A polynucleotide targeting line1

Abstract

The present invention relates to a polynucleotide targeting Long Interspersed Nuclear Element 1 (LINE1 or L1) or a composition comprising said polynucleotide. Further, the invention relates to medical uses of said polynucleotide and to an in vitro method for modulating LINE1 expression in a cell.

Description

[0001] DESCRIPTION TITLE

[0002] A POLYNUCLEOTIDE TARGETING LINE1

[0003] Technical field

[0004] The present invention relates to a polynucleotide targeting Long Interspersed Nuclear Element 1 (LINE1 or L1 ) or a composition comprising said polynucleotide.

[0005] Backg ound art

[0006] Long Interspersed Nuclear Element 1 (LINE1 ) is a type of retrotransposon that comprises a significant portion of the human genome. These elements have the ability to replicate and insert themselves into new locations within the genome, potentially impacting gene expression and cellular function. In recent years, research has revealed that LINE1 transcripts are expressed in various cell types, including T cells, and may influence cellular processes and disease states independently of their retrotransposition activities.

[0007] T cells, particularly tumor-infiltrating lymphocytes (TILs), are crucial components of the immune system's response to cancer. However, in many cases, these cells become dysfunctional within the tumor microenvironment, limiting their ability to effectively combat cancer cells. Understanding the factors contributing to T cell dysfunction is essential for developing new therapeutic approaches in cancer immunotherapy.

[0008] Recent studies have shown that LINE1 transcripts are over-expressed in dysfunctional T cells, including TILs, and contribute to their exhausted phenotype. The majority of expressed LINE1 elements consist of 3' untranslated region (3'UTR) portions, which have been partially targeted by previously designed antisense oligonucleotides (ASOs). However, the current approaches for designing ASOs to target LINE1 elements have limitations in their ability to effectively and specifically modulate LINE1 expression in dysfunctional T cells.

[0009] Furthermore, over-expression of LINE 1 may lead to T-cell dysfunction causing inflammatory and / or infectious diseases. In fact, a decreased function of the cells of the immune system may expose an individual to an increased susceptibility to infections.

[0010] One challenge in targeting LINE1 elements with ASOs is the high degree of sequence variability among different LINE1 subfamilies. This variability makes it difficult to design ASOs that can effectively target a broad spectrum of LINE1 elements while minimizing off-target effects on other genes or cellular processes. Additionally, the complex structure of LINE1 transcripts, including their tendency to form secondary structures, can impede the accessibility and efficacy of ASOs.

[0011] Another problem associated with current LINE1 -targeting approaches is the potential for unintended effects on cellular function. As LINE1 elements are widely distributed throughout the genome and may play roles in normal cellular processes, broad suppression of LINE1 activity could have unintended consequences on cell behaviour and gene expression.

[0012] Furthermore, the delivery of ASOs to specific cell populations, such as TILs within the tumor microenvironment, presents additional challenges. Ensuring that the ASOs reach their intended cellular targets and achieve sufficient intracellular concentrations to effectively modulate LINE1 expression remains a significant hurdle in developing LINE1 - targeted therapies.

[0013] It has been recognised that improved antisense oligonucleotides and design approaches are needed to overcome one or more of these problems.

[0014] Summary of the invention

[0015] In a first aspect, the present invention relates to a polynucleotide targeting Long Interspersed Nuclear Element 1 (LINE1 or L1 ) or a composition comprising said polynucleotide. Preferably, the polynucleotide is complementary to a sequence having at least 80% identity with a sequence selected from the group consisting of SEQ ID NOs: 1 and 2.

[0016] In some embodiments, the polynucleotide is complementary to a sequence having at least 90% identity with a sequence selected from the group consisting of SEQ ID NOs: 1 and 2.

[0017] In a preferred embodiment, the polynucleotide comprises a sequence having at least 80% of identity with a sequence selected from the group consisting of: SEQ ID NO: 3-11 .

[0018] Preferably, the polynucleotide comprises a sequence having at least 90% of identity with a sequence selected from the group consisting of: SEQ ID NO: 3-11 , more preferably, the polynucleotide consists of a sequence selected from the group consisting of: SEQ ID NO: 3-11.

[0019] In some embodiments, the polynucleotide comprises a sequence having at least 90% of identity with a sequence selected from the group consisting of: 3, 8-11 .

[0020] In some embodiments, the polynucleotide comprises a sequence having at least 80% of identity with a sequence selected from the group consisting of: SEQ ID NO: 4-9 and 11 or consists of a sequence selected from the group consisting of: SEQ ID NO: 3 and 10. Preferably, the polynucleotide comprises a sequence having at least 90% of identity with a sequence selected from the group consisting of: SEQ ID NO: 4-9 and 11. Preferably, the polynucleotide consists of a sequence selected from the group consisting of: SEQ ID NO: 4-9 and 11 . Preferably, the polynucleotide consists of a sequence selected from the group consisting of: SEQ ID NO: 3, 8-11 .

[0021] In a second aspect the present invention relates to the polynucleotide for use as a medicament. In a third aspect the present invention relates to the polynucleotide for use in the treatment or in the prevention or in a follow-up of a cancer or of a viral disease.

[0022] In some embodiments, the cancer is a blood or a solid cancer.

[0023] Preferably, the blood cancer is selected from a group consisting of: a leukaemia, preferably B lineage leukaemia, a lymphoma, preferably Non-Hodgkin lymphoma and a myeloma, preferably multiple myeloma and the solid cancer is selected from the group consisting of lung cancer, liver cancer, brain cancer, bladder cancer, kidney cancer, colon cancer, cervical cancer, uterine cancer, head and neck cancers, skin cancer and oesophageal gastric cancer.

[0024] In a fourth aspect the present invention relates to an in-vitro method for modulating LINE1 expression in a cell comprises at least one step of delivery of the polynucleotide as above disclosed to said cell.

[0025] In some embodiments, the cell is an immune selected from a group consisting of: an immune cell expressing a chimeric antigen receptor (“CAR”), an immune cell expressing an artificial T-cell receptor (“TCR”) subunit, a CD4+ or CD8+ T-cell, preferably exhausted and pre-exhausted CD3+ TILs, and a B cell.

[0026] Preferably, the immune cell is selected from the group consisting of: a CAR-T cell, a CAR-NK cell, a CAR-macrophage and a TCR-T cell.

[0027] In some embodiments, the delivery of the polynucleotide is achieved through nanoparticles, preferably lipid nanoparticles, transfection, electroporation or viral transduction.

[0028] Brief description of drawings

[0029] Figure 1 . Evaluation of killing activity of in vitro exhausted T cells treated with LINE1 ASO. Real-time monitoring of killing activity of T cells treated with LINE1 ASO from the previous patent (a) or four new LINE1 ASO sequences in this patent (b, c, d and e), compared to scramble ASO (scrASO). Killing curves represent the average of at least 3 independent replicates. The overall killing activity was quantified as the area under the curve (AUC) to showcase the difference in killing activity between LINE1 ASO (from previous or in the current patent) and scrASO (a, b, c, d and e - right panels). Error bars correspond to standard-error mean. Results of two-way ANOVA test (killing curves in left panels) or two- tailed t test (barplots in right panels) are showcased on the graphs. *p-value < 0.05, Upvalue < 0.01 , ***p-value < 0.001 .

[0030] Figure 2. Evaluation of killing activity of ex vivo TILs treated with LINE1 ASO. a) Realtime monitoring of killing activity of T cells treated with LINE1 ASO, compared to scramble ASO (scrASO) in one non-small cell lung cancer (NSCLC) patient sample, b) Overall killing activity, quantified as area under the curve (AUC), of TILs sorted from different patients and treated with LINE1 ASO sequences, compared to scramble ASO (scrASO). Error bars correspond to standard-error mean. Results of two-tailed t test test are showcased on the graphs. **p-value < 0.01 , ***p-value < 0.001 .

[0031] Detailed description of preferred embodiments of the invention

[0032] In a first aspect, the invention relates to a polynucleotide designed to target LINE1. The polynucleotide is designed with specific sequences and parameters to enable efficient and selective targeting of LINE1 . The use of the polynucleotide provides a valuable tool for studying LINE1 function and developing potential therapeutic interventions.

[0033] In some embodiments, the polynucleotide comprises 10 to 25 nucleotides, preferably 15 to 22 nucleotides. Preferably, the polynucleotide has a CG percentage between 30 and 70%, more preferably between 40 and 60%.

[0034] This range of CG content is selected to balance the stability of the polynucleotide-RNA duplex and the specificity of the polynucleotide for its target RNA. A higher CG content can increase the stability of the duplex, enhancing the efficacy of the polynucleotide. However, a too high CG content can also increase the risk of off-target effects, as the polynucleotide may bind to non-target RNAs with similar sequences. Therefore, a CG content between 40 and 60% is considered optimal for achieving effective and selective targeting of LINE1 elements.

[0035] In a preferred embodiment, the polynucleotide is an isolated inhibitory nucleic acid targeting LINE1 , preferably, said inhibitory nucleic acid is an antisense oligo (ASO), more preferably said inhibitory nucleic acid is modified, and / or comprises one or more modified bonds or bases. One example of modified inhibitory nucleic acid is 2’-fluoro- ribonucleotides (2’F-ASO).

[0036] In some embodiments, the polynucleotide is modified to enhance its stability, specificity, and overall efficacy. Preferably, the ASO is modified by the incorporation of phosphoroth ioate bonds and flanking 2’-fluoro-ribonucleotides (2’F-ASO) or 2'-deoxy-2'- fluoro-p-D-arabinonucleic acid (FANA) into the ASO.

[0037] In other embodiments, the polynucleotide modifications can include, but are not limited to, phosphoroth ioate bonds, 2'-O-methyl modifications, locked nucleic acids (LNAs), and peptide nucleic acids (PNAs). Each of these modifications can provide specific advantages. For instance, phosphorothioate bonds can enhance the nuclease resistance of the ASOs, increasing their stability in biological environments. 2'-O-methyl modifications can enhance the binding affinity of the ASOs for their target RNAs, potentially increasing their specificity. LNAs can enhance both the stability and the specificity of the ASOs, while PNAs can provide a unique backbone structure that can enhance the overall performance of the ASOs.

[0038] In some embodiments, the choice of modifications can be guided by the specific requirements of the application. For instance, if high stability is required, modifications such as 2’F-ASO or phosphorothioate bonds may be preferred. If high specificity is required, modifications such as 2'-O-methyl or LNA may be preferred.

[0039] In other embodiments, a combination of modifications may be used to achieve a balance of stability, specificity, and overall efficacy. The use of these modifications provides a versatile and flexible approach to the design of polynucleotide for targeting LINE1 elements.

[0040] In a preferred embodiment, the polynucleotide is an ASO.

[0041] The polynucleotide designed to target at least one LINE1 element is preferably characterized by its specific sequences and parameters, can selectively target LINE1 transcripts or elements, thereby modulating the expression of these elements in cells.

[0042] Through the identification of variants in their sequences accumulated in the evolutionary heritage, subfamilies of LINE-1 elements can be subcategorized. LINE-1 has been classified into three main groups during early primate evolution including L1 M (mammalian-specific, oldest), L1 P (primate-specific, intermediate), and L1 H (humanspecific, youngest) subfamilies.

[0043] In a preferred embodiment, the polynucleotide is complementary to at least an Untranslated Region (UTR) of L1 or to at least one Open Reading Frame (ORF), preferably the polynucleotide is complementary and targets at least one portion of the 3’ UTR and / or to ORF2 of L1 .

[0044] In a preferred embodiment, the polynucleotide is complementary to a sequence having at least 80%, preferably at least 90% identity with a sequence selected from the group consisting of SEQ ID NOs: 1 -2.

[0045] In some embodiments, the polynucleotide is complementary to a sequence that has 91 , 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 1 -2.

[0046] Preferably, the polynucleotide sequence has at least 70%, preferably at least 80%, more preferably at least 90% identity to a sequence selected from the group consisting of SEQ ID NOs: 3-11.

[0047] Preferably, the polynucleotide sequence consists of a sequence selected from the group consisting of SEQ ID NOs: 3-11 .

[0048] In a preferred embodiment, the polynucleotide sequence has at least 70%, preferably at least 80%, more preferably at least 90% identity to a sequence selected from the group consisting of SEQ ID NOs: 3, 8-11 .

[0049] Preferably, the polynucleotide sequence consists of a sequence selected from the group consisting of SEQ ID NOs: 3, 8-11 .

[0050] In some embodiments, the polynucleotide comprises a sequence having at least 80% of identity with a sequence selected from the group consisting of: SEQ ID NO: 4-9 and 11 or wherein the polynucleotide consists of a sequence selected from the group consisting of: SEQ ID NO: 3 and 10. Preferably, the polynucleotide comprises a sequence having at least 90% of identity with a sequence selected from the group consisting of: SEQ ID NO: 4-9 and 11 .

[0051] In some other embodiments, the polynucleotide consists of a sequence selected from the group consisting of: SEQ ID NO: 3, 8-11 .

[0052] In some embodiments, the polynucleotide is complementary to a sequence having at least 80%, preferably at least 90% identity with SEQ ID NO: 1 .

[0053] Preferably, the polynucleotide sequence has at least 70%, preferably at least 80%, more preferably at least 90% identity to a sequence selected from the group consisting of SEQ ID NOs: 3-7.

[0054] In some embodiments, the polynucleotide is complementary to a sequence having at least 80%, preferably at least 90% identity with SEQ ID NO: 2.

[0055] Preferably, the polynucleotide sequence has at least 70%, preferably at least 80%, more preferably at least 90% identity to a sequence selected from the group consisting of SEQ ID NOs: 8-11.

[0056] The Applicant surprisingly found that the ASO described above can effectively target a broad spectrum of LINE1 elements. This high binding ability enhances the inhibitory effects of the ASO on LINE1 expression, especially in immune cells, thereby increasing their efficacy against cancers and viral infections. In particular, the ASO described above are able to bind LINE1 , with particular specificity for the P and M subfamilies.

[0057] A second aspect of the present invention relates to a composition comprising the polynucleotide disclosed above and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be any suitable carrier known in the art that can facilitate the delivery of the polynucleotide to a subject's cells or tissues.

[0058] In some embodiments, the composition is formulated as a solution, suspension, emulsion, or other suitable formulation that can facilitate the administration of the polynucleotide. The composition may be prepared using standard techniques known in the art and may be administered to the subject using various methods, including but not limited to, injection, infusion, inhalation, or other suitable administration methods.

[0059] A third aspect of the present invention relates to the polynucleotide or to the composition comprising said polynucleotide for use as a medicament.

[0060] Preferably, the polynucleotide or a composition comprising said polynucleotide is administered to an individual in need thereof, more preferably an individual diagnosed with and / or affected by a cancer.

[0061] Preferably, the polynucleotide or a composition comprising said polynucleotide is administered to an individual in need thereof, more preferably an individual diagnosed with and / or affected by a viral disease.

[0062] In some embodiment, the composition comprising the polynucleotide is formulated for enteral or parenteral administration, preferably for parenteral administration.

[0063] Preferably, the composition is formulated for parenteral administration, more preferably as: a solution a suspension, an injectable preparation, an infusion, a concentrate for injectable preparations, or a powder for injectable preparation.

[0064] In some embodiments, the polynucleotide or the composition is administered in a single dose or in multiple doses over a period of time, depending on the needs of the patient and the type of disease being treated.

[0065] In some embodiments, the polynucleotide is used in immunotherapy, preferably in cancer immunotherapy. Preferably, the polynucleotide is used for improving the persistence of an immune cells, preferably of TILs within the tumor microenvironment and its anti-tumor activity , or of an immune cell expressing a chimeric antigen receptor (“CAR”) or artificial T-cell receptor (“TCR”) subunit.

[0066] A fourth aspect of the present invention relates to the polynucleotide or a composition comprising said polynucleotide for use in the treatment or in the prevention or in a followup of a cancer.

[0067] Preferably, the treatment comprises the administration of the polynucleotide or of a composition comprising said polynucleotide to an individual diagnosed with cancer, with the aim of destroying the cancer cells and reducing the size of the tumor.

[0068] Preferably, the prevention comprises the administration of the polynucleotide or of a composition comprising said polynucleotide to an individual at risk of developing cancer, with the aim of enhancing the patient's immune response and reducing the likelihood of cancer development.

[0069] Preferably, the follow-up comprises the administration of the polynucleotide to a patient who has undergone treatment for cancer, with the aim of monitoring the patient's response to treatment and detecting any signs of cancer recurrence. The follow-up may be conducted at regular intervals, such as every few months or once a year, depending on the type of cancer and the patient's risk of recurrence. The follow-up may involve various tests and procedures, such as blood tests, imaging studies, and biopsies, in addition to the administration of the polynucleotide.

[0070] In some embodiments, the polynucleotide is used in the treatment of blood cancers. Preferably, the blood cancer is selected from the group consisting of a leukaemia, preferably B lineage leukaemia, a lymphoma, preferably Non-Hodgkin lymphoma and a myeloma, preferably multiple myeloma. The polynucleotide may be administered to a patient diagnosed with a blood cancer, with the aim of destroying the cancer cells and reducing the disease burden. In other embodiments, the polynucleotide is used in the treatment of a solid cancer. Preferably, the solid cancer is selected from the group consisting of lung cancer, liver cancer, brain cancer, bladder cancer, kidney cancer, colon cancer, cervical cancer, uterine cancer, head and neck cancers, skin cancer and oesophageal gastric cancer.

[0071] The polynucleotide is administered to a patient diagnosed with a solid cancer, with the aim of destroying the cancer cells and reducing the size of the tumor.

[0072] In some embodiments, the polynucleotide or the composition comprising it is administered alone.

[0073] In other embodiments, the polynucleotide or the composition comprising it is administered in combination or in association with at least one therapy for the treatment of cancer. Preferably, the at least one therapy for the treatment of cancer is selected from surgery, radiation therapy, chemotherapy, thermal ablation or other forms of immunotherapy.

[0074] A fifth aspect of the present invention relates to the polynucleotide or a composition comprising said polynucleotide for use in the treatment or in the prevention or in a followup of a viral disease.

[0075] Preferably, the treatment comprises administering of the polynucleotide or of a composition comprising said polynucleotide to an individual diagnosed with and / or affected by a viral disease, with the aim of destroying the viral cells and / or boosting the immune system of the individual against the viral cells.

[0076] Preferably, the viral disease is selected from the group consisting of immunodeficiencies due to Human Immunodeficiency Virus (HIV), Lymphocytic choriomeningitis virus (LCMV), Hepatitis B (HBV), Hepatitis C (HCV), Epstein-Barr virus (EBV), Human papillomavirus (HPV) and Cytomegalovirus (CMV).

[0077] In some embodiments, the polynucleotide or the composition comprising it is administered alone.

[0078] In other embodiments, the polynucleotide or the composition comprising it is administered in combination or in association with at least one therapy for the treatment of the viral disease.

[0079] A sixth aspect of the present invention relates to a method for modulating LINE1 expression in a cell comprises at least one step of delivery of the polynucleotide to said cell.

[0080] Preferably, said method is an in-vitro method.

[0081] In some aspects, the polynucleotide is delivered into cell to modulate LINE1 expression. This can be achieved through various delivery methods, including lipid-based formulations that can enhance cellular uptake and delivery efficiency.

[0082] The target cells for the delivery of polynucleotide can include various types of cells, depending on the specific requirements of the application. In some embodiments, the target cells are CD4+ or CD8+ T cells, including exhausted and pre-exhausted CD3+ TILs. These cells are known to express LINE1 elements and may play a significant role in the immune response, making them a valuable target for the modulation of LINE1 expression.

[0083] In other aspects, the target cells are B cells, which also express LINE1 elements and play a crucial role in the immune response.

[0084] In some embodiments, the immune cell is an immune cell expressing a chimeric antigen receptor (“CAR”) or artificial T-cell receptor (“TCR”) subunit.

[0085] Preferably, the immune cell is selected from the group consisting of: a CAR-T cell, a CAR-NK cell, a CAR-macrophage or a TCR-T cell.

[0086] In a preferred embodiment, the immune cell is a cell expressing a CAR, preferably a CAR-T or a CAR-NK cell, more preferably a CAR-T cell.

[0087] In some embodiments, the delivery of the polynucleotide is achieved through nanoparticles, preferably lipid nanoparticles, transfection, electroporation or viral transduction, small molecules and antibody conjugated with the polynucleotide. The delivery of the polynucleotide may result in the stable or transient alteration of L1 expression in the cell, potentially enhancing their persistence and anti-tumor activity within the tumor microenvironment.

[0088] In some embodiments, the delivery of the inhibitor is achieved through direct injection of the inhibitor, preferably of the polynucleotide, more preferably of the polynucleotide in the cell.

[0089] These methods may involve delivering the polynucleotide into the cells, potentially using lipid-based formulations to enhance cellular uptake and delivery efficiency.

[0090] The polynucleotide and methods provided in this disclosure may have potential applications in treating various conditions associated with LINE1 expression, including cancer and a viral disease In some cases, the polynucleotide may be used to modulate immune cell function in cancer immunotherapy, potentially enhancing the efficacy of these therapies.

[0091] In some cases, the modulation of LINE1 expression in immune cells may provide a novel approach to cancer treatment. For instance, the polynucleotide to target LINE1 elements may help to restore normal T cell function, potentially enhancing the body's anti-tumor response. In other cases, the modulation of LINE1 expression may be used in combination with other cancer treatments, such as immunotherapy, to enhance the overall efficacy of these treatments.

[0092] In some aspects, the polynucleotide is delivered into cells using lipid-based formulations. These formulations can enhance the cellular uptake of the polynucleotide, increasing their delivery efficiency. The lipid-based formulations can encapsulate the polynucleotide, protecting them from degradation and facilitating their transport across the cell membrane. Once inside the cell, the polynucleotide can hybridize to their target LINE1 sequences, interfering with the processing and function of the LINE1 RNA and leading to a decrease in the expression of LINE1 elements.

[0093] In other aspects, the polynucleotide is delivered into cells using electroporation. This technique involves the application of an electric field to the cells, creating temporary pores in the cell membrane through which the polynucleotide can enter. Electroporation can provide a rapid and efficient method for delivering polynucleotide into cells, potentially enhancing the efficacy of the methods for modulating LINE1 expression.

[0094] In some cases, the polynucleotide is delivered into cells using viral vectors. These vectors can be engineered to carry the polynucleotide and deliver them into the cells. The use of viral vectors can provide a highly efficient method for delivering polynucleotide into cells, potentially enhancing the efficacy of the methods for modulating LINE1 expression.

[0095] Sequences

[0096] SEQ ID NO: 1

[0097] TGCAGTGGTGGCATAAAGAGGCCACACACACACNCCACAGCAAAATANACNNAANT ANTAATAATAAGATTATAAAANACAAAGNNAANACAAAAGTAAAATGTATGGNTTNAT TNCAACAACAATAGAAAANGAAAAANNATTTANTAAATAAACNCTGCAAACATNTGNN AAGAAAAATANATCTTATTACATATAAANTTAAATCANTATAAACAATATCCTACAGCC AAAGTGAGNNTGTATCTAATCAATACTACCACTTTTTTANTANTTCCTAAAAAAAAACA AGAATCNCATACAAACATNANAAAGGTTAGAAAAAAAATANATAACNGAAGNATGAAT ANTTAAAAGCTTNCCTTTTCCTACATATTAATGNGTACACCAANCTAAACTAAATTTTT CAATTTATTTTATTTTAGATNGCAATATAAGTTTGTTGAAACAAAGGGAGAAAANGTTT AANTGNAGGTTTTAACCTTNTACATAAATTTAATTCCAAGAGACTANNGANANTTGCT TAATTTATTTAGCNGNAACAATNAGAATATTATANAACAACTATACCATGANATATGAA AAATAAAAATGTAATTAACTTNTTTTTATTAATTATANTTAAATTATTATATTATAAATGA AAAAAATAAACTAATTTTACAATNAATTTTTTTATTTAAGTAATAAAAAAAAAAATATTG AAAG G AAAATTAATNTTACTAATATAAAAATAACTATGTTC AAAAAAAATG ATATTTAAT TAAAATGTTAATTTCCAATAAAANAATTTTTGTAAATACTGATTTCATTTTCTTTANNTA TTGGGTATTTAAAAGAGTATAATNANAAATGATTNATAAATGAAAAAAATAATTTCAAT GTAATGTTCATTTATGCNTTATNAGAATTAAAGTAGATTATTAATAAATTAACTTTATAT AANGTTAAAAAAGAATAAATTTTTTATATTNTATNANGANCAATATTTTTAAGATATGTA AATAAAAAAATTTTTTTACAAGCAATAACNTATGAAAANAAAATTTTATTAAAATTTTTT ATGATTTTTTAAAATTTAATATNTTTTATANATAACTACAATTATTNTTTTATTTATTTNT ATAANTATTTTAAATTTAATATCATTTTAAAAATATTATTAATAAGCTNCTTATAAAAATT TACTCTAATTATGATTATATTTATACCTTATCATAAATAAAATAAAAAANTTTTAAGTTA TANTATCTTATTTAAACAAATNCTTCTTAAGTTTTTTTACTATTTATNACCTATTNGGAN

[0098] ANTATTTAATTTTGTTTNTTTAAACTTAGATNTAGATTNATGAATTTTGAAAATATTTTA

[0099] TTTATTGTTAAATTTAATAAAAAGAANAAAAAAATTACATACTGTATTATTCAATTTATA

[0100] TGTCTATTTTTTAAAAATAANAAAAAAAATCANATCCATGGAGNTCATAATATGCATAA

[0101] TAANNANATAGTTACTTATGTTTTTTATACTGAATAAATTTGTTTGCGAAGGTNATTTA

[0102] ACATAAATATGTNTTGAGATTGGGAAANTGTATATTAAAAAGAATAGNAAGGNAAAAA

[0103] ATATTTGCAAATTTTTGTGANTGATTGAAATTTTTTTATATANATTATATAATTGANNGT

[0104] GTTGGTTGTATAANTTTTTATNAAANTTATATTAAATNTGTTTAAAAAGTCATTAAACTN

[0105] TACAATTTTTAAATAGTAAAAATGNTTAAATTTATTAAAAATTGTTTTTTATATAAATTTT

[0106] ATCTAAGAANAAAANATAATTTNTNAATAAACATATGAAAATTGTCGAGTTNTTCAACA

[0107] TCATTAGNTAATTAAATGAAAATGTAATTTAAAACNATATTGAGANATNTTGTCNCTTT

[0108] TAAATAATAAGAATGGCTAAAATTTAAATTATAAACTATATTTAAATATTTTTTGTTAGT

[0109] ATGTNGAGNANATGGTATTTTTATTGTTCTTTTAATCTTCTNTGNTAGAAANATTAACA

[0110] TTTTNNTTTTAATTTNNGTGTTGGGAATGTAAAATGTTATAACCACTTTGGAAAAACAG

[0111] TTTGGAATTTTCTTATTAAAAGTTAAACATAATTTTGTCATATTAATTACNATATTGATC

[0112] ATTCTATTTNAATTATTTAGAATTAGATTTTNAATTTAAAANAAAAATTAAATTAACTTN

[0113] TAATTTTTACAAATATAAAAATGTATATCTTTANATANTAAATTTTATATTAGCTTAATTT

[0114] TATAATAGCCAAAAANTAGAAANACAACCAAAATATNTTTCAATNTGTGAATGGATGA

[0115] TNCATTTCTTTNTTAATAAATTGTGGGATATCTCTTCAATAAATTTAATNNTACTATTCA

[0116] TGNAATGAATAGAATTGTTTTATTTTTCAGTTTTGCTNCACTTATTTNGTAAGNAATAA

[0117] AATACTGATAANTTNATTNTAAAACTTNTAGTANATTAAGNATATATATAAAAATGTTT

[0118] GANTNTAATAAATCAATTTACATAAAGAAAAATANTATTTTNATTATTCAANTTATATGA

[0119] AAGTATAACTAGNATATATAAATTCATATAAATTTTAAAAAAAGTCNTTTTTTCTGAAAC

[0120] TATTTAGATTGAATTTAGTANATTTGGCATTGGCNACTTTTAGANGCTTTGTGTAAAAT

[0121] TGATTNACTANTTATTTNATANAAGATGATAGAANTGTTTTTATACCTTCTCTTNNTTG

[0122] GTAATTANAATACATTAAAGTATGTTTCACTTGTTNANNNTNNTAGTATACATNTATTA

[0123] AATANCAANTAAAAANAATATATAATCTAACAAGGATAATGTAAATATTTTTTTTTATAA

[0124] ANAAATTTNAAAGNATNNTAACCNCAATTAACAACAATCATTGNNTATAAAGAAAAAT

[0125] CNTTAATGTGAAATCTGATTTTATTTTTATNTCAGCAAAATAAATAAACTTTTCCTTTNA

[0126] AATTTTTTCTGATACTTNCAAAAAAAGTAAAAGTTATGTTNATGATTTGAAATNAGCAT

[0127] NNGTTGNTTANAATGAGTTATATTTGCATTTAATATGTATANTAGTANCAAGAGNGAA

[0128] AANAGNAGNAAAACACANTCTANAACCTTATGNAGTNAAAATAATNTTTTTATAACNT

[0129] CTGNTNATATAAAATTNAAATTATTTCCAAATCTTTAGTANATATTTTATAATTTCTGGT

[0130] TTNTAAATTAAGAAATTAAAAAAAAAAAAGAAAAAAAAANTAATAAAANNGTATAAATA

[0131] TAAATTAGAAGGAAATATAAANNTAANACAAAAAAGTTATTGAGGTAAAAAATATATAA

[0132] AAATAAAAAAAGANANAACATATAAANTACAAAACTGGTTNTTTTAAAGAATATNAAAA

[0133] ATCTATAAGATTGAATATATAATTCAGTGAGATTAANNTATTTANAAANAAAAACAAAA GNAAAAAAAGTTNTGCATNACCATCACCACTATAAATNCANNAACAGCAGNCCNTAT GATNNTGANTNATGAAATCGATNNCAAAAACTTTTTGATCATCCCNAAAAACTGAAAC ACTGTACCGATTAAAAAATNCCATNAACTTCCCATTAAAAGGATGAAGACCTCTCNAC CCAGCNCCTGGCAACCCCTGAAAAGAGNAATTNTACTTTTTNTANCNNNNNNNNANN AATGTAANTNCTGTCTCTATGAATTAGACTATTNTAGCCAATATCTTATATAAATAGAA GTCATACAATATNTATGTATTTTGTATTGNTTGGCTTATTTAACACTTAGCATAATGTN

[0134] TTCAAGGTTCATACATGTNGTANCATATATCAGAATTTCATTCCTTTTTACANNGGAN NATTTATTCCATTCCTTTACAGAAATAAANCAAATTTTGTTTATCCATTCATTTACTAAN NAANAAAACAATNANTNNTAAGATTNNCAAGGATATNGNNCANNNANANAACTACTG ATNGACATTTGGGTTNTTTCCATNTTATAAAANACTTTTTGGCAATTTCAAATAAGGCT GCTATGAACATTCCTGTACATATGTCTCTTTGTAGTGATACNTAAGATATTCTNAAGT GAGNNTAAGAAAAATAAANAAATNTNAAAGGACTAATAANATNTTTATTATTTNCGTTA

[0135] ATACCTANNAGNAGAAATTCTGANATCANAGAACAGATAAANTAGGGAAATTTATAAA TTNTTG G AAANTAAATAAC AC AGTGTTTTCTAAC ANTAG GTATAC AAN ATAACTCTC AT ACNANCAGTGNATAATGTTTTGTATTTAATTTANATGNAGAACTAAAANNAATCAACAT TNTGNNAGTCANTCANTGNAATGNTCCNAGNGAANATGTATCTATAAATANGTNTNTT AAAAANNAATCAGACATNGTGNTTGTAANNTNNANTNNCNATTAACATGGNCNAAAA NNAG

[0136] SEQ ID NO: 2

[0137] GGATCNTGGNTTCCAAGTTTTCTTNTTTTATTTTTTTTTTTTTTACTTATAAANCTTTAT TTAAGTTCTAGGGTACATGTGCACACTGCATTACGTGCAGGAAGATTTGTTACATGC CAACTTATGTATACATGTGCCATGTTGGGTGTTTGTTTTCCTTTNGCTGCACCCATCG GAGGGNAANCCGAAGCANTGCAGGAACTTACAAGAATTAACGTTTNAAGTGAACAN GGNANNTCGTCCNCTNCCCAACNNANNCTCCCATTTAGGAAATTAACATAGTACCTT TTAGGTATATCTCCTAATGCTATCCCTCCCCACAGCTCCNTCCTCCCCAGCCCCACT

[0138] CCNAAAGACAGTCTNATCCCCGNGGTGTGATGTTCCCCTCTCTGTGTCCATGTGTTC TCATTGTNTTCAACTCCCACCTATGAGTGAGAACATGTGGGTGTTGCATTCTNGATTC ATGGTTTTCTGTTCTTGTGATAGCTTTGCAAGGNATTGCNTCACTTGCTGAGAATAAT GGTTTCCAGCTTCATCCATAGTCCCTGCAAAGGACATGAATTCATGTTTTTTTTGTGA TTTTCCTTTTTTTATGGCTGCATAGTATTCCATGGTGTATATGTGCCACATTTGCGCC TTTTCTTTATCCAGTCTATCATTGATGGACATTTGGGTTGGTTCCAAGTCTTTGCTATT

[0139] GTGAATATTGCNGCAATAAACATCCACAAACGTGTGCATGTGTCTTTTTAGTAGCATG ATTTCAATATTCCTTTGGGTATATACCCAGTAATGGAACTTGCTGGGTCGAGANGNA AATGGTATTTCTAATTCTTGATCNTTGAGGAATCTCCACACTGTCTTCCACAAGATGC CAGGAAGGTTGAACTAGTTTACANTCCCACCAGCTCAGTGTAATTTTACCTTGTNTTT NCTTCCTCAGTATATAAGTGTTCCTATTTCTCCACATCCTCAGATTAAAAGNTTNATCT CCAGTCACTTTAAAGCATCTGTTGTTTCCTTGACCTTTTTAATGATGGCCATTCTAACT GGTGTGAGATGGTNGTATTCTCATTGTGGTTTTGATTTGCATTTCTCTGATGACCAGT G ATG ATG AG C ATTTG GTTCTTTC ATC ATGG CTTTTG GCTGTTATN NG C ATATGTC AAA TGTCTTAATAGCTGNCAGTCTGTTGCCTAATTTTGAAGAAGTGTCTGTTCATTTCCTT TGCCCACTTTTTGATGGGGTGATCTTTNTTTGTGTTNCTTGTAAGTTTGTGTTGAGTT CATTGATAGATTCTTGGAGTATTAGCCANTTGTCAGATGAATAGTTTCCAACTATTTT CTCTTCTTCTGTAGGGTTGGATCAGGCNACTACAGTGGNTTTTTTNTTTTNNTANTGG AATGTTCACTCTGATGATAGTNAATTCTTTTATTGCGCNTGATAATCCTGTGCAGAAA AAGCTTTTTAGTTTGATCAGTTCAGATAACTTCAGTAAAACTGTAGCTTTTGTTGCCAT TTNTTTTAAGTGAAATTTTAAAAATGAAGTCTTTGACTACCCATACCTAAGTATTGAAT GTTTTTNCATATGTTTTAAATCTTCTAGGGTTTTTATGGCTTTAAGTNTTACATTGAAG TCTTCCTTAAATACATCTTGAAATTATTTTTTCTATATNTTAAGACCAAAGGATCCTATT TCCACNTAGATGATATTGCTAAATAGAATTGATTTTTAAATTTGNNGGGTAAAAAACTT TTCCCAGAACCATTTATTGAATAGGGTAAAAANCAGAGTGCCTATCCTTTTCCCAATG ATCGCAGCTCCTCTCCAGCAACTTTTCAAAGTTGGGTTGAAAATCAGATTGTTGTTG GTATATAGGAATGCTTCTGATGTTTGTANATTAATTTTGTTCCCTGAGACTATGGATC AGTTTTTATACCAATGCTAGGAAGCTTAAAACAGAAACTTGAAAAAGATTTTATAAGTA TAGCCTTCTAGTATAAGTNANAAGTCAGATAGTATGATNCCTCCAGCTTTCTGGANCT GTATACCTTGGATTGANTTCTCTATGATGGCTGCATTNGCNTTCCATGTNANTTTTTT TTCTTTATCTCCTCTCAGTAGCATTAAANANACANNTTANAATTAAAAANAGTAATTAT TTCCAATTCTGTGAAGAATATCATTGGTGGTTAAAGAGGAAATTGTTGTAATTTATGT AAATTGACATGGGCAGTACATAACAGTTTAAAAAGATTTGAACATAACCTTCTAGCAA TNTGTGACTATGTGNACATAATTCTAAATCTTTTGGGAGTNTGTTTCCTTGATGTTTN NTTATCTAATTCAGTGGTGGTTTATTTCATGGAACAATGTTGTAAAATTTTCTTTAAAN AGGACATTTTCCTGCATCACTTCCCCAACATAGTTATATAGTTCTACATTTTTTTATTC TTTTTCAGCAAATACGAATGGAATACAACTCATGATNTACCTCTCTGGTTGAATGATC ATTGCCTACACACATTATNGTCTGATTCATCAAGGTTGATTATGATATAATGATATGTT ACTGGAAGTCAGAGAATAAGGTTTGGTAATCTTTTAAAGGGAAGCCCATCAGACTAA TAGGGGNTTTCTCAGTAGAAACTCATCGAAAGCCAGCAAAGAGATTGAGTTTGACTT CCTCTCTTCACCATTNAAAACCAATAGACTTACTTTTTCTTTCCTGANTNCTATTAGCC AGAATTTCGTAATCCAGCACTATACTAAAAACATCATAAATGAAAGAGCGGAATACAT TCCTTTACAGATGTTCT

[0140] TTTGGGGACATAGCTTCCAGCTTTTTTNAATTAAGAATAATGTCTTCAAGGNGCTTCT NATATTAAGCTCTAAAATATTTNGAAACATGTACCAGCAATACCTTTTTTTTTAAANGG CTGAATAATATTAAATTGTGTTGAATTTTGTCAAAGGCNTTTAAAGAATCTATNAAAAT AATCATGTGGTTTTTGTTCTTTGGTTATGAAGAAGCTATGAATTACATTTATTTANTGG GCTAAATAAACACCGCATTATCTTNATGAAATGATCAAANCTTGATAGACAAGTATTA NCCTTTAATGTTAATGGGCTANATCCACCTTTTGGCTATTATAAATAGAATTGCTAAAT GGATAAAGTGTCAAGAAATATTTGTTTTGAATNCTGTATTTTTNTCTGGTTACNCATCT

[0141] CACGGGCTATAGACTCACAAAAGTTCAAATTTAAAGGGATGANATAGGAAAAAAGCT

[0142] CTATTTTTCAATTAATTGGAAAACAAAAAAANAAAAAAAAAGCAGAAGTAGCAGTCCT

[0143] AATTTATATTTCTACTAGAATTAAGCTATGAAAGATCAAAAATCCATCCAGTCCAGGG

[0144] CATTTTATAATGGGAAAGCTTTTATAAGCTNTTAATTATTGACTCAATTTAACAACAAG

[0145] ATATNGGTCTCTTCAATATTTATATTTCTTCCTTGGTTCAATATAGGAGGGTTCGTATT

[0146] TTTCCAGCAATTTCTCCATTACTNTCTAAATTTTCTAATTTATAAATTGTATAGACTTCT

[0147] TCATAATATTCTCAAATCAAATCTTAGGATACTTTTATATTCCTGTGGCAANATTAGAC

[0148] GTATTATCGAGTTTGAAATTTTATATTATGATAATTTGGATTTGCACTCTTCTTTTTTTA

[0149] TCTATTAGACCTAATATAGCTCTTAAGTGATATCTACANNTTTTTCAACCTCCAAAAAA

[0150] ACAACAGAATGTATTCGTTTACATTCTTCTTATGGAAGCATCACAAAATCTCAATTTCT

[0151] TTAAATTCTTCTATATNATTGGAAATATCAAACTTTTCTTCTTCAGCAATTTTTAAAGAT

[0152] NGTTTGCTCTTGATTTTTTCT

[0153] AGTTCTTTTAATTGTGATATTAGAATTTTAATTTAATATCTTTCTAGCTTTTCTATGTAA

[0154] GNAATATAGTGCAATAAAATTATATCTCTTTACAAAGAATTACATGTATCACAAATACA

[0155] ATCAAATANATGGTATCTTTGTTTTGAATAGTTTCAAAAACTAGCTCTTGAATTCTGCC

[0156] TGATTCATTTNGACATTTAAAGCTAAATAAAGTAAATNAAGGACAAAANTAAAGATTG

[0157] NTAGTTATTTAATTTCCATGTATTTGTNTGGTTTTGAAATAGTTTCTTATTAGAAATGAA

[0158] TTCAATTTATAATTGAATTAAGGTATAAAAATATCTTGGATACAATTTAAGCTCTTTTTG

[0159] TTTATAGTGAGAATTGTTTAATCCAGTACCTAAATGAGGTATATTTTGAAAGTAAGAAA

[0160] GATGTGATATAAAATTGAACAGATTCTAAATTATGTTCATACTATTGGGGTGAAAAGA

[0161] ACTATAGAAGTCTGAATAAGATACATTTGCAAAAGGGTAACTAGTAGAAGACAAGAA

[0162] NAAACTAAGATAAATTTGCCTNGAGCCGAAGATCTGAAAAATATTGAAAGTAAAAAAG

[0163] AACTTTNTAAAANAANGTCTCCCAAAAATATTTCAATGAATTCAGAAGATTTATTTTTT

[0164] GAAAAAAATAAAAACTTGGCTTTAAAAATAATTGACCACTAGTACCNCTGNNCTTATA

[0165] CTAGATTACATAAATAAAAAAAAAAAAAGAAAATTTTCAAATTTTATTAAATTTAAAAAT

[0166] GATAAAGGGTATATTACTACATATCCCGACCTTCTTACAGAAATANAAACTNTCATCA

[0167] TAGAAGTGTACTATNTTTTATCACCTATAAGAATAAAAACTAGAAAATTTTTTTTGTTTT

[0168] AAATATAAATTGANAATGGANAAATTTTTTCAAANCATTTNNTTTAGAACCATAAACAA

[0169] TAAACTCTCCCAATATCTNTAATTAGGAAGAAATTGAATCTCTGAAATAGAGCAAAAA

[0170] CAGGCTCTGAAATTGAGTCAATAATCAATAACTTACCAACCAATGTCTGACAAAAAAT

[0171] GTCCATAATACCAGAAGGATTTAAACCATTTNATTAAATNCAAAATTTATTCCAGATAT

[0172] ATAAAGAGAATTTGGTACCATTATTACTAANATTAGTTTTATCTNAACTATTTTTTCCAA

[0173] TTTTAAATAGTAAAG GTAG C AN AATG GTATATCTTCTCCTTATTC AT

[0174] TTTATGAGGCCAGCNTCATCCTGATACCAAAACCTGGGCATATTTTATATACAATAGT

[0175] TAAAAACAAAAAAAGAGAANTTTAGACCAATTTGNATATCCTTGATGAAAATGGATGC

[0176] AAACAATCCTTTATTTCNCCATCTTTAAAATTTTCCTTGCAAACCAATTCTAACAGCAT ATCAAAAAGCTAATCCACCATGATCAAGTGAGGGATTAAATCCCTAAGATTCAAGGC

[0177] TGTTTGAAAATATGTTCAAATTTATAAATGTAATTCATAAAATAAACAAAAACTAAATTT

[0178] AAACAACCACATGANTATATAATTATTTGAATGAAGAAATTTTCTTCTATAAAATTTAA

[0179] CATTATTCCTTTTCNTTTCATTATTAATACTTTCTTCTAGAAAGATCTATATATTGAAGG

[0180] AACATTTCTATCTGCTCAATATAATAATATCCATTTATGACAAACTNNNACAGAAAATA

[0181] TTTTTCATTNTAACTGAAATGGACAAAGTNATTAACTTGAAAGCATTTTACCTTTATTG

[0182] AAANCTTNTTTGAACAATACAACNATGATGTGTCTNCTTTCTACTCTCTCATCNCTCC

[0183] TATTNAATATATTATTGGAAGTTCTGCTCAATTGCCATGGAACTATCATGNAAGAAAN

[0184] GAATTTTATTTCTTTATNATAAAGAAATAAAAAGTTTTCAAATTTTTTTAAGAAAAGAGA

[0185] AAGTAAAATTTTCTCTGTTCTGCTTGCAGTTTACATAGCTTAATTTTTTCTTTATCTAGA

[0186] AGAAAANCTCATAATCTTTACATATCATCACCATAAACATTTTNCTCATTAAGNATTAT

[0187] AAACNAAAACTTCAGTTAAAGTCTTAACGTATACAAACATNAATTTTTAAAAATTTGTA

[0188] GAATTCCCTATTCTATCTAACANCAGTATTCTTAAAAATCTATGANAGGAGTGTATTCA

[0189] ATACAGAAAAATGTTTTAGTTTTTACTATTNATTTTTAGATTTAAAAAAANATATTTCGA

[0190] AAATTACTGAATTTTTTAATCTAGTAAAATCTTTTATATANNTTTGTGTTTTTCTTATTAT

[0191] TTTNATTTNTATTTCATTAATGAAAAATAAATTTGTAAACATTTTTTCATTCCATATAATT

[0192] TNAATTAACTGANGAGAAATATATTTTCTTATCATTGCANCCTCATTGACAATTGTTAC

[0193] TAAAANTTTCTTCATATAGTAATTAAAATACTTAGGTATTCAGCTATCTAATATTCAANT

[0194] ATATAAAAAATTACTCTAATAATATAAACAAAATTCNTAAAACACTGCTCTAAAATTATA

[0195] TAAATTTAAATATATAAAAATTATATAATATTAGGATAATATTTTATAATACTCAATATG

[0196] GATAAGAAAAATCAATATACATTAAAATTGTCCATATTACTNATAAAANTAGTAAAATT

[0197] TACATTAANTTAATATATTTAAGTACAATAATANTGGTTCAATATATCAATATAATTTTC

[0198] ATTCAACATAATTCAAATATTTTAAAANAGATTAAAATAATNTAAAAAATAATATTTATT

[0199] AATATTTATTATATTAAAGTCTTTAAATTGTAATGAAAAAAAAAAATATTTTAATTTTTTA

[0200] CATATGCATAAAAGTATCTTACAAAANATTNATGATTGAATATAGAATTTCCAAAATTA

[0201] AATTTTAAAAAAAATAACAATNTAAGAACAAATCATGTTAGAAGATAATCATATANTAT

[0202] TCTCTAA

[0203] TATGATTTTTTAAATTATATATTTACAATACAATGCTATTAGTAAATTAAAATTCTNAAA

[0204] ACAGTATTGTTATTAAATATACNAAAACAGACATTTATAAAATAAGAATTATAGTTGAC

[0205] CAATGGAACAGCACTCACAAAAGAGTCCTTAGAAATTTTAATTGATATCCANCACCAT

[0206] GTACATCTAATTTGATTTTTGACAAACCTGACATGAAAAAATATTNAATAGATAAATGA

[0207] ACACATACGTTTATTGAAATATCTTTTTTATNGGGGAAATAGAGGTTTNTCTATTCAAT

[0208] AAATAAATTTGGTGCTGGGAAAATATAGGATAGGCATTTCCACAATAATAATACATAC

[0209] CACTTTNCTANATGCAGATTTGAAAATTGAAACTTTATTTTGGAACCTTTNCTTNNTAC

[0210] ACCATATACAAAAATTAACTCAAGATGGATTAAAGACTTTAAAATTGTAAGACTTAATA

[0211] TCTNCAAACANCNTGAAAAAAATATGCTCCTTTATGATTTAACTANNTANAANCCAAC

[0212] CATAAAAATCCTAGAATATTTAGAAAACCTAGGCTAAAACCATTCAGGACATAGCAAA NAGCATTGGGCAAAGANTTCTCTTGACTAAAAACACCAAAAGCAGNANCAAAAGCAA

[0213] TGGCAACAAACAGGCAAAAATTGACAAATGGGATTTTAAAATTAAACTAAAGAGCTTC

[0214] TGCACAGCAAAAGAAACTATCATAACAGAGTGAANAGACAACCTACAGAATGNCGAG

[0215] AGAAAATTTTTTANAAAATATTTGCAAACTATTCATGCTGACCCANCTAAAGGNCTAAT

[0216] ATCCAGCTTAAGAAAATCTATAATGAACTCAAACAAATTTACAATAAACCACCAAAAAA

[0217] AAACAACCCCATCAAAAAGTGGGCAAAGGATATGAATAGACACTTCTCAAAAGAAGT

[0218] GANATTGTTTCACATTTAGATNGTTATCCAAAAATCTACACATGAAAAAATGCTCATCA

[0219] TCACACTGGTCATCAGAGAAGAATAGANATCCAAAGAAAATGCAAATCAAAACCACA

[0220] ATGAGATACCATTCCTCACACCAGTTAGAATGGCTATAATTAAAGCAAAAAAAATAAC

[0221] CNACTATAAAAAAGTCAAGAAATAACAAATGCTGGAGAGGATGTGGAAGAAATAGGA

[0222] ACTCTCTTATACACTGTTGGTGGGAATTGTCAATGAAAACTAATATATCGTTACAACC

[0223] ATNTTNTGTGGAAAACAGTATGGCNATTCCTCAAAAAAATAAATAGAGAACTAAAAAT

[0224] ATGCGAATTACCATTTGACCCAGCAATCCCATTACTGGGTATNATATATACCCAAAGG

[0225] ATTATAAATCATTCTGCTCATCACTACTAAAAGAATATGAACAAATAAGCACACNTATG

[0226] TCTCTTATTGCAGCACTATTCACAATAGCCCAGNGCAAAGATATGGAATCAACCCAA

[0227] ATGANTATTTTTATCCATCCAACAATGATAGANTGGTGATATAACGAAAATGTGGCAC

[0228] ACAAAAATACNTAAACACCATGGAATATTATTTCAGCCATAAAAAANAAAGTGAAATC

[0229] ATGCTCCTANTCACTTTGTGCAGGAAGATTACCCCACNNCATGGATTGAACTGGCTG

[0230] GAAAATGGTANGACTCATTAAAAAATTTCTTCAGTAAANTAAAAAACGCAGGAACAAA

[0231] AAAACCAAACACCGCTTCTTATGTTCTCACCAATACTCATATGTGGGAGCTAAACAAT

[0232] NGAGAACACATGGAATAATAAAAAACACANGAAGGGGAACATCACACACCGTGGGG

[0233] ACTGTTGGAAGGGTGGGGGGTGGGCAGGAGGGATAGTTCATTAGGAGAAATATACC

[0234] TAATGGGTATGGCAAAATGCAAAAATAGTTNTGTAAATGAANTGAGTTAGATGGGTG

[0235] CAGCANAACNACAACCTGAATANANTATACTTATNTTTGGCACATGTATACACATATG

[0236] TAACAAACCTGCACATTANGCACATGTACCAAAACCCTAAAACTTGATAAAAGTATAA

[0237] TAAAAAAAAAATATACTTTCCCGTGCTGTTATTATTCAGCCATCNNTTAACNTACCTAT

[0238] ACATNAAATNAACAATAAAAATAAATACAACTNTNATNTATNAATAAAAAT

[0239] SEQ ID NO: 3

[0240] TGTCCATCAACAGATGAATG

[0241] SEQ ID NO: 4

[0242] GGGTTTCTTTTTGGGGTGATG

[0243] SEQ ID NO: 5

[0244] AGTGGTGATGGTTGCACAAC

[0245] SEQ ID NO: 6

[0246] GTGACTGCTAATTGGAACAGGG

[0247] SEQ ID NO: 7

[0248] GAGAAGAAGAATTACTAGGAG SEQ ID NO: 8

[0249] TACACTGTTGGTGGGAATTG

[0250] SEQ ID NO: 9

[0251] ATTCATTTTATGAGGCCAGC

[0252] SEQ ID NO: 10

[0253] AACAACCCAAATGTCCATCA

[0254] SEQ ID NO: 11

[0255] CC A ATG G A AT ACTATG C AG C

[0256] EXAMPLE

[0257] A recent study reports that LINE1 transcripts are expressed in T Infiltrating Lymphocytes (TIL), contributing to T cell dysfunctional features. The majority if LINE1 expressed consists of LINE1 3’UTR portions.

[0258] In order to increase the targeting of the 3’UTR portion of LINE1 elements, two main approaches have been put in place. The first one relies on the technique of Multiple Sequence Alignment (1 ) while the second relies on the use of online tools to identify over- represented sequences within input sequences (2). For these approaches the sequences used as input were LINE1 elements imbedded in transcripts reconstructed from RNA-seq data generated on naive CD4+T cells (generating SEQ ID 1 ) or in exhausted and preexhausted CD3+TILs (sorted from 4 non-small cell lung cancer patient samples - generating SEQ ID 2), called LINE1 input sequences.

[0259] 1) Multiple Sequence Alignment (MSA)

[0260] LINE1 input sequences were aligned using ClustalW (https: / / www.ebi.ac.uk / jdispatcher / msa / clustalo). This tool enables the alignment of multiple sequences. The output was then visualized using Jalview. By using the details provided by Jalview (the consensus sequence and the number of sequences aligning at each individual base) it has been focused on regions with high homology among input sequences. In such regions 30 candidate ASOs have been designed by hand around, 20- nulcoetide long and with CG percentage between 40 and 60%. Predicted targets of candidate ASOs were then checked, both coding genes / transcripts (classic nucleotide [n]Blast) and all transposable element subfamilies (including LINE1 - this is an in-house analysis, transposable element-specific nBlast) The nBlast was conducted applying a filter of 90% ASO sequence coverage and 90% ASO sequence identity. For each individual ASO the number of predicted targets is computed (for both coding transcripts and transposable elements) and used to select best candidate ASOs. More specifically, ASOs with the lowest predicted coding targets and the highest predicted LINE1 targets. SEQ ID 3, SEQ ID 8 and SEQ ID 9 showed the best predicted effect with less than 15 predicted coding targets and more than 8600 and up to 14495 predicted LINE1 targets and were therefore selected for empirical validations. These ASOs were used to treat in vitro exhausted CD8+T cells or ex vivo derived CD3+TILs to evaluate LINE1 knock-down efficiency and re-activation of T cell effector functions, such as capability to kill cancer cells. Compared to previously designed LINE1 ASOs, these ASOs are not only designed following a different methodology but they are also predicted to target other LINEIs (Figure targeting, Table targeting).

[0261] 2) Identification of over-represented sequences

[0262] A tool commonly accepted in the scientific community is MEME for motif discovery (https: / / meme-suite.org / meme / tools / meme), belonging to MEME-suite. LINE1 input sequences were used to discover the top 10 new motifs (over-represented sequences) by using default parameters. Since the identified sequences were longer than 20 nucleotides and / or with a CG percentage lower than 40%, a first set of 5 hand-picked candidate ASOs was selected and checked as previously described (nBlast on canonical transcripts or genes and on transposable element subfamilies, including LINE1 ). ASOs with the lowest predicted coding targets and the highest predicted LINE1 targets have been selected. SEQ ID 10 and SEQ ID 11 showed the best predicted effect with less than 5 predicted coding targets and more than 2700 and up to 21274 predicted LINE1 targets and were therefore selected for empirical validations (as described above). As a whole, also in this case with a completely different methodology, LINE1 ASOs to target a broad spectrum of LINE1 subfamilies have been identified, such as evolutionary old LINE1 subfamilies like L1 MA5A, L1 MB4 and L1 MB7.

[0263] METHODS

[0264] In vitro exhausted T cells

[0265] Naive CD8+T cells were isolated from the blood of healthy donors and culture in vitro under continuous TCR stimulation through anti-CD3 / CD28 beads, which led to T cells exhaustion. After 5 days T cells were treated two times with 5pM ASOs every 48h.

[0266] Ex vivo TILs

[0267] Tumor-infiltrating lymphocytes (TILs) were FACSsorted from human tumor tissue after enzymatic dissociation. TILs were then treated with two times with 5pM ASOs every 48h.

[0268] Killing assay

[0269] In vitro cultured exhausted T cells or patient-derived, ex vivo isolated TILs, were treated for 5 days with 5pM ASOs and then co-cultured with GFP+target cells at a 5:1 ratio. Target cells killing was monitored in real time for several days using a live-cell imaging system (Incucyte, Sartorius) with a cytotoxicity dye to detect dead cells. Killing activity was quantified by measuring the number of remaining live target cells in the co-culture every 3 hours relative to target-cells-only controls. The area under the curve (AUC) for each ASO treatment was calculated and compared with scrASO-treated cells.

[0270] Results

[0271] Across both in vitro exhausted T cells (Figure 1 ) and patient-derived, ex vivo isolated TILs (Figure 2), treatment with LINE1 ASOs consistently enhanced cytotoxic activity compared to scramble controls. In exhausted T cells, LINE1 inhibition supported a marked recovery of effector function, while in ex vivo TILs from multiple patients it boosted their ability to eliminate target cells in clinically relevant settings.

Claims

CLAIMS1. A polynucleotide targeting Long Interspersed Nuclear Element 1 (LINE1 or L1 ) or a composition comprising said polynucleotide, wherein the polynucleotide is complementary to a sequence consisting of SEQ ID NOs: 1 or 2, wherein the polynucleotide comprises a sequence having at least 80% of identity with a sequence selected from the group consisting of: SEQ ID NO: 4-9 and 11 or wherein the polynucleotide consists of a sequence selected from the group consisting of: SEQ ID NO: 3 and 10.

2. The polynucleotide according to claim 1 , wherein the polynucleotide comprises a sequence having at least 90% of identity with a sequence selected from the group consisting of: SEQ ID NO: 4-9 and 11 .

3. The polynucleotide according to claim 1 or 2, wherein the polynucleotide consists of a sequence selected from the group consisting of: SEQ ID NO: 4-9 and 11 .

4. The polynucleotide according to claim 4 or 5, wherein the polynucleotide consists of a sequence selected from the group consisting of: SEQ ID NO: 3, 8-11 .

5. The polynucleotide according to anyone of claims 1 -4, for use as a medicament.

6. The polynucleotide according to anyone of claims 1 -5, for use in the treatment or in the prevention or in a follow-up of a cancer or of a viral disease.

7. The polynucleotide for use according to claim 6, wherein the cancer is a blood or a solid cancer.

8. The polynucleotide for use according to claim 7, wherein the blood cancer is selected from a group consisting of: a leukaemia, a lymphoma, and a myeloma, and wherein the solid cancer is selected from the group consisting of lung cancer, liver cancer, brain cancer, bladder cancer, kidney cancer, colon cancer, cervical cancer, uterine cancer, head and neck cancers, skin cancer and esophageal gastric cancer.

9. The polynucleotide for use according to claim 8, wherein the leukaemia is a B lineage leukaemia, the lymphoma is a Non-Hodgkin lymphoma, and the myeloma is a multiple myeloma.

10. An in-vitro method for modulating LINE1 expression in a cell comprises at least one step of delivery of the polynucleotide according to anyone of claims 1 -4 to said cell.11 . The method according to claim 10, wherein the cell is an immune selected from a group consisting of: an immune cell expressing a chimeric antigen receptor (“CAR”), an immune cell expressing an artificial T-cell receptor (“TCR”) subunit, a CD4+ or CD8+ T-cell ot a B cell.

12. The method according to claim 11 , wherein the immune cell is an exhausted or preexhausted CD3+ TILs.

13. The method according to claim 11 or 12, wherein the immune cell is selected from the group consisting of: a CAR-T cell, a CAR-NK cell, a CAR-macrophage and a TCR-T cell.

14. The method according to anyone of claims 10-13, wherein the delivery of the polynucleotide is achieved through nanoparticles, transfection, electroporation or viral transduction, small molecules or antibodies conjugated with the polynucleotide.

15. The method according to claim 14, wherein the nanoparticles are lipid nanoparticles.

Images