CN-114729364-B - Antisense oligonucleotide sequences for silencing human L1-MET transcripts in tumors

Abstract

The present invention relates to the use of antisense oligonucleotides to induce the death of several types of human cancer cells by silencing human L1-MET, a non-coding transcript that is specifically transcribed in tumor cells.

Inventors

• Tiziana Venecio

Assignees

• 皮埃蒙特肿瘤学基金会

Dates

Publication Date

20260508

Application Date

20201113

Priority Date

20191115

Claims (7)

1. 1. An antisense oligonucleotide targeting the region of the L1-MET transcript encoded by SEQ ID No.1, wherein said antisense oligonucleotide consists of SEQ ID No. 4 or SEQ ID No. 5.
2. 2. The antisense oligonucleotide of claim 1, wherein each of the antisense oligonucleotides comprises a ribonucleotide, a combination of ribonucleotides and deoxyribonucleotides, and/or a nucleotide with a modified ribose and/or deoxyribose, wherein a phosphate group is modified or unmodified.
3. 3. A pharmaceutical composition comprising as active ingredient one or more antisense nucleotides as defined in any of claims 1-2, and one or more excipients.
4. 4. The pharmaceutical composition of claim 3, further comprising one or more anticancer drugs.
5. 5. Use of an antisense oligonucleotide as defined in any one of claims 1-2 or of a pharmaceutical composition as defined in any one of claims 3-4 in the manufacture of a medicament for the treatment of a tumor expressing L1-MET, wherein when the antisense oligonucleotide consists of SEQ ID No. 4, the tumor expressing L1-MET is lung adenocarcinoma and when the antisense oligonucleotide consists of SEQ ID No. 5, the tumor expressing L1-MET is lung adenocarcinoma and breast cancer.
6. 6. Use of one or more antisense oligonucleotides as defined in any one of claims 1-2 in combination with one or more anti-cancer drugs in the manufacture of a medicament for the treatment of a tumor expressing L1-MET, wherein when the antisense oligonucleotide consists of SEQ ID No. 4, the tumor expressing L1-MET is lung adenocarcinoma and when the antisense oligonucleotide consists of SEQ ID No. 5, the tumor expressing L1-MET is lung adenocarcinoma and breast cancer, and wherein the one or more antisense oligonucleotides are used alone or sequentially with the one or more anti-cancer drugs.
7. 7. The use of claim 6, wherein the breast cancer is a triple negative breast cancer.

Description

Antisense oligonucleotide sequences for silencing human L1-MET transcripts in tumors Technical Field The present invention relates to antisense oligonucleotide sequences useful for silencing human L1-MET transcripts in tumors. Background In particular, the present invention relates to the use of antisense oligonucleotides to induce the death of several types of human cancer cells by silencing human L1-MET, a non-coding transcript that specifically transcribes in tumor cells. The focus of research today is to find new therapies for the treatment of cancer, in particular for cancer cells that are selective. In fact, it is well known that anticancer therapies such as chemotherapy lead to death of both cancer and normal cells. Death of normal cells can lead to some undesirable side effects. To address this problem, over the last 20 years, new therapeutic strategies have been developed to target specific molecules that are more expressed in cancer cells. The personal molecular landscape (molecular landscape) of the patient solves the physical problem of specific drugs and reduces off-target. However, the molecules targeted by such drugs are present not only on cancer cell membranes, but also in some normal cells. Furthermore, the presence of mutations in other genes may lead to inefficiency, i.e. in colorectal cancer, the presence of mutations in the KRAS gene leads to inefficiency of using drugs that bind to EGFR and block this pathway. In lung cancer, the presence of mutations in EGFR can lead to a better response to EGFR inhibitors, but when resistant mutations rise, the drug becomes ineffective. In view of the above, there is therefore a clear need to provide new anti-cancer therapies that can overcome the drawbacks of known anti-cancer therapies. As is well known, the long interspersed nuclear element (LINE-1) is a retrotransposable element that accounts for approximately 20% of the human genome. Line-1 retains the ability to transpose itself to new chromosomal region sites when they are activated by hypomethylation of CpG islands located in its promoter region [1]. Only a few of these sequences (usually located in the non-coding region) have retrotransposable capacity, but usually remain inactive for almost the entire life [2]. When demethylated, the LINE-1 promoter may act as a sense promoter, direct transcription of both open reading frames (ORF-1 and ORF-2), or as an antisense promoter [3]. The activity of an antisense promoter to drive transcription of the opposite strand relative to the LINE-1 direction can lead to the appearance of transcripts that include a neighborhood sequence [4]. In this regard, the novel primate-specific open reading frame (ORF-0) recently found within the 5' UTR of the LINE-1 sequence was demonstrated to be the origin of the proximal exon fusion transcript, using two splice donor sites [5]. The LINE-1 sequence, called L1-MET (FIG. 1), located within intron 2 of the human MET gene, belongs to the subfamily primate and is not capable of retrotransposition. However, the promoter region has been fully retained, thus allowing the antisense promoter to be activated by hypomethylation and producing an alternative transcript derived from the ORF-0 region and comprising the neighborhood MET sequence. L1-MET transcripts were first described in 2002 [6], but their full length characterization was only achieved in 2018, as described by Miglio et al (2018) (FIG. 1) [7]. In the latter study, transcripts were shown to start from ORF-0 and end at MET 3' utr, comprising 6 different splice variants derived from a combination of two splice donor sites with three different acceptor sites, two of which are located in intron 2 of the MET gene. The length of the L1-MET transcript and the absence of the coding open reading frame indicate its function as a long non-coding RNA. It has also been demonstrated that although L1-MET does not encode a functional protein, the presence of the 3' utr and polyA regions confers the ability of the transcript to be transported from the nucleus to the cytoplasm. This feature, along with its length, suggests its role as a long non-coding RNA. To date, only two studies have attempted to investigate the biological function of L1-MET. Among them, weber et al observed reduced MET protein levels after inducing L1-MET expression by knocking down DNA methyltransferase proteins and promoting transcription by hypomethylation [8]. On the other hand, wolff et al reported the presence of a truncated MET isomer after transfection of L1-MET in the cell line [9]. However, in Miglio et al, 2018[7], neither western blot nor informatics prediction tools demonstrate the presence of truncated MET proteins. Disclosure of Invention Activation of the L1-MET antisense promoter has been shown to be a tumor-specific mechanism, as experimental studies and in silico analysis clearly show that there is no evidence for L1-MET expression in normal tissues [7]. According to the present invention, i