Genetic reprogramming by re-expression of ESE3 / EHF for the treatment of advanced prostate cancer

Abstract

The present invention relates to a nucleic acid sequence encoding the transcription factor ESE3 / EHF or an isoform thereof, for use in the treatment of a cancer in a patient in need thereof and can be a mRNA or a DNA, in particular for use in the treatment of prostate cancer. The invention also relates to a plasmid, a viral vector or a pharmaceutical composition comprising, such nucleic acid sequence. In particular, the present invention is based on the use of plasmid DNA coding the full-length sequence of the gene ESE3 / EHF and to the use of in vitro transcribed (IVT) mRNA of ESE3 / EHF as gene therapy in aggressive prostate cancerESE3 / EHF is a transcription factor that is expressed in normal prostate but is lost in prostate tumors, particularly during the progression from indolent to aggressive tumors. The aim is to replace the ESE3 / EHF transcription factor using gene therapy approaches at the time the gene is reduced or lost. The invention also relates to the use of ESE3 / EHF replacement in combination therapy with androgen deprivation therapy (ADT), the standard treatment for metastatic prostate cancer, and other therapies, such as androgen receptor signalling inhibitors (ARSI), chemotherapeutics, molecular-targeted therapeutics, and immunotherapeutics.

Description

[0001] SIB BW1361 R

[0002] “GENETIC REPROGRAMMING BY RE-EXPRESSION OF ESE3 / EHF FOR THE TREATMENT OF ADVANCED PROSTATE CANCER”

[0003] TECHNICAL FIELD OF THE INVENTION

[0004] The present invention relates to a nucleic acid sequence encoding the transcription factor ESE3 / EHF or an isoform thereof, for use in the treatment of a cancer in a patient in need thereof and can be a mRNA or a DNA, in particular for use in the treatment of prostate cancer (Figure 1). The invention also relates to a plasmid, a viral vector or a pharmaceutical composition comprising such nucleic acid sequence. In particular, the present invention is based on the use of plasmid DNA coding the full-length sequence of the gene ESE3 / EHF and the use of in vitro transcribed (IVT) mRNA of ESE3 / EHF as gene therapy in aggressive prostate cancerESE3 / EHF is a transcription factor that is expressed in normal prostate but is lost in prostate tumors, particularly during the progression from indolent to aggressive tumors. The aim is to replace the ESE3 / EHF transcription factor using gene therapy approaches at the time the gene is reduced or lost. The invention also relates to the use of ESE3 / EHF replacement in combination therapy with androgen deprivation therapy (ADT), the standard treatment for metastatic prostate cancer, and other therapies, such as androgen receptor signalling inhibitors (ARSI), chemotherapeutics, molecular-targeted therapeutics, and immunotherapeutics.

[0005] BACKGROUND OF THE INVENTION

[0006] Prostate cancer (PC) is the second most common cancer in men. Despite the improvement in the treatment of localized disease, PC remains a leading cause of cancer-related mortality. Androgen deprivation therapy (ADT) remains the first-line therapy for patients with metastatic PC. However, ADT in heavily treated patients is frequently followed by resistance and aggressive disease characterized by broad transcriptional reprogramming and increased phenotypic plasticity with the acquisition of stem-like properties, AR independence and neuroendocrine differentiation.

[0007] ESE3 / EHF is a transcription factor highly expressed in the normal prostate but is repressed in prostate tumors. In particular, ESE3 / EHF expression is reduced during the transition from primary to metastatic and castration-resistant prostate cancer (CRPC) (Figure 2). Thus, the technical problem that we intend to solve with this invention is to replace ESE3 / EHF using a gene therapy strategy at the time the gene is reduced or lost. Figure 2 highlights the evolution of prostate cancer from hormone-sensitive to hormoneinsensitive stages, showcasing the loss of ESE3 / EHF and the potential therapeutic window for intervention. Despite multiple pieces of evidence supporting that ESE3 / EHF SIB BW1361 R constitutes a barrier against dedifferentiation and malignant transformation in epithelial cancers, current medical practices lack effective strategies to restore the function of downregulated transcription factors such as ESE3 / EHF in PC. This gap in treatment underscores the need for novel therapeutic approaches that directly target the underlying genetic changes driving prostate cancer progression.

[0008] SUMMARY OF THE INVENTION

[0009] The Authors of the present invention surprisingly found that ESE3 / EHF3-based gene therapy is highly effective for the cure of cancer, in particular prostate cancer, and more in particular in castration-resistant prostate cancer as a single active ingredient (agent) or in combination with other treatments. As will be further detailed in the experimental section of the present specification, the inventors have surprisingly found that stable reexpression of ESE3 / EHF in aggressive prostate cancer cells, such as LNCaP-ABL that are ESE3 / EHF negative and CRPC cells (Figure 3A), causes a reversion of the aggressive phenotypes, reducing growth of tumor organoids, the fraction of tumorinitiating stem-like cells (cancer stem cells), and tumor growth in mice (Figure 3B-D).

[0010] All these effects were not necessarily expected due to the highly aggressive phenotype of these prostate cancer cells, reflecting a very advanced stage of the disease. It was also unexpected that ESE3 / EHF replacement caused such a broad and beneficial reprogramming of the transcriptome, reducing expression of pro-proliferative gene sets and reactivating the androgen signalling toward a normal-like state (Figure 3E-F).

[0011] Indeed, ESE3 / EHF-supplemented prostate cancer cells become more like normal prostate epithelial cells. Specifically, the upregulated gene signature in ESE3- supplemented cells vs control, was associated with primary tumors when applied to prostate cancer tumor transcriptomic datasets (Figure 3F). Consistently, ESE3- supplemented castration-resistant cells became sensitive to the AR antagonist enzalutamide (MDV-3100) (Figure 3G-H). These beneficial effects of ESE3 / EHF were related to the ability of this transcription regulatory factor to control directly a wide network of genes responsible for cell differentiation and phenotypic plasticity, as indicated by the analysis of the genomic distribution of ESE3 / EHF binding sites by CUT&RUN assays (Figure 3I). Collectively, these data indicated that ESE3 / EHF preferentially activated prodifferentiation genes, while repressing an extensive network of genes and other transcriptional regulators, enhancing cell plasticity and loss of cell fate determination. By being upstream of fundamental regulatory pathways enforcing epithelial cell commitment, therefore, ESE3 / EHF re-expression could overcome the deleterious effects of the multiple heterogeneous genetic and epigenetic alterations in different tumor contexts.

[0012] Collectively, the data provided in the present application support the beneficial effects of ESE3 / EHF re-expression in reducing the aggressive malignant traits of cancer cells. This SIB BW1361 R aspect was highly relevant for proposing the use of ESE3 replacement gene therapy in combination with other treatments, such as androgen deprivation therapy (ADT) , although it remained to be demonstrated that transient supplementation of ESE3 / EHF could be practical and equally effective in a therapeutic setting.

[0013] Multiple additional pieces of evidence strongly support the fact that transient supplementation of ESE3 / EHF reverses the aggressive malignant phenotypes of CRPC cells. As will be further detailed in the experimental section of the present specification, the inventors have also surprisingly found that DNA-based ESE3 / EHF gene replacement reduces in cell culture systems the growth of prostate tumor organoids generated from a human cell line (LNCaP-ABL), a CRPC patient-derived model, and a mouse-derived model with genetic deletion of ESE3 / EHF (EHF-KO) (Figure 4A-F). ESE3 / EHF supplementation was equally effective in murine tumor organoid models with combined ESE3 / EHF deletion and overexpression of the oncogene ERG (Figure 5A). Notably, the ESE3 / EHF protein was expressed efficiently in murine tumor organoids following the DNA-based supplementation (Figure 5B), reduced organoid numbers (Figure 5C-D), and induced a significant phenotypic reversion of the malignant organoids toward a normal-like (cystic) morphology (Figure 5E-F). Moreover, the systemic administration of the DNA-based construct by intravenous injection to mice resulted in the relevant expression of ESE3 / EHF protein in tumor xenografts and a substantial delay of tumor growth with the induction of tumor cell death (cleaved caspase 3) in the human CRPC LNCaP-ABL model (Figure 6). Thus, systemic administration of the DNA construct and transient expression of ESE3 / EHF reproduced the effects of the stable expression on this ESE3 / EHF-negative prostate cancer model.

[0014] The supplementation of ESE3 / EHF using an RNA-based system (such as in vitro transcribed - IVT mRNA encoding ESE3 / EHF protein) also resulted in efficient delivery of ESE3 / EHF mRNA and expression of ESE3 / EHF protein in LNCaP-ABL cells (Figure 7A-C). mRNA-based delivery also reduced cancer stem cell proliferation and tumor cell migration (Figure 7D-E), two malignant properties evaluated in vitro by the sphere formation assay (SFA) and the Boyden chamber assay, respectively.

[0015] Moreover, mRNA-based supplementation led to efficient delivery of ESE3 / EHF mRNA as shown by qRT-PCR and significantly reduced the growth of tumor organoids generated from mice with genetic deletion of the ESE3 / EHF gene (EHF KO and EHF / R26ERGmice) (Figure 8). Notably, ESE3 / EHF protein was effectively and persistently expressed in both murine and human tumor organoids as demonstrated by fluorescence confocal microscopy, resulting in a significant reduction of their growth and reversion of their malignant phenotype (Figure 9 and Figure 10).

[0016] Moreover, the inventor found that a single systemic administration of ESE3 / EHF mRNA to mice in vivo by tail vein injection resulted in efficient delivery of ESE3 / EHF mRNA in the tumors and very rapid and effective reduction of the tumor size, with significant SIB BW1361 R regression of LNCaP-ABL tumor xenografts both at high and low tumor volumes (Figure 11 and Figure 12). These data support the feasibility of ESE3 / EHF re-expression driven by a DNA- and RNA-based approach as a suitable therapeutic strategy for prostate cancer.

[0017] The RNA-based delivery approach was further tested in an additional model, EPG2 cells, a murine prostate cancer cell line derived from genetically engineered mice with combined overexpression of the oncogene ERG and deletion of the tumor suppressor PTEN, as frequently seen in prostate cancer patients. EPG2 cells, therefore, represented a complex and challenging testing ground to verify the impact of ESE3 / EHF overexpression. EPG2 cells have low levels of ESE3 / EHF, and supplementation of pESE3 DNA and mRNA led to a significant burst of ESE3 / EHF expression, as shown by fluorescence confocal microscopy and immunohistochemistry (Figure 13). Moreover, systemic administration of ESE3 / EHF mRNA in vivo significantly reduced the growth of EPG2 tumor xenografts in syngeneic C57BI / 6 mice.

[0018] To further demonstrate the deliverability of IVT mRNA by intravenous injection, we administered mRNA encoding the luciferase gene, a reporter protein that can be detected by bioluminescence imaging, to mice bearing subcutaneous tumor xenografts of EPG2 cells. Luciferase activity was detected at 24 h posttreatment by bioluminescence imaging in the tumor xenografts of mice that had received the injection of luciferase IVT mRNA and not in mice receiving control mRNA (Figure 14). Moreover, systemic administration of IVT mRNA by tail vein injection led to delivery and efficient reporter protein expression in metastatic lesions in mice (Figure 15). Relevantly, luciferase reporter activity was detected by ex vivo imaging at 24 h posttreatment in liver metastasis in mice with a metastatic xenograft model generated by intravenous injection of mCherry-labeled LNCaP-ABL cells, indicating that the ESE3 / EHF replacement strategy could be an effective treatment for advanced and metastatic prostate cancer spread to multiple organs.

[0019] To address the potential toxicity of ESE3 / EHF overexpression in normal prostate epithelial cells, we supplemented with ESE3 / EHF DNA (Figure 16) or mRNA (Figure 17) to 3D organoids established from normal prostate epithelial cells from wild-type mice (WT). Notably, WT murine organoids have predominantly cystic structures and express ESE3 / EHF. Supplementation of ESE3 / EHF DNA or RNA resulted in a significant increase of ESE3 / EHF protein but did not affect the number or morphology of WT organoids that remained prevalently cystic. To further test this issue, we supplemented ESE3 / EHF to prostate cancer cells (LNCaP) that retain endogenous expression of ESE3 / EHF (Figure 18). The increased level of ESE3 / EHF did not impact significantly tumor sphere or organoid formation by LNCaP cells. Collectively, these data show the lack of side effects of supraphysiological levels of ESE3 / EHF in normal prostate epithelial cells and ESE3 / EHF expressing tumor cells, further supporting the specificity and safety SIB BW1361 R of this approach acting only in the context of tumors lacking ESE3 / EHF expression.

[0020] Collectively, for ESE3 / EHF gene replacement therapy, the inventors implemented several experimental human and murine models, including 2D cell cultures, 3D human and murine normal and tumor organoids, tumor spheres, patient-derived xenografts, and genetically engineered mouse models, providing supportive data on the feasibility, efficacy and specificity of this approach in several in vitro and in vivo prostate cancer models.

[0021] Therefore, the objects of the present invention are: a nucleic acid sequence encoding the transcription factor ESE3 / EHF or an isoform thereof for use in the treatment of a cancer in a patient in need thereof, preferably said nucleic acid sequence is a mRNA or DNA sequence, more preferably said cancer is prostate cancer. a plasmid comprising the nucleic acid sequence for use according to the present invention, a viral vector comprising a nucleic acid sequence for use according to the present invention, a pharmaceutical composition comprising a nucleic acid sequence according to the present invention, or a plasmid according to the present invention or a viral vector according to the present invention, for use in the treatment of cancer in a patient in need thereof, preferably wherein said cancer is prostate cancer.

[0022] In vitro use of a nucleic acid sequence according to the present invention, or a plasmid according to the present invention or a viral vector according to the present invention for the expression of the transcription factor ESE3 / EHF in a cancer cell, in particular wherein said cancer is prostate cancer.

[0023] DESCRIPTION OF THE FIGURES

[0024] Figure 1. Synthesis, encapsulation and delivery of ESE3 / EHF DNA and mRNA molecules. A) Schematic representation of the full-length ESE3 / EHF used for cloning in plasmid DNA and IVT mRNA production. B-C) Schematic of cloned circular DNA (B) and IVT mRNA (C) for ESE3 / EHF. D-E) Packaging of plasmid DNA and mRNA for ESE3 / EHF gene replacement therapy in nanoparticle delivery systems for systemic administration by intravenous injection in mice.

[0025] Figure 2. Evolution of prostate cancer and the predicted therapeutic window for ESE3 / EHF replacement therapy. Progression of primary tumors to castration-resistant (CRPC) and neuroendocrine (NEPC) prostate cancer is associated with loss of ESE3 / EHF expression, acquisition of cell plasticity and sternness, and deep transcriptional and epigenetic reprogramming. ESE3 / EHF re-expression can reverse this SIB BW1361 R broad phenotypic and transcriptomic reprogramming, restoring a normal-like / indolent tumor phenotype and sensitivity to concurrent treatments like ADT and ARSI.

[0026] Figure 3. Transcriptional and phenotypic reversion to a normal-like state by stable ESE3 / EHF re-expression in aggressive castration-resistant prostate cancer cells.

[0027] A) Left, immunoblot of LNCaP parental and castration-resistant LNCaP-ABL. Right, Immunoblot of LNCaP-ABL expressing ESE3 / EHF (pESE3) and Ctrl (pCTRL). B) Sphere formation assay (SFA) in the indicated cell lines. C) 3D organoid formation assay in the indicated cell lines. D) Growth curve of xenografts established with pESE3 and pCTRL LNCaP-ABL cells. Right, tumor volume. E) Hallmark enrichment analysis of the indicated comparisons. DOWN (light gray), UP (black). F) Cumulative gene expression level of upregulated genes in LNCaP-ABL pESE3 cells versus LNCaP-ABL pCTRL in PC patient cohorts. G-H) SFA (G) and 3D organoid assay (H) in indicated cell lines treated with increasing doses of MDV-3100. I) Genomic distribution of ESE3 / EHF chromatin binding sites by CUT&RUN analysis in pESE3 LNCaP-ABL cells. Percentage of peak distribution in the indicated genomic regions, t-test was used for panels B, C and D. One-way- Anova was used for panel F. Two-way Anova was used for panels G and H.

[0028] Figure 4. Transient DNA-based ESE3 / EHF supplementation impairs tumor organoid formation in human and murine models. A) Cartoon showing phenotypic differences between ESE3 / EHF-positive and negative models. B) Number (left) and diameter (right) of 3D tumor organoids of LNCaP-ABL cells upon supplementation with pESE3 and pCTRL DNA at the indicated time. C-D) Schematic representation of the ex vivo generation of 3D tumor organoids from patient-derived xenografts and organoid growth upon supplementation with pESE3 and pCTRL DNA. E-F) Schematic representation of the ex vivo generation of 3D prostate tumor organoids from mice with genetic deletion of ESE3 / EHF and organoid growth upon supplementation with pESE3 and pCTRL DNA. Two-way Anova was used for panels B, D and F (number of organoids). One-way Anova was used for panel B, D and F (diameter).

[0029] Figure 5. Re-expression of ESE3 / EHF by DNA-based supplementation impairs tumor organoid formation and reverses their malignant morphology. A) Schematic representation of the experimental plan to obtain ex vivo 3D tumor organoids from the prostates of GEMM models (cEHF-KO and cEHF-KO / ERG) with ESE3 / EHF genetic deletion. B) ESE3 / EHF re-expression detected by immunofluorescence in cEHF-KO and cEHF-KO / ERG murine organoids (described in A) after supplementation of pCMV and pESE3 DNA. Note the strong positive immunostaining for ESE3 / EHF in pESE3-treated samples. C-D) Number of tumor organoids in indicated GEMM models after supplementation of pCMV and pESE3 / EHF. E-F) Phenotypic changes in GEMM-derived SIB BW1361 R tumor organoids upon supplementation with pCMV and pESE3 DNA, showing concomitant increase of normal-like cystic organoids and decrease of irregular, malignant morphologies, t-test was used for panels C-F.

[0030] Figure 6. Re-expression of ESE3 / EHF by systemic DNA-based delivery impairs the growth of LNCaP-ABL tumor xenografts in mice. A) Schematic of the experimental plan. B) Growth of LNCaP-ABL tumor xenografts in mice receiving a single tail vein injection (Arrow) of ESE3 / EHF encoding plasmid DNA (pESE3) and control plasmid (pCTRL). Note the significant reduction of tumor growth in mice receiving pESE3 DNA compared to pCTRL-injected mice. C) Representative tissue sections of tumor xenografts processed by immunohistochemistry to detect ESE3 / EHF protein level. D) Immunoblot of tumor xenografts from mice treated with pCTRL and pESE3 to detect ESE3 / EHF and cleaved caspase 3 protein level. Tubulin, loading control, t- test was used for panel B.

[0031] Figure 7. mRNA-based ESE3 / EHF re-expression restrains LNCAP-ABL aggressive phenotypes. A) Schematic representation of the experimental plan. B-C) Delivery of ESE3 / EHF mRNA and ESE3 / EHF protein expression in LNCaP-ABL cells determined by qRT-PCR (B) and immunoblotting (C). D) Tumor sphere formation (SFA assay) upon ESE3 / EHF mRNA supplementation. Bottom, representative images of tumor-spheres. E) Cell migration assay (Boyden chamber assay) upon ESE3 / EHF mRNA supplementation. Bottom, representative images of migrated cells, t-test was used for panels B, D and E.

[0032] Figure 8. Re-expression of ESE3 / EHF by RNA-based supplementation impairs murine organoid formation. A) Schematic representation of the experimental plan to obtain ex-vivo 3D organoids from indicated GEMM models. B) Schematic representation of ESE3 / EHF mRNA supplementation in GEMM-derived tumor organoids. C) ESE3 / EHF expression in tumor organoids determined by qRT-PCR. D) Number of GEMM-derived organoids upon supplementation with CTRL or ESE3 / EHF mRNA. t-test was used for panels C and D.

[0033] Figure 9. Efficient delivery and persistence of EHF / ESE3 mRNA reduce GEMM- derived organoid growth and malignant morphology. A-B) Schematic representation of the experimental plan to obtain ex vivo 3D organoids and ESE3 / EHF mRNA supplementation. C) ESE3 / EHF re-expression detected by immunofluorescence in murine organoids (described in A) after supplementation of CTRL or ESE3 mRNA. Note the positive immunostaining for ESE3 / EHF in ESE3 mRNA-treated samples. D) Quantification SIB BW1361 R of the fluorescence signal at different time points. E) Organoid number (left) and morphology (right), t-test was used for panels C and D.

[0034] Figure 10. Persistent expression of ESE3 / EHF and impaired organoid formation by RNA-based supplementation in LNCaP-ABL derived organoids. A) Schematic representation of the experimental plan. B) ESE3 / EHF protein detected by immunofluorescence microscopy in 3D organoids after supplementation of CTRL or ESE / EHF mRNA. C) Quantification of the fluorescence signal intensity. D) Number and morphometric analysis of tumor organoids after supplementation of CTRL or ESE / EHF mRNA. t-test was used for panels C and D.

[0035] Figure 11. Re-expression of ESE3 / EHF by systemic RNA-based delivery impairs the growth of LNCaP-ABL tumor xenografts. A) Schematic representation of the experimental plan. B) Growth of LNCaP-ABL tumor xenografts upon systemic administration of CTRL or ESE3 / EHF mRNA. Note the rapid regression of tumor size in mice receiving a single administration of ESE3 / EHF mRNA compared to control-injected mice. C) Efficient delivery of ESE3 / EHF mRNA upon systemic administration of mRNA by intravenous injection in mice carrying LNCaP-ABL tumor xenografts determined by qRT-PCR. t-test was used for panels B and C.

[0036] Figure 12. Re-expression of ESE3 / EHF by systemic RNA-based delivery led to rapid tumor regression. A) Schematic representation of the experimental plan. B) Growth of LNCaP-ABL tumor xenografts upon systemic administration of CTRL or ESE3 / EHF mRNA. Note the rapid regression of tumor size in mice receiving a single administration of ESE3 / EHF mRNA compared to control-injected mice.

[0037] Figure 13. Efficient delivery of ESE3 / EHF DNA and mRNA in EPG2 cells and tumor growth inhibition. A) ESE3 / EHF expression levels in control and ESE3 / EHF-treated EPG2 cells detected by immunofluorescence microscopy. B) Schematic representation of the in vivo experimental plan. C) Growth (left) and tumor weight (right) of tumor xenografts established from EPG2 cells. D) Representative immunohistochemistry images of the indicated tumor xenografts.

[0038] Figure 14. Delivery of luciferase reporter mRNA to subcutaneous tumor xenografts of EPG2 cells. A) Mice with subcutaneous EPG2 tumors were injected intravenously with luciferase encoding IVT mRNA (luciferase mRNA) or control mRNA. B) Luciferase reporter activity was assessed at 24 h posttreatment by in vivo (left) and ex vivo (right) bioluminescence imaging. Note the positive signal detected in subcutaneous tumor lesions in vivo and tumor explants ex vivo in mice injected with luciferase mRNA. SIB BW1361 R

[0039] Figure 15. Efficient delivery of IVT mRNA in metastatic tumor xenograft models. A) Schematic representation of the experimental plan to generate and treat with luciferase-encoding IVT mRNA (luciferase mRNA) or control mRNA mice with multiple metastases of LNCaP-ABL cells. B) Liver metastases of mCherry-labeled LNCaP-ABL cells in the liver detected by ex vivo fluorescence imaging on the explanted organs. C) Luciferase reporter activity in liver metastases at 24 h posttreatment detected by ex vivo bioluminescence imaging of the explanted organs. Bright field, fluorescence or bioluminescence (signal), and overlay images are shown. Note that the positive bioluminescent signals are detected in correspondence with the mCherry-positive metastatic lesions in mice injected with luciferase mRNA.

[0040] Figure 16. EHF / ESE3 DNA supplementation to normal prostate cells does not affect organoid growth and morphology. A) Schematic of the experimental plan. B- C) ESE3 / EHF expression by immunofluorescence microscopy (B) and signal intensity (C) in WT murine organoids. D) Number of WT organoids after supplementation of Control and ESE3 / EHF DNA.

[0041] Figure 17. EHF / ESE3 mRNA supplementation to normal prostate cells does not affect organoid growth and morphology. A) Schematic of the experimental plan. B- C) ESE3 / EHF expression by immunofluorescence microscopy (B) and signal intensity (C) in WT murine organoids. D) Number of WT organoids after supplementation of Control and ESE3 / EHF DNA.

[0042] Figure 18. EHF / ESE3 DNA supplementation to ESE3 / EHF expressing LNCaP cells has no phenotypic impact on sternness and organoid growth. A) Schematic of the experimental plan. B) Detection of ESE3 / EHF by immunofluorescence microscopy following supplementation of control or ESE3 DNA. C-D) Tumor sphere formation (C) and organoid growth (D) following supplementation with control and ESE3 / EHF DNA. SIB BW1361 R

[0043] GLOSSARY

[0044] Unless otherwise defined herein, scientific, and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0045] Generally, nomenclature used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated.

[0046] In any point of the present specification or of the claims, the expression “comprising” or “comprise(s)” can be replaced by “consisting of” or “consist(s) of”.

[0047] DETAILED DESCRIPTION OF THE INVENTION

[0048] Nucleic acid sequence encoding the transcription factor ESE3 / EHF

[0049] The present invention relates to a nucleic acid sequence encoding the transcription factor ESE3 / EHF or an isoform thereof for use in the treatment of a cancer in a patient in need thereof. As also explained in the summary of the invention, the Authors surprisingly found that stable re-expression of ESE3 / EHF in aggressive prostate cancer cells causes a reversion of the aggressive phenotypes, reducing growth of tumor organoids, the fraction of tumor-initiating stem-like cells (cancer stem cells), and tumor growth in mice, as well as that ESE3 / EHF replacement causes a beneficial reprogramming of the transcriptome, reducing expression of pro-proliferative gene sets and reactivating the androgen signalling toward a normal-like state. Therefore, in one embodiment the nucleic acid sequence encoding the transcription factor ESE3 / EHF or an isoform thereof is for use in the treatment of prostate cancer.

[0050] In one embodiment, said nucleic acid sequence is a mRNA or DNA sequence. Preferably, the nucleic acid sequence encodes for the full-length ESE3 / EHF transcription factor. In one embodiment, to synthesize and formulate DNA and mRNA molecule, the full length ESE3 / EHF is encapsulated and protected from degradation in the extracellular environment. For example, for the preparation of a mRNA, a linearized DNA template is first prepared and, based on the DNA template, the mRNA is transcribed. In one embodiment, the mRNA is in vitro transcribed (I T) in the presence of an RNA polymerase and ribonucleoside triphosphates. In the following step, the residual DNA template is removed by DNAse digestion, and the mRNA molecules are capped by chemical or enzymatic methods. The mRNA can also be further purified, for example SIB BW1361 R using microbeads-based precipitation or chromatographic methods to remove the enzymes, free nucleotides, truncated nucleic acid fragments and double-stranded RNA. In one embodiment, said chromatographic method is HPLC. In one embodiment, the purified mRNA can be dissolved in a storage buffer, and then filtered for sterilization and frozen for long-term storage. In one embodiment, the in vitro transcribed mRNA comprises or consists of a start sequence with a cap structure, followed by an untranslated region (5’IITR) up to the start codon. Between the start and stop codon is placed in the coding sequence. The latter coding sequence is followed by the 3’IITR and a poly-A tail.

[0051] In one embodiment, the encoded transcription factor has an aminoacidic sequence of SEQ ID NO:2, or an aminoacidic sequence having an identity of at least 95%, preferably 99% of SEQ ID NO:2 and substantially the same activity. The term 'substantially the same activity' means that the encoded transcription factor retains at least 90% of the specific biological activity of the protein having SEQ ID NO:2, more preferably at least 99% of the specific biological activity of the protein having SEQ ID NO:2. This activity can be measured using methods such as a DNA-binding assay, a transcription activation assay, an electrophoretic mobility shift assay (EMSA), chromatin immunoprecipitation (ChIP), or reporter gene assays. The key functions or properties include, but are not limited to, the ability to bind a specific promoter, activate the transcription of a target gene, and achieve stable re-expression of a gene, in the present case ESE3 / EHF transcription factor, that is lost in cancer cells but present in normal cells. For example, the re-expression can be verified by restoring the expression levels of the target gene to at least 90% of the levels observed in healthy cells, as measured by quantitative PCR (qPCR), Western blot analysis, or immunohistochemistry (IHC).

[0052] In one embodiment, said nucleic acid sequence has SEQ ID NO:1 or at least 95%, preferably at least 99% sequence identity to SEQ ID NO:1.

[0053] The term "percent sequence identity" in the context of nucleotide or aminoacidic sequences means the residues in two sequences that are the same when aligned for maximum correspondence. The length of sequence identity comparison may be over a stretch of at least about nine nucleotides, usually at least about 18 nucleotides, more usually at least about 24 nucleotides, typically at least about 28 nucleotides, more typically at least about 32 nucleotides, and preferably at least about 36, 48 or more nucleotides. There are a number of different algorithms known in the art that can be used to measure nucleotide sequence identity. For instance, polynucleotide sequences can be compared using FASTA, Gap or Bestfit, which are programs available, provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson, Methods Enzymol. 183:63-98 (1990); Pearson, Methods Mol. Biol. 132:185-219 (2000); Pearson, Methods Enzymol. 266:227-258 (1996); Pearson, J Mol. Biol 276:71-84 (1998); incorporated herein by reference). The SIB BW1361 R term "substantial similarity" or "substantial sequence similarity," when referring to a nucleic acid or fragment thereof, or aminoacidic means that when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 85%, preferably at least about 90%, and more preferably at least about 95%, 96%, 97%, 98% or 99% of the nucleotide bases, as measured by any well-known algorithm of sequence identity, such as FASTA, BLAST or Gap, as discussed above. As applied to polypeptides, the term "substantial identity" means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights as supplied with the programs, share at least 70%, 75% or 80% sequence identity, preferably at least 90% or 95% sequence identity, and more preferably at least 97%, 98% or 99% sequence identity.

[0054] In one embodiment, the isoform of said nucleic acid sequence encoding the transcription factor ESE3 / EHF has a sequence selected from SEQ ID NO:3 or SEQ ID NO:5.

[0055] In one embodiment, the nucleic acid sequence encoding the transcription factor ESE3 / EHF or an isoform thereof is for use in the treatment of aggressive prostate cancer. More preferably, said prostate cancer is castration-resistant prostate cancer (CRPC). In particular, the nucleic acid sequence is particularly efficient in the treatment of any cancer where the cancer cells of the cancer in the patient have a reduced or no expression of the transcription factor ESE3 / EHF or an isoform thereof, and in particular in castration - resistant prostate cancer (CRPC).

[0056] In one embodiment, the treatment of cancer comprises a step of analyzing the expression of the transcription factor ESE3 / EHF in the cancer cells of the prostate cancer in said patient.

[0057] Vectors

[0058] The present invention also relates to a vector comprising a nucleic acid sequence for use according to the present invention.

[0059] The term "vector", as used herein, means a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. In some embodiments, the vector is a plasmid, i.e., a circular double-stranded piece of DNA into which additional DNA segments may be ligated. In some embodiments, the vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. In some embodiments, the vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). In other embodiments, the vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. SIB BW1361 R

[0060] Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors"). Expression vectors include plasmids, retroviruses, adenoviruses, lentiviruses, adeno-associated viruses (AAV), EBV-derived episomes, and the like.

[0061] In one embodiment, the vector comprising a nucleic acid sequence for use according to the present invention is a plasmid. In one embodiment, said plasmid contains the full- length ESE3 / EHF encoding sequence or an isoform thereof. Preferably, full-length ESE3 / EHF or an isoform thereof is subcloned into an expression vector, such as a mammalian expression vector pRc, with a promoter. In one embodiment said promoter is a CMV promoter or any other tissue-specific promoters that can drive expression in prostate tumor cells. In one embodiment, the promoter is operably linked to the nucleic acid sequence.

[0062] In one embodiment, the vector of the present invention is encapsulated in nanoparticles. Examples of nanoparticles are polymeric or lipid nanoparticles. In particular, for DNA systemic delivery, the plasmid is complexed with polymeric nanoparticles. For example, the complex is prepared by in vivo jetPEI, Polyplus Transfection, in vivo jetPEI is a transfection reagent comprising polyethylenimine (PEI), a cationic polymer with a high density of amino groups that facilitate complexation with negatively charged nucleic acids such as DNA or RNA. The PEI is dissolved in a physiological buffer solution, which maintains the pH and osmolarity necessary for stability and effective complex formation. The composition may also include stabilizers and preservatives to enhance the reagent's stability and prevent microbial contamination. This formulation ensures efficient delivery and internalization of nucleic acids into cells or tissues in vivo.

[0063] For in vitro delivery (for example for experimental testing), ESE3 / EHF mRNA is formulated using Lipofectamine™ MessengerMAX mRNA Transfection Reagent (Invitrogen). Lipofectamine™ MessengerMAX mRNA Transfection Reagent (Invitrogen) is a specialized reagent designed for the efficient delivery of mRNA into cells. It comprises proprietary lipid nanoparticles formulated to form stable complexes with mRNA. These lipid nanoparticles facilitate the encapsulation and cellular uptake of the mRNA, promoting its effective delivery and expression within target cells. The reagent is prepared in a buffer solution that maintains optimal pH and osmolarity to ensure the stability and activity of the lipid-mRNA complexes. Additionally, the formulation includes stabilizers to enhance the stability of the reagent and ensure its effectiveness over time. For in vivo delivery, in v / vo-jetRNA®+ liposomes (Polyplus Transfection) can be used, in vivo-jetRNA®+ liposomes (Polyplus Transfection) is a transfection reagent specifically engineered for the in vivo delivery of RNA molecules. The reagent consists of lipid-based SIB BW1361 R liposomes designed to encapsulate RNA and facilitate its delivery into cells within living organisms. The liposomes are formulated to form stable, non-toxic complexes with RNA, enhancing its cellular uptake and subsequent gene expression. The formulation includes a buffer solution to maintain physiological conditions and stabilizers to ensure the longevity and efficacy of the liposomes. This composition enables efficient and targeted RNA delivery for various research and therapeutic applications in vivo.

[0064] In one embodiment the ESE3 / EHF3-expressing nucleic acid sequence packaging is further optimized. In one instance, for example for DNA delivery, it is possible to reduce the plasmid size by exploiting the Nanoplasmid technology and generate mini-plasmids comprising a suitable promoter and the nucleic acid sequence according to the present invention. This technology involves the modification of plasmid DNA to create smaller, more efficient plasmids that retain essential elements for gene expression while minimizing non-essential sequences. The reduced plasmid size enhances the delivery efficiency and stability of the plasmid DNA, facilitating improved transfection performance in various applications. Furthermore, it is possible to optimize synthetic mRNA produced for mRNA delivery. Synthetic mRNA can also be modified to generate linear, circular, or self-replicative forms of mRNA encoding ESE3 / EHF according to the present invention with improved stability, intracellular delivery, and translation efficiency. Various options for improving mRNA / DNA formulation can be used, including polyplex and lipoplex design and conjugation to tumor-specific ligands for active targeting. Polyplexes involve the formation of complexes between nucleic acids and polycationic polymers to enhance stability and cellular uptake. Lipoplexes refer to lipid-based formulations that facilitate the delivery of nucleic acids into cells. Additionally, conjugating these formulations to tumorspecific ligands (e.g., small molecules, peptides, antibodies) enables targeted delivery, improving the specificity and efficacy of gene transfer to tumor cells.

[0065] Specifically, for the present case, the cationic poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA), a water-soluble polycation widely used for polyplex formation and nucleic acid delivery, can be used. Furthermore, controlled-living polymerization techniques would also allow for the production of polymers with controlled chain length, low molecular weight dispersity, and the possibility of adding functionality by copolymerization with other functional monomers for further modification. Click chemistry techniques can also be used to conjugate specific ligands to the polyplexes (peptide, saccharide moieties) to enhance the selectivity and efficacy of DNA / RNA delivery to cancer cells. Lipid-based and polymeric-based nanocarriers can be be designed for the SIB BW1361 R encapsulation of nucleic acids, including for example a PEG shell to minimize opsonization / immune activation. Moreover, ligands (e.g., small molecules, peptides, antibodies) can be grafted on the nanocarriers using click-chemistry approaches, similar to polyplexes, to increase tumor-targeted and intracellular delivery.

[0066] Pharmaceutical composition

[0067] The present invention also relates to a pharmaceutical composition comprising a nucleic acid sequence according to the present invention or a vector according to the present invention, for use in the treatment of cancer in a patient in need thereof. In one embodiment, said cancer is aggressive prostate cancer, and in particular metastatic and castration-resistant prostate cancer (CRPC).

[0068] In one embodiment, the pharmaceutical composition is for systemic administration, preferably by intravenous injection.

[0069] Uses and combination treatments.

[0070] Advantageously, the nucleic acid sequence, the vector or the pharmaceutical composition for use according to the present invention is used, in the treatment of cancer, and in particular prostate cancer, in combination with other treatments such as androgen deprivation therapy (ADT), androgen receptor signalling inhibitors (ARSI), chemotherapeutics, molecular-targeted therapeutics, and immunotherapeutics. Androgen deprivation therapy consists in the administration of leuprorelin (leuprolide), goserelin, triptorelin, histrelin, buserelin and degarelix. Androgen receptor signalling inhibitors (ARSI) are abiraterone or androgen receptor antagonists such as enzalutamide, darolutamide or apalutamide. Moreover, suitable chemotherapeutic drugs for use in combination with the nucleic acid sequence or the vector or the pharmaceutical composition of the present invention are docetaxel or cabazitaxel. Molecular targeted anticancer drugs that can be combined with the nucleic acid sequence or the vector or the pharmaceutical composition of the present invention are PARP inhibitors, such as Olaparib, or kinase inhibitors, such as the AKT inhibitors Ipatasertib and Capivasertib. Immune checkpoint inhibitors, such as the anti-PD1 and anti-CTLA-A antibodies nivolumab, pembrolizumab and ipilimumab, and other immunological therapies can also be used in combination with the present invention composition.

[0071] In one embodiment, for in vitro delivery, from 0,01 pg to 100 pg of vector, preferably a plasmid, are incubated with cancer cells. Preferably, the weight of vector incubated with cancer cells is from 1 to 3 pg. SIB BW1361 R

[0072] In one embodiment, for in vivo delivery, 0,001 mg / Kg of body weight to 100 mg / Kg of body weight of vector, preferably a plasmid, are injected to the patient. Preferably, the weight of vector incubated with cancer cells is from 1 to 3 mg / Kg of body weight, preferably 2 mg / Kg of body weight.

[0073] Another object of the present invention is the in vitro use of the nucleic acid sequence or of the vector or of the pharmaceutical composition of the present invention in a cancer cell. In one embodiment said cancer is prostate cancer, preferably aggressive prostate cancer, more preferably CRPC. In particular, the nucleic acid sequence or of the vector or of the pharmaceutical composition is used in vitro in order to carry out a stable reexpression of ESE3 / EHF in ESE3 / EHF negative cancer cells.

[0074] Also disclosed herein is a method of treating cancer in a cancer in need thereof, comprising a step of administering the nucleic acid sequence or of the vector or of the pharmaceutical composition of the present invention to a patient in need thereof. The method may further comprise an initial step of determining the expression of the transcription factor ESE3 / EHF or an isoform thereof in cancer cells, in order to determine if the cells have a reduced or no expression of the transcription factor ESE3 / EHF or an isoform thereof. In one embodiment said cancer is prostate cancer, preferably aggressive prostate cancer, more preferably castration-resistant prostate cancer (CRPC).

[0075] In compliance with Art. 170bis paragraph 2 of the C.P.I. and in accordance with Art. 21 paragraph 2 of the Implementation Regulation of the C.P.I. adopted with Ministerial Decree 13.1.2010 n.33, it is declared that the invention does not concern plant material and the material of animal origin used in the experiments, i.e. LNCaP-ABL ESE3 / EHF negative CRPC cells, LuCaP 35 castration-resistant (CR) and GEMMs ESE3 / EHF-KO mice have been generated in the Inventors laboratories from material of animal origin (e.g., tumor cells, cell lines, mouse strains) that are commercially available..

[0076] EXAMPLES

[0077] The data provided herein indicated that stable re-expression of ESE3 / EHF in LNCaP- ABL ESE3 / EHF negative CRPC cells (Figure 3A) causes a reversion of the aggressive phenotype, reducing cancer stem cells, organoid growth, and in vivo tumor growth (Figure 3B-D). ESE3 / EHF replacement causes the beneficial reprogramming of the transcriptome, including reduced pro-proliferative gene sets and reactivation of the androgen signalling toward a normal-like state (Figure 3E). Indeed, the signature of SIB BW1361 R upregulated genes in ESE3-supplemented cells vs control, when applied to PC datasets, was associated with indolent tumors (Figure 3F). Consistently, CRPC cells become more sensitive to the AR antagonist enzalutamide (MDV-3100) (Figure 3G-H).

[0078] ESE3 / EHF DNA was supplemented in multiple preclinical models of castration-resistant ESE3 / EHF negative models, including LNCaP-ABL cells, LuCaP 35 castration-resistant (CR) and GEMMs ESE3 / EHF-KO mice (Figure 4A-C-E). Following transient supplementation of ESE3 / EHF DNA, a significant reduction in the number of organoids formed from multiple aggressive models of prostate cancer (Figure 4B-D-F, left panels) was observed. Moreover, ESE3 / EHF restrained the 3D organoids aggressive phenotype by significantly reducing their diameter (Figure 4B-D-F, right panels). Considering the aggressive phenotype of the tested models, these results are highly relevant and support the ESE3 / EHF gene replacement as an efficient therapeutic tool. Indeed, it was not predictable that ESE3 / EHF supplementation was able to reverse the organoid growth and phenotypes of these aggressive prostate cancer models.

[0079] Notably, by performing immunofluorescence of ESE3 / EHF it was possible to demonstrate that ESE3 / EHF was efficiently delivered and re-expressed in tumor organoids (Figure 5B).

[0080] Next, systemically injected ESE3 / EHF expressing plasmid DNA was packaged with in vivo jetPEI (Figure 6A). Surprisingly, ESE3 / EHF significantly reduced tumor growth (Figure 6B). Importantly, IHC and western blots showed that the plasmid DNA reached the tumors, and ESE3 / EHF protein was expressed in tumor cells (Figure 6C-D). ESE3 / EHF mRNA supplementation have also been tested.

[0081] ESE3 / EHF IVT mRNA was efficiently delivered into LNCaP ABL cells (Figure 7A-B-C) and concomitantly impaired tumor spheroid growth and migration in functional assays (Figure 7D-E). ESE3 / EHF mRNA was also efficiently supplemented to tumor organoids (Figure 8A-B-C) and impaired organoid formation in GEMM-derived models with ESE3 / EHF knockout (Figure 8D). Moreover, systemic delivery of ESE3 / EHF IVT mRNA by tail vein injection impaired the growth of LNCaP-ABL tumor xenografts in mice, resulting in rapid and persistent regression of tumors compared to the control group (Figure 9A-B). This effect was associated with the fast and massive uptake of ESE3 / EHF IVT mRNA in tumor xenografts (Figure 90).

[0082] Methods

[0083] Plasmid description and formulation for in vitro and in vivo studies. SIB BW1361 R

[0084] The ESE3 / EHF-expressing plasmid (pRc / CMV-EHFb-FLAG, pEHF) contains the full- length (J) (ESE3 / EHF3b) subcloned into the mammalian expression vector pRc with a CMV promoter. It has a FLAG epitope either at the 5’ or at the 3’ ends. The pRc / CMV- ESE3b-FLAG plasmid has an ampicillin resistance and the selection marker Neomycin (selectable with G418). A control plasmid with the same backbone but not containing the insert is used as control. For experiments of supplementation ex-vivo and in vivo, the EE3 / EHF-expressing vector is packaged in polymeric nanoparticles (jetPEI, Polyplus Transfection) and supplemented to the cells before the functional assays. For in vitro studies, 1-3 pg of plasmid will be incubated with cells. For in vivo studies, the plasmid will be complexed with polymeric nanoparticles suitable for systemic delivery in vivo (in vivo jetPEI, Polyplus Transfection).

[0085] EHF mRNA synthesis and delivery.

[0086] The synthesis and formulation are done in collaboration with experts in mRNA synthesis and delivery. Briefly, in vitro transcribed (ivt) EHF mRNA will be produced and purified by using a HPLC-based purification method. For in vitro delivery of EHF mRNA ESE3 / EHF mRNA will be formulated by using Lipofectamine™ MessengerMAX mRNA Transfection Reagent (Invitrogen).

[0087] In vivo delivery

[0088] For systemic treatment, mice with subcutaneous tumor xenografts are injected intravenously with pEHF or control plasmid formulated with in vivo jetPEI (Polyplus Transfection) at the dose of 2 mg / kg / body weight. For mRNA EHF injection, in vivo- jetRNA®+ liposomes (Polyplus Transfection) have been used at the dose of 1 mg / kg / body weight.

[0089] SEQ ID NO:1 - ESE3 / EHF isoform 1

[0090] Nucleotide sequence (969 nt):

[0091] ATGGGGTTGCCGGAGAGAAGAGGATTGGTCCTGCTTTTAAGCCTAGCTGAAATTC TTTTCAAGATCATGATTCTGGAAGGAGGTGGTGTAATGAATCTCAACCCCGGCAA CAACCTCCTTCACCAGCCGCCAGCCTGGACAGACAGCTACTCCACGTGCAATGTT TCCAGTGGGTTTTTTGGAGGCCAGTGGCATGAAATTCATCCTCAGTACTGGACCA AGTACCAGGTGTGGGAGTGGCTCCAGCACCTCCTGGACACCAACCAGCTGGATG CCAATTGTATCCCTTTCCAAGAGTTCGACATCAACGGCGAGCACCTCTGCAGCAT GAGTTTGCAGGAGTTCACCCGGGCGGCAGGGACGGCGGGGCAGCTCCTCTACA GCAACTTGCAGCATCTGAAGTGGAACGGCCAGTGCAGTAGTGACCTGTTCCAGTC CACACACAATGTCATTGTCAAGACTGAACAAACTGAGCCTTCCATCATGAACACCT SIB BW1361 R

[0092] GGAAAGACGAGAACTATTTATATGACACCAACTATGGTAGCACAGTAGATTTGTTG

[0093] GACAGCAAAACTTTCTGCCGGGCTCAGATCTCCATGACAACCACCAGTCACCTTC

[0094] CTGTTGCAGAGTCACCTGATATGAAAAAGGAGCAAGACCCCCCTGCCAAGTGCCA

[0095] CACCAAAAAGCACAACCCGAGAGGGACTCACTTATGGGAATTCATCCGCGACATC

[0096] CTCTTGAACCCAGACAAGAACCCAGGATTAATAAAATGGGAAGACCGATCTGAGG

[0097] GCGTCTTCAGGTTCTTGAAATCAGAGGCAGTGGCTCAGCTATGGGGTAAAAAGAA

[0098] GAACAACAGCAGCATGACCTATGAAAAGCTCAGCCGAGCTATGAGATATTACTAC

[0099] AAAAGAGAAATTCTGGAGCGTGTGGATGGACGAAGACTGGTATATAAATTTGGGA

[0100] AGAATGCCCGAGGATGGAGAGAAAATGAAAACTGA

[0101] SEQ ID N0:2 - ESE3 / EHF isoform 1

[0102] Protein translation (322 aa):

[0103] MGLPERRGLVLLLSLAEILFKIMILEGGGVMNLNPGNNLLHQPPAWTDSYSTCNVSSG

[0104] FFGGQWHEIHPQYWTKYQVWEWLQHLLDTNQLDANCIPFQEFDINGEHLCSMSLQE

[0105] FTRAAGTAGQLLYSNLQHLKWNGQCSSDLFQSTHNVIVKTEQTEPSIMNTWKDENYL

[0106] YDTNYGSTVDLLDSKTFCRAQISMTTTSHLPVAESPDMKKEQDPPAKCHTKKHNPRG THLWEFIRDILLNPDKNPGLIKWEDRSEGVFRFLKSEAVAQLWGKKKNNSSMTYEKLS RAMRYYYKREILERVDGRRLVYKFGKNARGWRENEN

[0107] SEQ ID NO:3 - ESE3 / EHF isoform 2

[0108] Nucleotide sequence (903 nt):

[0109] ATGATTCTGGAAGGAGGTGGTGTAATGAATCTCAACCCCGGCAACAACCTCCTTC

[0110] ACCAGCCGCCAGCCTGGACAGACAGCTACTCCACGTGCAATGTTTCCAGTGGGTT

[0111] TTTTGGAGGCCAGTGGCATGAAATTCATCCTCAGTACTGGACCAAGTACCAGGTG

[0112] TGGGAGTGGCTCCAGCACCTCCTGGACACCAACCAGCTGGATGCCAATTGTATCC

[0113] CTTTCCAAGAGTTCGACATCAACGGCGAGCACCTCTGCAGCATGAGTTTGCAGGA

[0114] GTTCACCCGGGCGGCAGGGACGGCGGGGCAGCTCCTCTACAGCAACTTGCAGC

[0115] ATCTGAAGTGGAACGGCCAGTGCAGTAGTGACCTGTTCCAGTCCACACACAATGT

[0116] CATTGTCAAGACTGAACAAACTGAGCCTTCCATCATGAACACCTGGAAAGACGAG

[0117] AACTATTTATATGACACCAACTATGGTAGCACAGTAGATTTGTTGGACAGCAAAAC

[0118] TTTCTGCCGGGCTCAGATCTCCATGACAACCACCAGTCACCTTCCTGTTGCAGAG

[0119] TCACCTGATATGAAAAAGGAGCAAGACCCCCCTGCCAAGTGCCACACCAAAAAGC

[0120] ACAACCCGAGAGGGACTCACTTATGGGAATTCATCCGCGACATCCTCTTGAACCC

[0121] AGACAAGAACCCAGGATTAATAAAATGGGAAGACCGATCTG

[0122] AGGGCGTCTTCAGGTTCTTGAAATCAGAGGCAGTGGCTCAGCTATGGGGTAAAAA

[0123] GAAGAACAACAGCAGCATGACCTATGAAAAGCTCAGCCGAGCTATGAGATATTAC SIB BW1361 R

[0124] TACAAAAGAGAAATTCTGGAGCGTGTGGATGGACGAAGACTGGTATATAAATTTG

[0125] GGAAGAATGCCCGAGGATGGAGAGAAAATGAAAACTGA

[0126] SEQ ID NO:4 - ESE3 / EHF isoform 2

[0127] Protein translation (300 aa):

[0128] MILEGGGVMNLNPGNNLLHQPPAWTDSYSTCNVSSGFFGGQWHEIHPQYWTKYQV

[0129] WEWLQHLLDTNQLDANCIPFQEFDINGEHLCSMSLQEFTRAAGTAGQLLYSNLQHLK

[0130] WNGQCSSDLFQSTHNVIVKTEQTEPSIMNTWKDENYLYDTNYGSTVDLLDSKTFCRA

[0131] QISMTTTSHLPVAESPDMKKEQDPPAKCHTKKHNPRGTHLWEFIRDILLNPDKNPGLI

[0132] KWEDRSEGVFRFLKSEAVAQLWGKKKNNSSMTYEKLSRAMRYYYKREILERVDGRR

[0133] LVYKFGKNARGWRENEN

[0134] SEQ ID NO:5 - ESE3 / EHF isoform 3

[0135] Nucleotide sequence (834 nt):

[0136] ATGATTCTGGAAGGAGGTGGTGTAATGAATCTCAACCCCGGCAACAACCTCCTTC

[0137] ACCAGCCGCCAGCCTGGACAGACAGCTACTCCACGTGCAATGTTTCCAGTGGGTT

[0138] TTTTGGAGGCCAGTGGCATGAAATTCATCCTCAGTACTGGACCAAGTACCAGGTG

[0139] TGGGAGTGGCTCCAGCACCTCCTGGACACCAACCAGCTGGATGCCAATTGTATCC

[0140] CTTTCCAAGAGTTCGACATCAACGGCGAGCACCTCTGCAGCATGAGTTTGCAGGA

[0141] GTTCACCCGGGCGGCAGGGACGGCGGGGCAGCTCCTCTACAGCAACTTGCAGC

[0142] ATCTGAAGTGGAACGGCCAGTGCAGTAGTGACCTGTTCCAGTCCACACACAATGT

[0143] CATTGTCAAGACTGAACAAACTGAGCCTTCCATCATGAACACCTGGAAAGACGAG

[0144] AACTATTTATATGACACCAACTATGGTAGCACAGTAGCAGAGTCACCTGATATGAA

[0145] AAAGGAGCAAGACCCCCCTGCCAAGTGCCACACCAAAAAGCACAACCCGAGAGG

[0146] GACTCACTTATGGGAATTCATCCGCGACATCCTCTTGAACCCAGACAAGAACCCA

[0147] GGATTAATAAAATGGGAAGACCGATCTGAGGGCGTCTTCAGGTTCTTGAAATCAG

[0148] AGGCAGTGGCTCAGCTATGGGGTAAAAAGAAGAACAACAGCA

[0149] GCATGACCTATGAAAAGCTCAGCCGAGCTATGAGATATTACTACAAAAGAGAAATT

[0150] CTGGAGCGTGTGGATGGACGAAGACTGGTATATAAATTTGGGAAGAATGCCCGAG

[0151] GATGGAGAGAAAATGAAAACTGA

[0152] SEQ ID NO:6 - ESE3 / EHF isoform 3

[0153] Protein translation (277 aa):

[0154] MILEGGGVMNLNPGNNLLHQPPAWTDSYSTCNVSSGFFGGQWHEIHPQYWTKYQV

[0155] WEWLQHLLDTNQLDANCIPFQEFDINGEHLCSMSLQEFTRAAGTAGQLLYSNLQHLK

[0156] WNGQCSSDLFQSTHNVIVKTEQTEPSIMNTWKDENYLYDTNYGSTVAESPDMKKEQ SIB BW1361 R

[0157] DPPAKCHTKKHNPRGTHLWEFIRDILLNPDKNPGLIKWEDRSEGVFRFLKSEAVAQL

[0158] WGKKKNNSSMTYEKLSRAMRYYYKREILERVDGRRLVYKFGKNARGWRENEN

Claims

SIB BW1361 RCLAIMS1 . A nucleic acid sequence encoding the transcription factor ESE3 / EHF or an isoform thereof for use in the treatment of a cancer in a patient in need thereof, preferably said nucleic acid sequence is a mRNA or DNA sequence.

2. The nucleic acid sequence according to claim 1 , wherein said cancer is prostate cancer.

3. The nucleic acid sequence for use according to any one of claims 1 or 2, wherein the encoded transcription factor having an aminoacidic sequence SEQ ID NO:2 or an aminoacidic sequence having an identity of at least 95%, preferably 99% of SEQ ID NO:2 and substantially the same activity.

4. The nucleic acid sequence for use according to any one of claims 1 to 3, wherein said nucleic acid sequence having SEQ ID NO: 1 or at least 95%, preferably at least 99% sequence identity to SEQ ID NO:1 .

5. The nucleic acid sequence for use according to any one of claims 1 to 4, wherein said nucleic acid sequence is selected from SEQ ID NO:3 or SEQ ID NO:5.

6. The nucleic acid sequence for use according to any one of claims 2 to 5, wherein said prostate cancer is aggressive prostate cancer.

7. The nucleic acid sequence for use according to any one of claims 2 to 6, wherein said prostate cancer is castration-resistant prostate cancer (CRPC).

8. The nucleic acid sequence for use according to any one of claims 2 to 7, wherein the cancer cells of the prostate cancer in said patient have a reduced or no expression of the transcription factor ESE3 / EHF or an isoform thereof.

9. A vector comprising the nucleic acid sequence for use according to any one of claims from 1 to 8, preferably said vector is a plasmid or a viral vector.

10. A pharmaceutical composition comprising the nucleic acid sequence according to any one of claim 1 to 8 or a vector according to claim 9, for use in the treatment of cancer in a patient in need thereof, preferably wherein said cancer is prostate cancer.

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