-factors binding sites.

1.4 SSO reconstitution

The reconstitution is made in sterile conditions in order to obtain lOOpM stock in sterile RNA-free water. SSO are stored in aliquots at -80°C. 1.5 SSO transfection

The day before the transfection, the A549 or H1650 cell line is plated homogeneously in 12- wells plates in 2 mL of the corresponding complete media.

The day of the transfection, cells is around 60% confluency. The media is changed for 500 pL of media without any antibiotic. SSO are administered in Lipofectamine™ 3000 (thermoFisher scientific) according to manufacturer’s instructions. Lipofectamine 3000 and Optimem is brought to room temperature. In a first mix, Lipofectamine 3000 and Optimem are mixed (60uL of Lipofectamine 3000 for ImL of Optimem). In a second mix, SSO are prepared in individual 1,5 mL ependorf tubes in an Optimem -P3000 reagent solution (40 pL of P3000 for ImL Optimem). Mixes are pooled, mixed by up-and-down pipetting, incubated for 10 minutes at room temperature and transferred to cells in a drop-by-drop fashion. Plates are incubated at 37°C.

24h or 5 days post-transfection, medium is removed, cells are washed with PBS and 350 pL of lysis buffer is added. Otherwise, fresh complete medium is added to maintain the cells in culture. Lysis buffer is composed of RLT buffer provided by Rneasy Mini Qiagen kits and B -mercaptoethanol (lOuL of B-mercaptoethanol for ImL of RLT).

1.6 RNA extraction

350 pL of 70% ethanol was added to the lysate. The whole volume (700 pL) was transferred to an Rneasy spin column (Qiagen) placed in a collection tube for each sample. RNA was extracted following manufacturer’s instructions, including the on-column digestion using RNAse-free DNAse set (Qiagen), optional in the Rneasy minikit protocol. Samples were eluted twice and submitted to digestion using TurboDNAse (Fisher Scientific) according to manufacturer’s instructions.

1.7 cDNA synthesis

1 pg RNA was used to perform first-strand synthesis using Superscript III, with oligodT primers, following manufacturer’s instructions. cDNA was diluted 5 or 10 times in nuclease free ddH20 for further use.

1.8 qRT-PCR

JET-specific Taqman probes were designed using the AllelelD 7 software (Premier Biosoft), using default parameters (or slight modifications in probe or primer lengths). Wherever possible, the inventors designed minor groove binding probes, and otherwise attempted to make regular Taqman probes. Custom Taqman probes and primers were ordered from Eurogentec. 20X concentrated Taqman assays were prepared by mixing 5pL of Taqman probe, 18pL of each primer, and 59pL of ddH20. B2M, actine B and HPRT1 Taqman assays (Fisher scientific) were used as housekeeping gene controls. qRT-PCR were performed in 384 well plates, using dTTTP MasterMix (Eurogentec), according to manufacturer’s instructions (for each well, 0.5pL Taqman assay, 5pL Mastermix and 4.5pL of cDNA). Amplification was performed as recommended by the manufacturer, on a Roche Lighcycler® 480, and the relative quantification tool from the analysis software was applied. The detection threshold was place manually in the first 1/3 of the linear amplification phase. Data were analyzed Microsoft Excel to calculate ACt, and further analyzed in Graphpad Prism.

2. Results

2.1 SSO targeting human JET 2242 expression in A549 cell line.

Following the method explained in the previous part, A549 cells were plated at 0,5.10⁵ cells/well in 12-wells plates. The transfection was made with SSO reaching a final concentration of 500nM. 24 hours later, cells were lysed, and RNA extraction was performed.

SSO HD(-49-69) (SEQ ID NO: 21) induces a 4-fold increase of JET 2242 expression (Figure 1A). In a previous assay this increase was 10 times higher than the basal expression. The expression of the normal junction (NJ) is constant (Figure IB).

To find which concentration of SSO induces the highest effect with the lowest cell mortality, a range of SSO concentration was tested, from 50nM to 500nM for Scramble, and HD(-49- 69) (Figure 2A). A viability staining was performed in the same experiment (Figure 2B). The optimal concentration seems to be lOOnM for viability and also for the HD(-49-69) effect.

2.2 SSO effect on 2242 JET expression is sustainable a least for 72 hours.

To determine the duration of the SSO effect on JET 2242 transcript expression, a transfection assay with SSO Scramble, and HD(-49-69) at lOOnM was done. 24h after transfection the media was removed and changed for complete media without antibiotics. Then, each day cells were lysed in lysis buffer. The promoting effect of SSO HD(-49-69) on JET2242 transcript expression was sustained for 72h (Figure 3).

2.3 SSO targeting human JET 3175 expression in A549 cell line.

A549 cells were plated at 0,75.10⁵ cells/well in 12-wells plates. The transfection was made with SSO reaching a final concentration of lOOnM. 24 hours later, cells were lysed, and RNA extraction was performed.

In this experiment, HA(-44-69) (SEQ ID NO: 22) and HD(-20-45) (SEQ ID NO: 23) showed an increased expression of 3175 JET (Figure 4).

2.4 SSO targeting human JET 3175 expression in H1650 cell line.

Hl 650 cells are human lung adenocarcinoma cells. Hl 650 cells were plated at 1.10⁵ cells/well in 12-wells plates. The transfection was made with SSO reaching a final concentration of lOOnM in 500 pL of media. 6 hours after the transfection the media was removed and replaced by fresh media. 24 hours after the transfection, cells were lysed, and RNA extraction was performed followed by RTqPCR.

In this experiment, HA(-44-69) (SEQ ID NO: 22) and HD(-20-45) (SEQ ID NO: 23) showed an increased expression of 3175 JET (2,6-fold and 1,6-fold respectively) (Figure 5).

2.5 SSO targeting human JET 3175 expression in H1650 cell line until at least 5 days

H1650 cells were plated at 1.10⁵ cells/well. The transfection was made at day 1 and at day 3 with SSO reaching a final concentration of 50nM. 5 days after the transfection, cells were lysed, and RNA extraction was performed followed by RTqPCR.

In this experiment, HA(-44-69) (SEQ ID NO: 22) showed an increased expression of 3175 JET (Figure 6).

2.6 SSO targeting human JET 148 expression in H1650 cell line.

H1650 cells were plated at 1.10⁵ cells/well in 12-wells plates. The transfection was made with SSO reaching a final concentration of 100 nM in 500 pL of media. 6 hours after the transfection the media was removed and replaced by fresh media. 24 hours after the transfection, cells were lysed, and RNA extraction was performed followed by RTqPCR.

In this experiment, HA(-94-74) showed an increased expression of PTEN JET (Figure 7). 2.7 SSO targeting human PTEN expression in H1650 cell line.

H1650 cells were plated at 1.10⁵ cells/well in 12-wells plates. The transfection was made with SSO reaching a final concentration of 50nM in 500 pL of media. 6 hours after the transfection the media was removed and replaced by fresh media. 24 hours after the transfection, cells were lysed, and RNA extraction was performed followed by RTqPCR.

In this experiment, HD(+13+38) and HD(+53+78) showed an increased expression of PTEN JET. HA(+56+78) showed a decreased expression of PTEN JET (Figure 8).

Claims

1. A Splice-switching antisense oligonucleotide (SSO) that modulates the expression of a tumor specific protein derived from an exon-transposable element splicing junction (JET) transcript resulting from the splice junction between a transposable element (TE) and exonic sequences, wherein said SSO comprises a complementary nucleic acid sequence to a splice modulator site localized within a pre-mRNA sequence encoding said JET transcript.

2. The SSO of claim 1 wherein said splice modulator site is localized within a TE sequence or a sequence adjacent to TE or exonic sequence of said pre-mRNA JET transcript, in particular within a sequence of up to 200 nucleotides adjacent to said TE or exonic sequence.

3. The SSO according to claim 1 or 2 wherein said complementary nucleic acid sequence is of 18 to 30 nucleotides in length.

4. The SSO according to any one of claims 1 to 3 comprising a chemically modified nucleic acid sequence, preferably a 2’-O-methyl (2’0me) modified phosphorothioate oligonucleotide.

5. The SSO according to any one of claims 1 to 4 wherein said JET transcript includes an open-reading frame coding for a non-canonical peptide or protein isoform.

6. The SSO according to any one of claims 1 to 5 wherein said tumor specific protein is a tumor specific antigen.

7. The SSO according to claim 6 wherein said SSO induces the expression of the tumor specific antigen and wherein said splice modulator site is a splice silencer site.

8. The SSO according to claim 7 wherein said JET transcript results from the splice junction between a TE and exonic sequence selected from the pairs consisting of: SEQ ID NO: 1 and 2; 3 and 4, 5 and 6, 7 and 8, 9 and 10, 11 and 12, 13 and 14, 15 and 16, and 17 and 18, SEQ ID NO: 24 and 25, preferably SEQ ID NO: 7 and 8, SEQ ID NO: 15 and 16 or SEQ ID NO: 24 and 25.

9. The SSO according to claim 8 wherein said JET transcript results from the splice junction between a TE sequence and exonic sequence of SEQ ID NO: 7 and 8 and preferably wherein said SSO comprises or consists of SEQ ID NO: 21.

10. The SSO according to claim 7 wherein said JET transcript results from the splice junction between a TE sequence and exonic sequences of SEQ ID NO: 15 and 16 and preferably wherein said SSO comprises or consists of SEQ ID NO: 22 or 23.

11. The SSO according to claim 7 wherein said JET transcript results from the splice junction between a TE sequence and exonic sequences of SEQ ID NO: 24 and 25 and preferably wherein said SSO comprises or consists of SEQ ID NO: 27.

12. The SSO according to any one of claims 1 to 5 wherein said tumor specific protein is a tumor suppressor protein.

13. The SSO according to claim 12 wherein said SSO induces the expression of the tumor suppressor protein and wherein said splice modulator site is a splice silencer site.

14. The SSO according to claim 13 wherein said JET transcript results from the splice junction between a TE and exonic sequence selected from the pairs consisting of: SEQ ID NO: 28 and 29, and preferably wherein said SSO comprises or consists of SEQ ID NO: 31 or 32.

15. The SSO according to claim 12 wherein said SSO inhibits the expression of the tumor suppressor protein and wherein said splice modulator site is a splice activator site.

16. The SSO according to claim 15 wherein said JET transcript results from the splice junction between a TE and exonic sequence selected from the pairs consisting of: SEQ ID NO: 28 and 29, and preferably wherein said SSO comprises or consists of SEQ ID NO: 33.

17. A pharmaceutical composition comprising the SSO according to any one of claims 1 to 16 and a pharmaceutical acceptable excipient.

18. The SSO according to any one of claims 1 to 16 or the pharmaceutical composition of claim 17 for use in modulating the expression of a tumor specific protein derived from JET transcript in a tumor cell of a subject in need thereof suffering from a cancer.

19. The SSO according to any one of claims 6 to 11 or the pharmaceutical composition of claim 18 for use in inducing the expression of a tumor specific antigen derived from JET transcript in a tumor cell of a subject in need thereof suffering from a cancer.

20. The SSO according to any one of claims 12-16 or the pharmaceutical composition of claim 17 for use in modulating the expression of a tumor suppressor protein derived from JET transcript in a tumor cell of a subject in need thereof suffering from a cancer.

21. The SSO or the pharmaceutical composition for use according to any one of claims 18 to 20 wherein said cancer is lung cancer.

22. The SSO or the pharmaceutical composition for use according to any one of claims 18 to 20, wherein said SSO is administered in combination with an immunotherapy.

23. The SSO or the pharmaceutical composition for use according to claim 22, wherein said immunotherapy treatment is selected from the group consisting of: immune inhibitory checkpoint inhibitors, T-cell transfer therapy, monoclonal antibodies and immune system activators.

24. A method for identifying a SSO that modulates the expression of a tumor specific protein derived from TE-exon junction (JET) transcript in a cell comprising the steps of: a) identifying a putative splice modulator site, preferably silencer site localized within a pre-mRNA encoding JET transcript, preferably said JET transcript results from the splice junction between a TE sequence and exonic sequence selected from the pairs consisting of: SEQ ID NO: 1 and 2; 3 and 4, 5 and 6, 7 and 8, 9 and 10, 11 and 12, 13 and 14, 15 and 16, 17 and 18, 24 and 25 and 28 and 29 preferably SEQ ID NO: 7 and 8, SEQ ID NO: 15 and 16, SEQ ID NO: 24 and 25, and SEQ ID NO: 28 and 29, b) introducing a SSO comprising a complementary sequence to said splice modulator site localized within a pre-mRNA encoding JET transcript into a cell, c) determining the expression level of the JET transcript in said cell, d) selecting said SSO when the expression level of the JET transcript in said cell is higher or lower than a control value.

25. A kit for use in inducing the expression of a tumor specific antigen derived from JET transcript comprising a SSO according to any one of claims 6 to 11, preferably said SSO comprises or consists of a sequence selected from the group consisting of: SEQ ID NO: 21, 22, 23, and 27.

26. A kit for use in inducing the expression of a tumor suppressor protein derived from JET transcript comprising a SSO according to any one of claims 12 to 14, preferably said SSO comprises or consists of SEQ ID NO: 31 or 32.

27. A kit for use in inhibiting the expression of a tumor suppressor protein derived from JET transcript comprising a SSO according to any one of claims 15 or 16, preferably said SSO comprises or consists of SEQ ID NO: 33.