EP-4735597-A1 - RNA INTERFERENCE MEDIATED THERAPY FOR NEOPLASTIC DISEASES

Abstract

A double- stranded small interfering RNA (siRNA) having a length of at least 19 nucleotides, targeting an exon of Trim28 gene mmRRNNAA (gene ID: ENST000000253024.10), wherein the exon is selected from the group consisting of exon 3, exon 4, exon 6 and exon 12, and wherein the double-stranded siRNA is capable of inhibiting TRIM28 protein, uusseess and compositions thereof.

Inventors

• SICA, ANTONIO
• PORTA, Chiara
• GARLATTI, Valentina
• CARRARO, Lorenzo
• MOLA, Silvia

Assignees

• Università degli Studi del Piemonte Orientale "Amedeo Avogadro"
• Humanitas Mirasole S.p.A.

Dates

Publication Date

20260506

Application Date

20240628

Claims (1)

1. CLAIMS 1. A double-stranded small interfering RNA (siRNA) having a length of at least 19 nucleotides, targeting an exon of Trim28 gene mRNA having the nucleotide sequence set forth in ENST000000253024.10, wherein the exon is selected from the group consisting of exon 3, exon 4, exon 6 and exon 12, and wherein the double- stranded siRNA is capable of inhibiting TRIM28 protein. 2. The double-stranded siRNA according to claim 1, wherein the double-stranded siRNA is able to induce selective degradation of Trim28 transcripts containing one of exons 3, 4, 6 and 12. 3. The double-stranded siRNA according to claim 1 or claim 2, wherein the double-stranded siRNA has a length comprised between 19 and 29 nucleotides. 4. The double-stranded siRNA according to any one of the preceding claims, wherein the exon is selected from the group consisting of exon 4, exon 6 and exon 12, preferably exon 12. 5. The double-stranded siRNA according to any one of the preceding claims, wherein at least one strand of the double-stranded siRNA has an overhang at the 3' terminus. 6. The double-stranded siRNA according to any one of the preceding claims, wherein the double-stranded siRNA has a content of G-C bases lower than 52%. 7. The double-stranded siRNA according to any one of the preceding claims, wherein the double-stranded siRNA has a content of G-C bases higher than 36%. 8. The double-stranded siRNA according to any one of the preceding claims, wherein at least one strand of the double-stranded siRNA has a melting temperature lower than 64 °C, wherein the melting temperature is measured as disclosed using the free software online tool OligoEvaluator TM (Merck). 9. The double-stranded siRNA according to any one of the preceding claims, wherein the sense strand of the double-stranded siRNA has a nucleotide sequence selected from SEQ ID No.: 1 to 4 and 21, wherein the antisense strand has a sequence complementary and reverse thereto. 10. A double-stranded siRNA according to any one of the preceding claims for use in treating and/or preventing a neoplastic disease associated with an abnormality of Trim28 gene encoded protein TRIM28. 11. The double-stranded siRNA for use according to claim 10, wherein the abnormality of Trim28 gene encoded protein TRIM28 is upregulation and/or hyperphosphorylation of protein TRIM28. 12. The double-stranded siRNA for use according to claim 10 or claim 11, wherein the neoplastic disease is a primary cancer and/or a metastatic cancer. 13. The double-stranded siRNA for use according to any one of claims 10 to 12, wherein the neoplastic disease is selected from stomach, pancreas, kidney, ovary, melanoma, prostate, testis, liver, lymphoma, lung, thyroid, glioma, cervical, gastric, ovarian, glioma, colorectal, breast, mesothelioma and prostate cancer. 14. The double-stranded siRNA for use according to any one of claims 10 to 13 in a combination therapy with a poly ADP ribose polymerase (PARP) inihibitor. 15. A pharmaceutical composition comprising at least one double-stranded siRNA according to any one of claims 1 to 9 and a pharmaceutically acceptable excipient. 16. A pharmaceutical composition according to claim 15 for use in treating and/or preventing a neoplastic disease associated with an overexpression of Trim28 gene encoded protein TRIM28.

Description

“RNA interference mediated therapy for neoplastic diseases” * * * FIELD OF THE INVENTION The present invention concerns RNA interference mediated therapy for primary and metastatic cancers. More specifically, the present invention concerns siRNA-mediated gene silencing of TRIM28 as therapy for neoplastic diseases. BACKGROUND OF THE INVENTION The discovery of RNA interference (RNAi), a natural mechanism controlling gene expression in mammalian cells, has recently led to the development of innovative methods to efficiently repress target genes by delivering synthetic small interfering RNA (siRNA) molecules into the host cells(1). siRNAs selectively bind to messenger RNA (mRNA), preventing them from being translated into proteins. As a result, a target gene can be efficiently silenced both in vitro and in vivo. Due to its flexibility and efficiency, the use of siRNA in therapy is increasingly considered as a promising therapeutic strategy and siRNA drugs have recently been approved for clinical use or are currently being evaluated for diseases like cancers (i.e. glioblastoma)(2). Colorectal cancer (CRC) is the third-most prevalent malignancy and the second leading cause of cancer-related deaths worldwide(3). Over the last two decades, the improved understanding of the pathogenic mechanisms and the risk factors has led to the development of surveillance programs and new treatment modalities. Nevertheless, CRC incidence has further increased and the mortality rate has remained unacceptable high(3). In the context of CRC, chronic inflammation is well recognized as a key trigger of intestinal carcinogenesis, but little is it known about the molecular sensors connecting chronic inflammatory signals to genomic instability, DNA mutations and consequent cell transformation. In the attempt to develop more effective therapeutic options, many studies have evaluated the possibility of targeting key oncogenic signaling pathways (e.g., Hedgehog, JAK/STAT, TGFβ, EGFR/MAPK, Notch, PI3K, Wnt/βcatenin, NF-κB) by using siRNA-based drugs(4, 5). This has brought about a large body of preclinical in vitro and in vivo evidence demonstrating that siRNA-mediated gene silencing can effectively inhibit tumor cell proliferation, survival, progression, invasion and treatment resistance(4, 5). For the development of siRNA therapeutics three central steps have to been taken: (i) identification of a cancer-related gene, (ii) design and synthesis of a specific siRNA and (iii) delivery of synthetic siRNA into the cancer cells controlling the absence of unspecific off-target effects. TRIM28, also known as KRAB (Kruppel-associated box)-associated protein (KAP1) or transcription intermediary factor 1β (TIF1β), is a pleiotropic protein involved in the dynamic organization of chromatin and in several aspects of cellular physiology, including gene expression, DNA repair, pluripotency, proliferation, differentiation and survival(6). Due to this diverse range of functions, TRIM28 exerts a complex role in cancer cell biology. While it may prevent neoplastic transformation by maintaining epigenetic stability and promoting repair of double strand DNA breaks (DSBs)(7, 8), in most cancers TRIM28 emerges as an oncogenic driver. Indeed, TRIM28 can promote cancer cell survival and proliferation by inhibiting p53-dependent apoptosis(9, 10), repressing of the cyclin-dependent kinase inhibitor p21(11, 12) and inducing mTOR signaling pathway(13, 14). Additionally, in different cancer cell types, such as pancreatic(15), lung(16), breast(17) and ovarian(18) cancer cells, TRIM28 favors invasion and dissemination by supporting epithelial-to-mesenchymal transition (EMT) and cell migration. Finally, being a key pluripotency gatekeeper of normal embryonic stem cells(19, 20) and inducible pluripotent stem cells(21, 22), not surprisingly, different studies have demonstrated that in different types of cancer (e. g. breast, melanoma and glioblastoma) overexpression of TRIM28 also promotes cancer stem cell maintenance(23- 26). Accordingly, TRIM28 is over-expressed in many human cancers (e. g. stomach, colon, pancreas, kidney, ovary, melanoma, prostate, testis, liver, lymphoma, lung, thyroid, glioma) and its association with worse clinical outcomes has been proven in cervical, gastric, ovarian, glioma, hepatocellular, colorectal, breast and prostate cancers(27). This conclusion was further consolidated by recent gene expression analyzes of TCGA and GTEx databases, which confirmed that TRIM28 expression is higher in various tumor tissues than in normal tissues and that higher TRIM28 expression is associated with poorer prognosis in multiple cancers(42). Additional histochemical analysis performed taking advantage of a specific antibody targeting TRIM28, revealed an higher TRIM28 intensity level at localized at the neoplastic lesions if compared to the healthy resected margin (The Human Protein Altas). Overall, this large body of evidence fosters the development of strate