Transgene cassettes and epigenetic silencers for treatment of disorders

Abstract

An epigenetic silencing factor (ESF) or a polynucleotide encoding the same for use in the treatment of cancer wherein the ESF comprises a transcription factor DNA binding domain operably linked to at least one epigenetic effector domain wherein the transcription factor is an oncogenic transcription factor or a cancer-related transcription factor, wherein the cancer is selected from the group consisting of glioma, glioblastoma, myeloblastoma, astroblastoma, neuroblastoma, ventricular tubuloma, meningioma, retinoblastoma, rhabdomyosarcoma, lung cancer, prostate cancer, breast cancer, liver cancer, pancreatic cancer (e.g., human pancreatic ductal adenocarcinoma), bladder cancer, oropharyngeal cancer, kidney cancer, colon cancer (e.g., human pancreatic ductal adenocarcinoma), bladder cancer, oropharyngeal cancer, kidney cancer, colon cancer (e.g., human pancreatic ductal adenocarcinoma). E.g., colonic adenocarcinoma), colorectal cancer (CRC), or metastases of any of the foregoing.

Description

Field of the Invention

[0001] The present invention relates to transgenic cassettes and polynucleotides for treating diseases or disorders. The polynucleotides of the present invention may comprise an epigenetic silencing factor (ESF) that mediates targeted gene silencing. Further, the polynucleotides of the present invention may promote cell-specific transgenic expression for improved targeting specificity and safety. Background of the Invention

[0003] Gene therapy involves the incorporation of genetic material into cells to treat or prevent disease. The genetic material can replenish defective genes with functional copies of those genes, inactivate genes that function inappropriately, silence genes that may be associated with a disease state (such as oncogenic or cancer-associated genes), or introduce new therapeutic genes into cells.

[0004] To date, two main targeted technologies have been used to silence gene expression: RNA interference (RNAi) with a single short hairpin RNA (shRNA); and gene targeting using artificial nucleases. Although promising preclinical and clinical data have been obtained using these technologies, the inefficiency of partial elimination of gene expression with shRNA and homozygous disruption in diploid mammalian cells may reduce the efficacy of these treatments. These shortcomings are particularly relevant in those applications where the residual level of gene activity is sufficient for biological function.

[0005] In addition, epigenetic mechanisms have been used to silence gene expression. Epigenetics refers to the mechanism of transmitting heritable changes in genome function without changing the primary DNA sequence. These changes can mediate short-term instructions that can respond to exogenous stimuli (e.g., histone post-transcriptional modification; HPTM) quickly. Alternatively, they can constitute long-term instructions (e.g., DNA methylation) that stabilize cell identity and memory.

[0006] Treatment of cancer can involve a range of approaches, including surgery, chemotherapy, and radiotherapy. However, even if surgical removal of the tumor is as thorough as possible, even a small amount of remaining cancer cells with tumor-initiating potential may be sufficient to regrow the tumor mass in the short term, which leads to recurrence of the disease. In particular, cancer stem cells (CSCs), which are defined as cells that can self-renew and initiate or regrow tumors, remain quiescent or at very low proliferative activity and may be able to resist certain adjuvant therapies. Therefore, there is an urgent need to achieve long-term remission through more effective targeting of cancer cells, especially after tumor resection.

[0007] Such methods may be particularly desirable for diseases such as glioblastoma multiforme (GBM), which is the most common and deadly brain cancer in adults, with 1-5 cases per 100,000 people per year and a median survival of 12-15 months. This poor outcome is due to a combination of the aggressiveness of the disease and the limited efficacy of current therapies, which can only slightly improve overall survival. Patients typically undergo surgical resection of the primary tumor mass, followed by adjuvant radiation and chemotherapy (temozolomide), although tumor regrowth may lead to cancer recurrence.

[0008] Attempts have been made to inhibit the development of cancers such as GBM by silencing the expression of one or more transcription factors (TFs) using different techniques. For example, TF inactivation has been attempted using a variety of techniques including shRNA, miRNA, and TALEN-based epigenetic repressors, but complete and long-term gene silencing has proven challenging. In addition, cancer cells can rearrange their genetic program to accommodate the silencing of a single gene, thereby maintaining an unchanged tumorigenic potential.

[0009] Therefore, there remains an urgent need to develop more effective cancer treatments, particularly treatments that are effective against aggressive cancers such as GBM, as well as treatments that can target CSCs.

[0010] A further limitation of gene therapy is the ability to selectively determine whether a transgene is expressed within the cells to which it is delivered. There is a continuing need for transgene expression cassettes that can provide cell type specific transgene expression to reduce off-target effects associated with transgene expression, which in turn can improve the specificity and safety of any such gene therapy. Summary of the invention

[0011] The inventors have engineered oncogenic and cancer-associated transcription factors that function as epigenetic repressors, called epigenetic silencing factors (ESFs), which, for example, enable silencing of downstream tumorigenic networks, thereby limiting CSC survival and proliferation and thereby reducing the chance of tumor regrowth. Herein, the inventors provide polynucleotides (e.g., transgenic expression cassettes) comprising said ESFs and miRNA target sequences that can regulate the expression of ESFs in a cell type-specific manner.

[0012] Herein, the inventors provide polynucleotides (e.g., transgenic expression cassettes) that utilize miRNA target sequences to regulate transgenic expression. The expression cassettes allow for regulation of transgenic expression, such that unwanted expression is reduced or eliminated, thereby improving safety and reducing off-target effects.

[0013] In a first aspect, polynucleotides are provided that comprise at least one miR-124 target sequence, and / or at least one miR-338-3p target sequence, and / or at least one miR-31 target sequence, wherein the miRNA target sequence is operably linked to a transgene.

[0014] In one embodiment, the polynucleotide comprises at least one miR-124 target sequence, wherein the target sequence is operably linked to a transgene.

[0015] In one embodiment, the polynucleotide comprises at least one miR-338-3p target sequence, wherein the target sequence is operably linked to a transgene.

[0016] In one embodiment, the polynucleotide comprises at least one miR-31 target sequence, wherein the target sequence is operably linked to a transgene.

[0017] In one embodiment, the polynucleotide comprises at least one miR-124 target sequence and at least one miR-338-3p target sequence, wherein the target sequences are operably linked to a transgene.

[0018] In one embodiment, the polynucleotide comprises at least one miR-124 target sequence and at least one miR-31 target sequence, wherein the target sequences are operably linked to a transgene.

[0019] In one embodiment, the polynucleotide comprises at least one miR-338-3p target sequence and at least one miR-31 target sequence, wherein the target sequences are operably linked to a transgene.

[0020] In one embodiment, the polynucleotide comprises at least one miR-124 target sequence, at least one miR-338-3p target sequence, and at least one miR-31 target sequence, wherein the target sequences are operably linked to a transgene.

[0021] In one aspect, polynucleotides are provided that comprise at least one miR-124 target sequence, at least one miR-338-3p target sequence, and at least one miR-31 target sequence, wherein the miRNA target sequence is operably linked to a transgene.

[0022] In one embodiment, the number of copies of each miRNA target sequence is independently selected from the group consisting of: one, two, three, and four.

[0023] In one embodiment, the polynucleotide comprises four miR-124 target sequences, four miR-338-3p target sequences, and four miR-31 target sequences, wherein the target sequences are operably linked to a transgene.

[0024] In one embodiment:

[0025] (a) the miR-124 target sequence comprises or consists of a nucleotide sequence having at least 90% sequence identity, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1;

[0026] (b) the miR-338-3p target sequence comprises or consists of a nucleotide sequence having at least 90% sequence identity, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 2; and / or

[0027] (c) the miR-31 target sequence comprises or consists of a nucleotide sequence having at least 90% sequence identity, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:3.

[0028] In one embodiment, the miR-124 target sequence comprises or consists of a nucleotide sequence having at least 90% sequence identity, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1. In one embodiment, the miR-338-3p target sequence comprises or consists of a nucleotide sequence having at least 90% sequence identity, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 2. In one embodiment, the miR-31 target sequence comprises or consists of a nucleotide sequence having at least 90% sequence identity, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 3.

[0029] In one embodiment:

[0030] (a) the miR-124 target sequence comprises or consists of: a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 1;

[0031] (b) the miR-338-3p target sequence comprises or consists of a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 2; and / or

[0032] (c) The miR-31 target sequence comprises or consists of a nucleotide sequence having at least 90% sequence identity to SEQ ID NO:3.

[0033] In one embodiment:

[0034] (a) the miR-124 target sequence comprises or consists of: a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 1;

[0035] (b) the miR-338-3p target sequence comprises or consists of a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 2; and / or

[0036] (c) The miR-31 target sequence comprises or consists of a nucleotide sequence having at least 95% sequence identity to SEQ ID NO:3.

[0037] In one embodiment:

[0038] (a) the miR-124 target sequence comprises or consists of: a nucleotide sequence having at least 99% sequence identity to SEQ ID NO: 1;

[0039] (b) the miR-338-3p target sequence comprises or consists of a nucleotide sequence having at least 99% sequence identity to SEQ ID NO: 2; and / or

[0040] (c) The miR-31 target sequence comprises or consists of a nucleotide sequence having at least 99% sequence identity to SEQ ID NO:3.

[0041] In one embodiment:

[0042] (a) the miR-124 target sequence comprises or consists of a nucleotide sequence having 100% sequence identity to SEQ ID NO: 1;

[0043] (b) the miR-338-3p target sequence comprises or consists of a nucleotide sequence having 100% sequence identity to SEQ ID NO: 2; and / or

[0044] (c) The miR-31 target sequence comprises or consists of a nucleotide sequence having 100% sequence identity with SEQ ID NO:3.

[0045] In one embodiment, the miRNA target sequence is located downstream of the transgene, i.e., 3'. In other words, in the 5' to 3' direction, the miRNA target sequence can be located after the transgene.

[0046] In one embodiment, the miRNA target sequence is located within the 3'-UTR of the transgene.

[0047] In one embodiment, the miRNA target sequence or the cluster of copies of the miRNA target sequence is arranged from 5' to 3' in the following order: one or more miR-124 target sequences, one or more miR-338-3p target sequences and one or more miR-31 target sequences. The target sequence or the cluster comprising one or more copies thereof can be arranged, for example, from 5' to 3', so that they form a group according to their target specificity, for example, in one embodiment, the polynucleotide comprises 5'-[miR-124 target sequence]4-[miR-338-3p target sequence]4-[miR-31 target sequence]4-3'.

[0048] Both single target sequences and clusters of target sequences can be adjacent to each other, separated by spacer sequences, or any combination thereof.

[0049] Thus, in one embodiment, the miRNA target sequences are separated by a spacer sequence.

[0050] In one embodiment, a polynucleotide is provided, wherein the polynucleotide comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 4. In one embodiment, the polynucleotide comprises a nucleotide sequence having at least 90%, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 4.

[0051] In one embodiment, the miRNA target sequence comprises the sequence shown in SEQ ID NO:4.

[0052] In one embodiment, the miRNA target sequence consists of the sequence shown in SEQ ID NO:4.

[0053] In one embodiment, the transgene encodes an epigenetic silencing factor (ESF) comprising a transcription factor DNA binding domain operably linked to at least one epigenetic effector domain, wherein the transcription factor is an oncogenic transcription factor or a cancer-associated transcription factor.

[0054] In some embodiments, the ESF is a fusion protein comprising a transcription factor DNA binding domain and at least one epigenetic effector domain.

[0055] In preferred embodiments, the transcription factor is an oncogenic transcription factor. In some embodiments, the transcription factor is a cancer-associated transcription factor.

[0056] In one embodiment, the transcription factor is selected from the group consisting of SOX2, MYC, MYCN, TEAD1, TEAD2, TEAD3, TEAD4, FOXA1, FOXA2, ELK1, ELK3, ELK4, SRF, FOXM1, FOXC1, FOXC2, TWIST1, SALL4, ELF1, HIF1A, SOX9, SOX12, SOX18, ETS1, PAX3, PAX8, GLI1, GLI2, GLI3, ETV1, ETV2, ETV3, RUNX1, RUNX2, RUNX3, MAFB, TFAP2C and E2F1.

[0057] In one embodiment, the transcription factor is SOX2.

[0058] In one embodiment, the transcription factor is TEAD1.

[0059] In one embodiment, the transcription factor is MYC.

[0060] In preferred embodiments, the ESF does not comprise a transcription factor activation domain.

[0061] In one embodiment, the epigenetic effector domain is selected from the group consisting of a KRAB domain, a DNMT3A domain, a DNMT3L domain, a ZIM3-KRAB (Z-KRAB) domain, a Chromo Shadow (CS) domain, a YAF2-RYBP (YR) domain, an Engrailed Repressor (En-R) domain, a MeCP2 domain, a GLI3RD domain, and a MAD1RD domain.

[0062] In one embodiment, the epigenetic effector domain is (a) a CS domain; (b) a YR domain; (c) a CS domain and a YR domain; (d) a KRAB domain; and / or (e) a DNMT3A domain and a DNMT3L domain.

[0063] The ESF according to the present invention may comprise a combination of a plurality of the aforementioned transcription factor DNA binding domains and epigenetic effector domains.

[0064] In one embodiment, the epigenetic effector domain is a CS domain.

[0065] In one embodiment, the epigenetic effector domain is a YR domain.

[0066] In one embodiment, the epigenetic effector domains are a CS domain and a YR domain.

[0067] In one embodiment, the epigenetic effector domain is a KRAB domain.

[0068] In one embodiment, the epigenetic effector domain is a DNMT3A domain.

[0069] In one embodiment, the epigenetic effector domain is a DNMT3L domain.

[0070] In one embodiment, the epigenetic effector domains are a DNMT3A domain and a DNMT3L domain.

[0071] In one embodiment, the epigenetic effector domain is a ZIM3-KRAB (Z-KRAB) domain.

[0072] In one embodiment, the epigenetic effector domain is a jagged repressor (En-R) domain.

[0073] In one embodiment, the epigenetic effector domain is a MeCP2 domain.

[0074] In one embodiment, the epigenetic effector domain is a GLI3RD domain.

[0075] In one embodiment, the epigenetic effector domain is the MAD1RD domain.

[0076] In one embodiment, the ESF comprises:

[0077] (a) KRAB domain, SOX2 DNA binding domain, DNMT3A domain, and DNMT3L domain;

[0078] (b) Chromo Shadow (CS) domain and SOX2 DNA binding domain;

[0079] (c) SOX2 DNA binding domain and YAF2-RYBP (YR) domain;

[0080] (d) KRAB domain, TEAD1 DNA binding domain, DNMT3A domain, and DNMT3L domain;

[0081] (e) KRAB domain, DNMT3A domain, DNMT3L domain and MYC DNA binding domain;

[0082] (f) Chromo Shadow (CS) domain and TEAD1 DNA binding domain;

[0083] (g) TEAD1 DNA binding domain and YAF2-RYBP (YR) domain;

[0084] (h) Chromo Shadow (CS) domain, TEAD1 DNA binding domain and YAF2-RYBP (YR) domain;

[0085] (i) Chromo Shadow (CS) domain and MYC DNA binding domain;

[0086] (j) YAF2-RYBP (YR) domain and MYC DNA binding domain; or

[0087] (k) Chromo Shadow (CS) domain, YAF2-RYBP (YR) domain and MYC DNA binding domain.

[0088] In one embodiment, the ESF comprises from 5' to 3':

[0089] (a) KRAB domain, SOX2 DNA binding domain, DNMT3A domain, and DNMT3L domain;

[0090] (b) Chromo Shadow (CS) domain and SOX2 DNA binding domain;

[0091] (c) SOX2 DNA binding domain and YAF2-RYBP (YR) domain;

[0092] (d) KRAB domain, TEAD1 DNA binding domain, DNMT3A domain, and DNMT3L domain;

[0093] (e) KRAB domain, DNMT3A domain, DNMT3L domain and MYC DNA binding domain;

[0094] (f) Chromo Shadow (CS) domain and TEAD1 DNA binding domain;

[0095] (g) TEAD1 DNA binding domain and YAF2-RYBP (YR) domain;

[0096] (h) Chromo Shadow (CS) domain, TEAD1 DNA binding domain and YAF2-RYBP (YR) domain;

[0097] (i) Chromo Shadow (CS) domain and MYC DNA binding domain;

[0098] (j) YAF2-RYBP (YR) domain and MYC DNA binding domain; or

[0099] (k) Chromo Shadow (CS) domain, YAF2-RYBP (YR) domain and MYC DNA binding domain.

[0100] In one embodiment, the polynucleotide comprises a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 114-119 and 126-131, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

[0101] In one embodiment, the polynucleotide comprises a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 114-119 and 126-131.

[0102] In one embodiment, the polynucleotide comprises a nucleotide sequence having at least 95% sequence identity to any one of SEQ ID NOs: 114-119 and 126-131.

[0103] In one embodiment, the polynucleotide comprises a nucleotide sequence having at least 99% sequence identity to any one of SEQ ID NOs: 114-119 and 126-131.

[0104] In one embodiment, the polynucleotide comprises or consists of the nucleotide sequence of any one of SEQ ID NOs: 114-119 and 126-131.

[0105] In one embodiment, the polynucleotide comprises a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 158-163, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

[0106] In one embodiment, the polynucleotide comprises a sequence according to any one of SEQ ID NOs: 158-163.

[0107] In one embodiment, the polynucleotide comprises a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 178-182, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

[0108] In one embodiment, the polynucleotide comprises a sequence according to any one of SEQ ID NOs: 178-182.

[0109] In one embodiment, the polynucleotide comprises a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 164-174, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

[0110] In one embodiment, the polynucleotide comprises a sequence according to any one of SEQ ID NOs: 164- 174. In one embodiment, the polynucleotide comprises a nucleotide sequence having at least 90% sequence identity, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOs: 136-141 and 153-157.

[0111] In one embodiment, the polynucleotide comprises a sequence according to any one of SEQ ID NOs: 136-141 and 153-157.

[0112] In one embodiment, the polynucleotide comprises a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 142-152, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

[0113] In one embodiment, the polynucleotide comprises a sequence according to any one of SEQ ID NOs: 142-152.

[0114] In one embodiment, the polynucleotide comprises a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 114-119, 126-131 and 136-174, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

[0115] In one embodiment, the polynucleotide comprises a sequence according to any one of SEQ ID NOs: 114-119, 126-131 and 136-174.

[0116] In one embodiment, the polynucleotide further comprises a promoter. In one embodiment, the promoter is operably linked to a transgene or nucleotide sequence encoding an ESF.

[0117] In one embodiment, the promoter is a constitutive promoter. In some embodiments, the constitutive promoter is the Ef1a promoter.

[0118] In one embodiment, the promoter is a tissue-specific promoter, preferably a cancer cell-specific promoter.

[0119] In one embodiment, the promoter is a proliferating cell-specific promoter.

[0120] In one embodiment, the polynucleotide further comprises a promoter operably linked to the transgene, optionally, wherein the promoter is a tissue-specific promoter or a constitutive promoter, optionally a cancer cell-specific promoter.

[0121] In one embodiment, the promoter is selected from the group consisting of Mki67 promoter, Ccnd1 promoter, Ccnb2 promoter, Ccna2 promoter, Cdc25c promoter, Cdc2 promoter, Cks1 promoter, PCNA promoter, CDC6 promoter, POLD1 promoter, CSK1B promoter, MCM2 promoter and PLK1 promoter.

[0122] In one embodiment, the promoter is the Mki67 promoter.

[0123] In one embodiment, the promoter is the Ef1a promoter.

[0124] In one aspect, a vector is provided, comprising a polynucleotide according to the invention.

[0125] In one embodiment, the vector is a viral vector.

[0126] In one embodiment, the vector is a lentiviral vector.

[0127] In one embodiment, the vector is an adeno-associated virus (AAV) vector.

[0128] In one embodiment, the AAV vector is serotype 2, 5 or 9. In a preferred embodiment, the vector is an AAV2 vector. In another preferred embodiment, the vector is an AAV5 vector.

[0129] In one embodiment, the vector is an mRNA vector.

[0130] In one aspect, a protein is provided, which is encoded by a polynucleotide or a vector according to the invention.

[0131] In one embodiment, a therapeutic protein is provided, which is encoded by a polynucleotide or a vector according to the present invention.

[0132] In one embodiment, there is provided an ESF encoded by a polynucleotide or a vector according to the present invention.

[0133] In one aspect, a protein (e.g., ESF) is provided comprising or consisting of an amino acid sequence having at least 70% sequence identity, such as at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOs: 109-113 and 120-125.

[0134] In one embodiment, the polynucleotide or transgene encodes a protein having at least 70% sequence identity, such as at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOs: 109-113 and 120-125, or a fragment thereof.

[0135] In one embodiment, the polynucleotide or transgene encodes a protein having at least 90% sequence identity to any one of SEQ ID NOs: 109-113 and 120-125, or a fragment thereof.

[0136] In one embodiment, the polynucleotide or transgene encodes a protein having at least 95% sequence identity to any one of SEQ ID NOs: 109-113 and 120-125, or a fragment thereof.

[0137] In one embodiment, the polynucleotide or transgene encodes a protein having at least 99% sequence identity to any one of SEQ ID NOs: 109-113 and 120-125, or a fragment thereof.

[0138] In one embodiment, the polynucleotide or transgene encodes a protein having 100% sequence identity to any one of SEQ ID NOs: 109-113 and 120-125, or a fragment thereof.

[0139] In one aspect, polynucleotides are provided comprising a transgene encoding an epigenetic silencing factor (ESF), wherein the transgene is operably linked to at least one micro RNA (miRNA) target sequence, wherein the ESF comprises a transcription factor DNA binding domain operably linked to at least one epigenetic effector domain, and wherein the transcription factor is an oncogenic transcription factor or a cancer-associated transcription factor.

[0140] The miRNA target sequence can be designed to be recognized by any suitable miRNA or selected from any suitable known miRNA target sequence, such as a miRNA expressed in a cell type in which transgene expression is undesirable.

[0141] In one embodiment, at least one miRNA target sequence is a target site of a miRNA selected from the group consisting of miR-124, miR-338-3p, and miR-31.

[0142] In one embodiment, the miRNA target sequence is a target site for miR-124 or consists of a nucleotide sequence having at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:1.

[0143] In one embodiment, the miRNA target sequence is a target site of miR-338-3p or consists of a nucleotide sequence having at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:2.

[0144] In one embodiment, the miRNA target sequence is a target site for miR-31 or consists of a nucleotide sequence having at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:3.

[0145] In one embodiment, at least one miRNA target sequence comprises SEQ ID NO: 1, 2 and 3, or a sequence having at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity thereto.

[0146] In a preferred embodiment, the at least one miRNA target sequence comprises target sites for miR-124, miR-338-3p, and miR-31, preferably wherein four copies of each target sequence are present.

[0147] The order of the miRNA target sequences may vary. In one embodiment, the miRNA target sequences or clusters of copies of sequences are arranged from 5' to 3' in the following order: one or more miR-124 target sequences, one or more miR-338-3p target sequences, and one or more miR-31 target sequences.

[0148] In one embodiment, the miRNA target sequence or cluster of copies of sequences are arranged from 5' to 3' in the following order: one or more miR-124 target sequences, one or more miR-31 target sequences, and one or more miR-338-3p target sequences.

[0149] In another embodiment, the miRNA target sequence or cluster of copies of sequences are arranged from 5' to 3' in the following order: one or more miR-338-3p target sequences, one or more miR-124 target sequences, and one or more miR-31 target sequences.

[0150] In another embodiment, the miRNA target sequences or clusters of copies of sequences are arranged from 5' to 3' in the following order: one or more miR-338-3p target sequences, one or more miR-31 target sequences, and one or more miR-124 target sequences.

[0151] In another embodiment, the miRNA target sequence or cluster of copies of sequences are arranged from 5' to 3' in the following order: one or more miR-31 target sequences, one or more miR-124 target sequences, and one or more miR-338-3p target sequences.

[0152] In another embodiment, the miRNA target sequence or cluster of copies of sequences are arranged from 5' to 3' in the following order: one or more miR-31 target sequences, one or more miR-338-3p target sequences, and one or more miR-124 target sequences.

[0153] Both the single target sequence and the cluster of copies of the sequence can be adjacent to each other, separated by a spacer sequence, or any combination thereof.

[0154] Thus, in one embodiment, the miRNA target sequences are separated by a spacer sequence.

[0155] In one embodiment, a polynucleotide is provided, wherein the polynucleotide comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 4. In one embodiment, the polynucleotide comprises a sequence having at least 90%, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 4.

[0156] In one aspect, an epigenetic silencing factor (ESF) is provided, comprising a transcription factor DNA binding domain operably linked to at least one epigenetic effector domain, or a polynucleotide encoding the same, wherein the transcription factor is an oncogenic transcription factor or a cancer-associated transcription factor, wherein the ESF comprises:

[0157] (a) Chromo Shadow (CS) domain and TEAD1 DNA binding domain;

[0158] (b) TEAD1 DNA binding domain and YAF2-RYBP (YR) domain;

[0159] (c) Chromo Shadow (CS) domain, TEAD1 DNA binding domain and YAF2-RYBP (YR) domain;

[0160] (d) Chromo Shadow (CS) domain and MYC DNA binding domain;

[0161] (e) YAF2-RYBP (YR) domain and MYC DNA binding domain; or

[0162] (f) Chromo Shadow (CS) domain, YAF2-RYBP (YR) domain and MYC DNA binding domain.

[0163] In one embodiment, an epigenetic silencing factor (ESF) is provided, comprising a transcription factor DNA binding domain operably linked to at least one epigenetic effector domain, wherein the transcription factor is an oncogenic transcription factor or a cancer-associated transcription factor, wherein the ESF consists of:

[0164] (a) Chromo Shadow (CS) domain and TEAD1 DNA binding domain;

[0165] (b) TEAD1 DNA binding domain and YAF2-RYBP (YR) domain;

[0166] (c) Chromo Shadow (CS) domain, TEAD1 DNA binding domain and YAF2-RYBP (YR) domain;

[0167] (d) Chromo Shadow (CS) domain and MYC DNA binding domain;

[0168] (e) YAF2-RYBP (YR) domain and MYC DNA binding domain; or

[0169] (f) Chromo Shadow (CS) domain, YAF2-RYBP (YR) domain and MYC DNA binding domain.

[0170] In one embodiment, ESF comprises a sequence or a fragment thereof having at least 70%, such as at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOs: 120-125.

[0171] In one embodiment, the ESF comprises a sequence or a fragment thereof having at least 90% sequence identity to any one of SEQ ID NOs: 120-125.

[0172] In one embodiment, the ESF comprises a sequence or a fragment thereof having at least 95% sequence identity to any one of SEQ ID NOs: 120-125.

[0173] In one embodiment, the ESF comprises a sequence or a fragment thereof having at least 99% sequence identity to any one of SEQ ID NOs: 120-125.

[0174] In one embodiment, the ESF comprises a sequence or a fragment thereof having 100% sequence identity to any one of SEQ ID NOs: 120-125.

[0175] In one embodiment, the ESF comprises or consists of a sequence according to any one of SEQ ID NOs: 120-125, or a fragment thereof.

[0176] In one embodiment, the polynucleotide encoding ESF comprises a sequence having at least 70%, such as at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOs: 126-131.

[0177] In one embodiment, the polynucleotide encoding ESF comprises a sequence having at least 70%, such as at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOs: 158-163.

[0178] In one embodiment, the polynucleotide encoding ESF comprises a sequence having at least 70%, such as at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOs: 178-182.

[0179] In one embodiment, the polynucleotide encoding ESF comprises a sequence having at least 70%, such as at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOs: 168-173.

[0180] In one embodiment, the polynucleotide encoding ESF comprises a sequence having at least 70%, such as at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOs: 136-141.

[0181] In one embodiment, the polynucleotide encoding ESF comprises a sequence having at least 70%, such as at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOs: 146-148 and 150-152.

[0182] The following aspects and embodiments may be applied to any of the aforementioned aspects and embodiments of the present invention.

[0183] In one aspect, a nanoparticle is provided, comprising a polynucleotide, a vector, an ESF or a protein according to the invention.

[0184] In one embodiment, the nanoparticle is a polymer nanoparticle, an inorganic nanoparticle, or a lipid nanoparticle. In some embodiments, the nanoparticle is a liposome.

[0185] In one aspect, a cell is provided, comprising a polynucleotide, a vector, a protein, an ESF or a nanoparticle according to the invention.

[0186] In one embodiment, the cell is a eukaryotic cell, such as a mammalian cell. In another embodiment, the cell is a human cell.

[0187] In one aspect, a composition is provided comprising a polynucleotide, a vector, a protein, an ESF, a nanoparticle or a cell according to the invention.

[0188] The composition can be a hydrogel. In some embodiments, the hydrogel is a poly(ethylene glycol) dimethacrylate (PEG-DMA) hydrogel. In some embodiments, the hydrogel further comprises hydroxyapatite nanoparticles.

[0189] In one aspect, a pharmaceutical composition is provided, which comprises a polynucleotide, vector, protein, ESF, nanoparticle or cell according to the present invention, and a pharmaceutically acceptable diluent, solvent, carrier, salt and / or adjuvant.

[0190] In one aspect, there is provided a polynucleotide, a vector, a protein, an ESF, a nanoparticle, a cell, a composition or a pharmaceutical composition according to the invention for use in therapy.

[0191] In one aspect, there is provided a polynucleotide, vector, protein, ESF, nanoparticle, cell, composition or pharmaceutical composition according to the invention for use in the treatment of a disease or disorder.

[0192] In one aspect, there is provided a polynucleotide, a vector, a protein, an ESF, a nanoparticle, a cell, a composition or a pharmaceutical composition according to the invention for use in the treatment of cancer.

[0193] In some embodiments, the cancer is a glioma, glioblastoma, medulloblastoma, astrocytoma, neuroblastoma, ependymoma, meningioma, retinoblastoma, rhabdomyosarcoma, lung cancer, prostate cancer, breast cancer, liver cancer, pancreatic cancer, bladder cancer, oropharyngeal cancer, or kidney cancer. In some embodiments, the cancer is a brain tumor. In some embodiments, the cancer is glioblastoma multiforme.

[0194] In one embodiment, the cancer is liver metastasis.

[0195] In one embodiment, the cancer is colon cancer, such as colon adenocarcinoma. In one embodiment, the cancer is colorectal cancer (CRC).

[0196] In some embodiments, treatment reduces tumor size.

[0197] In some embodiments, the treatment is as adjuvant therapy, optionally in combination with surgery. The treatment can reduce the risk of recurrence of cancer (eg, GBM).

[0198] In some embodiments, the ESF, polynucleotide, vector, cell, or composition (eg, hydrogel) is administered topically.

[0199] In one aspect, there is provided a use of a polynucleotide, a vector, a protein, an ESF, a nanoparticle, a cell, a composition or a pharmaceutical composition according to the present invention for reducing the transcription and / or expression of at least one target gene in a cell.

[0200] In one aspect, a method for reducing the transcription and / or expression of at least one target gene in a cell is provided, the method comprising introducing a polynucleotide, vector, protein, ESF, nanoparticle, cell, composition or pharmaceutical composition according to the present invention into the cell.

[0201] In one embodiment, the aforementioned method is an in vitro method.

[0202] In one embodiment, the aforementioned method is an in vivo method.

[0203] In one embodiment, the aforementioned method is an ex vivo method.

[0204] In one embodiment, at least one target gene is silenced, preferably permanently.

[0205] In another aspect, the present invention provides use of a polynucleotide, a vector, a protein, an ESF, a nanoparticle, a cell, a composition or a pharmaceutical composition according to the present invention in the preparation of a medicament for therapy.

[0206] In another aspect, the present invention provides use of a polynucleotide, a vector, a protein, an ESF, a nanoparticle, a cell, a composition or a pharmaceutical composition according to the present invention in the preparation of a medicament for treating cancer.

[0207] In one aspect, an epigenetic silencing factor (ESF) or a polynucleotide encoding the same is provided for use in the treatment of cancer, wherein the ESF comprises a transcription factor DNA binding domain operably linked to at least one epigenetic effector domain, wherein the transcription factor is an oncogenic transcription factor or a cancer-associated transcription factor, and wherein the cancer is selected from the group consisting of glioma, glioblastoma, medulloblastoma, astrocytoma, neuroblastoma, ependymoma, meningioma, retinoblastoma, rhabdomyosarcoma, lung cancer, prostate cancer, breast cancer, liver cancer, pancreatic cancer (e.g., human pancreatic ductal adenocarcinoma), bladder cancer, oropharyngeal cancer, kidney cancer, colon cancer (e.g., colon adenocarcinoma), or metastasis of any of the above.

[0208] The ESF or polynucleotide may, for example, be in the form of or contained in a vector, nanoparticle, cell or composition.

[0209] In some embodiments, the cancer is a glioma, glioblastoma, medulloblastoma, astrocytoma, neuroblastoma, ependymoma, meningioma, retinoblastoma, rhabdomyosarcoma, lung cancer, prostate cancer, breast cancer, pancreatic cancer (e.g., human pancreatic ductal adenocarcinoma), bladder cancer, oropharyngeal cancer, colon cancer (e.g., colon adenocarcinoma), or kidney cancer. In some embodiments, the cancer is a brain tumor. In some embodiments, the cancer is glioblastoma multiforme.

[0210] In one embodiment, the cancer is glioblastoma, prostate cancer, human pancreatic ductal adenocarcinoma, colon cancer, or colon adenocarcinoma.

[0211] In one embodiment, the cancer is a metastasis of a primary cancer, wherein the primary cancer is a glioma, a glioblastoma, a medulloblastoma, an astrocytoma, a neuroblastoma, an ependymoma, a meningioma, a retinoblastoma, a rhabdomyosarcoma, a lung cancer, a prostate cancer, a breast cancer, a pancreatic cancer (e.g., a human pancreatic ductal adenocarcinoma), a bladder cancer, an oropharyngeal cancer, a colon cancer (e.g., a colon adenocarcinoma), or a kidney cancer. In some embodiments, the primary cancer is a brain tumor. In some embodiments, the primary cancer is a glioblastoma multiforme.

[0212] In one embodiment, the cancer is glioma.

[0213] In one embodiment, the cancer is human pancreatic ductal adenocarcinoma.

[0214] In one embodiment, the cancer is a liver metastasis of colon cancer. In one embodiment, the cancer is a liver metastasis of pancreatic adenocarcinoma. In one embodiment, the cancer is a liver metastasis of colorectal cancer (CRC).

[0215] In one embodiment, the cancer is colon cancer.

[0216] In some embodiments, the metastasis is a liver metastasis (preferably derived from colon cancer).

[0217] In some embodiments, the treatment is as adjuvant therapy, optionally in combination with surgery. The treatment can reduce the risk of recurrence of cancer (eg, GBM).

[0218] In some embodiments, the ESF, polynucleotide, vector, cell, or composition (eg, hydrogel) is administered topically.

[0219] In one embodiment, the vector is a viral vector.

[0220] In one embodiment, the vector is a lentiviral vector.

[0221] In one embodiment, the vector is an adeno-associated virus (AAV) vector.

[0222] In one embodiment, the vector is an mRNA vector. BRIEF DESCRIPTION OF THE DRAWINGS

[0223] Figure 1 .Generation and testing of SOX2 epigenetic silencers (SES).

[0224] (a) Constructs generated based on the human SOX2 transcription factor and the epigenetic domains KRAB, DNMT3a (3A) and DNMT3L (3L), with V5 added as a tag. (b) Infection efficiency of lentiviruses carrying the indicated constructs in SNB19 cells. (c) Left, growth curves of SNB19 cells infected with the indicated constructs show that SES is able to kill cells after 12 days of culture, ****p<0.0001; statistical comparison with two-way ANOVA; right, micrographs of cells at the indicated time points from infection with mock (GFP) or SES. (d) Western blot (WB) of V5, SOX2 and H3 (as loading control) in SNB19 cells uninfected or infected with lentiviruses carrying GFP or SES. (e) Left, growth curves of U87 cells, ****p<0.0001; statistical comparison with two-way ANOVA; right, micrographs of cells before treatment and after 9 days of infection with mock (GFP) or SES. f) Left, growth curves of U251 cells, ***p<0.001; statistical comparisons were performed with two-way ANOVA; right, micrographs of cells before treatment and 10 days after infection with mock (GFP) or SES. (g) Quantification of the indicated SOX2 targets in SNB19 cells 3 days after infection with GFP or SES. ****p<0.0001, ***p<0.001; statistical comparisons were performed with unpaired t-test. (h) Growth curves of the indicated cancer cell lines. ***p<0.001, ns=not significant; statistical comparisons were performed with two-way ANOVA.

[0225] Figure 2 .Efficacy of the SOX2 epigenetic silencer on patient-derived cancer stem cells in vitro.

[0226] (a) Micrographs, growth curves, and percentage of dead cells of patient-derived stem cells (CSCs) of classical (left) and mesenchymal (right) GBM subtypes infected with GFP (both subtypes), SES (both), or binding-deficient SES (R74P-L97P) (classical subtype only). ****p<0.0001; ***p<0.001; statistical comparisons were performed with two-way ANOVA. The clonogenic potential of CSCs (classical type) uninfected or infected with lentivirus carrying GFP or SES was determined using the number and size of spheres (percentage of spheres with a diameter below 100 μm) at the indicated time points as parameters. ****p<0.0001; statistical comparisons were performed with two-way ANOVA.

[0227] Figure 3 .Molecular consequences of SES.

[0228] (a) SES leads to dysregulation of a large number of genes in both U87 and SNB19 cells, as assessed by RNA-seq. (b) IGV snapshots of RNAseq tracks within the Sox2 locus in both cell lines under each condition (mock and SES infection) indicating the initial portion of SOX2 in the SOX2-negative (U87) and SOX2-positive (SNB19) cell lines (included in the SES construct, see Figure 1 (a) Overexpression of SES. (c) Gene ontology analysis showed that genes associated with apoptosis (up-regulated) and cell cycle regulation (down-regulated) were impaired by SES expression. (d) Genes predicted to be regulated by SOX2 were affected, with most genes down-regulated. (e) Density plot of ChIP-seq normalized signals (SOX2 and SES) on SOX2 peaks showed that SES bound to the same region at the genome-wide level. (f) Density plot of MeDIP-seq normalized signals (mock and SES) on SOX2 peaks showed that SES was able to increase DNA methylation levels.

[0229] Figure 4 .SES functionality in vivo: heterotopic xenotransplantation.

[0230] (a) Heterotopic xenografts were performed by subcutaneous injection of 1 million GBM cells in NSG mice pre-infected with mock (GFP) or SES. (b) 4 weeks after injection, mock U87 cells always generated huge clumps, while only in one case did we recover a small nodule from SES-infected cells. (c) Evaluation of the volume of tumors generated using the indicated cells after the indicated time windows (Mo = month).

[0231] Figure 5 .SES functionality in vivo: orthotopic xenotransplantation.

[0232] (a) Orthotopic xenografts were performed in the brains of NSG mice pre-infected with mock (GFP) or SES by injecting 300,000 cells in the striatum. (bc) 25-30 days after injection, mock U87 cells always generated large GFP-positive tumors that were also able to invade the cortex, while we never detected any tumors in brains transplanted with SES U87 cells by Nissl histological staining (b) or V5 antibody (c). (d) Tumors generated by mock cells were formed by human nuclear (HuN)-positive, proliferating (PH3-positive) cells, while almost no human cells were recovered in brains injected with SES cells. It is noteworthy that the few PH3-positive cells present were located at the level of the lateral ventricles and are probably actively dividing mouse neural progenitor cells. (e) Estimation of tumor volume. (f) Kaplan-Meier curves show that mice injected with mock cells died within one month after surgery, while animals receiving SES were in good health and in good health at the time of sacrifice (n = 5 animals per group). (g) Xenografts of mock-infected CSCs produced large tumors, whereas mice injected with SES CSCs showed small tumors 6 weeks after surgery. (h) Estimation of tumor volume. (i) Kaplan-Meier curves showing that mice injected with mock cells died within 6 weeks after surgery, whereas animals receiving SES were in better condition at the time of sacrifice (n = 5 animals per group).

[0233] Figure 6 .GBM cortical organoids.

[0234] (a) Early patterned cortical organoids were seeded with GBM suspension spheroids (labeled with RFP) to achieve confluence. (b) Confluent GBM-cortical organoids were fixed, cut, and stained using mock- or SES-infected spheroids (1 week after seeding). Notably, SES restricted the growth and infiltration of GBM cells (RFP positive) within the normal cortical parenchyma (DAPI staining only).

[0235] Figure 7 .SES functionality in vivo: processing of preformed orthotopic xenografts.

[0236] (a) Orthotopic xenografts were generated by injecting 75,000 initial U87 cells into the striatum of NSG mice; 4 days later, animals were reoperated to inject lentivirus carrying mock (GFP) or SES and evaluated 26 days later (total 30 days). (b) Histological staining at the time of lentivirus injection (pre-treatment) and at the end of the regimen (post-treatment) shows that tumor growth is restricted in SES-treated animals. (c) Estimation of tumor volume 26 days from lentivirus injection (treatment). (d) Kaplan-Meier curves show that mice whose tumors were injected with mock virus died within two months after cell injection, while all but one of the animals that received SES reached 3 months (n = 4 animals per group). (e) Immunohistochemistry shows that the resulting tumors were GFP-positive in the case of mock treatment and V5-negative in the case of SES injection (asterisk), indicating a negative selection effect on those tumor cells infected by the SES virus. Notably, V5 labeling (arrows) is present in the parenchyma of the mouse brain surrounding the tumor. (f) Orthotopic xenografts were generated by injecting 100,000 naive CSCs in the striatum of NSG mice; 7 days later, the animals were reoperated to inject lentivirus carrying mock (GFP) or SES and evaluated by MRI scans and sacrificed 6 weeks after treatment (7 weeks after cell transplantation). (g) Examples of MRI scans at 3, 4 and 5 weeks post infection (pi) (two sections from one mock treated mouse and one SES treated mouse). (h) Histological staining at the end of the protocol (endpoint) shows that tumor growth is restricted in SES treated animals. (i) Evaluation of tumor volume as measured by high intensity T2 weighted imaging (MIPAV software). (j) Tumor volume estimated by histological measurements at the end point and therefore 6 weeks after lentiviral injection (treatment) and 7 weeks after CSC injection.

[0237] Figure 8 .Effects of SES on cultured neurons.

[0238] (a) Evaluation of infection and death of primary mouse hippocampal neurons infected with mock or SES shows that SES does not induce neurodegeneration. (b) SES only causes dysregulation of marginal genes in mouse primary neurons as assessed by RNA-seq. (c) Human iPSC-derived neurons were infected with mock or SES. (d) Evaluation of neuronal loss by staining for PI, V5, and MAP2 shows that the presence of SES does not increase neuronal death at least 21 days after infection.

[0239] Fig. 9 .Effects of SES on the normal mouse brain.

[0240] (a) Mock or SES lentiviral injection in the hippocampus of WT c57bl / 6 mice. (b) Example of viral transduction in the mouse hippocampus using GFP as a reporter gene 4 weeks after injection. (c) Quantification of both viral genome and exogenous transgenic mRNA in infected hippocampus by qPCR showed no differences between conditions. (d) Quantification of cleaved caspase 3-positive cells within infected hippocampus showed no differences between conditions, indicating that SES was not toxic to mouse neural cells in this condition. (e) Spontaneous alternation test showed no differences between mock and SES injected mice as assessed by the percentage of entries in different arms, as well as the percentage of spontaneous alternation performance (SAP), the percentage of alternate arm returns (ARR), and the percentage of same arm returns (SAR) as a percentage of total entries. Statistical comparisons were performed with the Mann-Whitney test. (f) Radial maze test showed no differences in the time to complete the task or the specific tendency to make errors throughout the test protocol in SES treated animals compared to mock injected animals. Statistical comparisons were performed with two-way ANOVA. (g) Morris water maze test. Left, the protocol we used and the protocol for positioning of the platform (black square). Right, graphs of the time taken to complete the task (top) and quantification of the time spent in the platform area or the opposite area (bottom) show no differences between conditions. Statistical comparisons were performed with the Mann-Whitney test.

[0241] Fig.10 .Carrier improvement.

[0242] (a) Scheme depicts the original SES (v1) and a further version (v1.1) carrying a different promoter (the KI67 promoter expressed in proliferating cells, v1.1). (b) Testing of SES v1.1 in U251 cells shows that the KI67 promoter directs expression of the transgene (GFP or SES) in a very high proportion of KI67-positive proliferating cells. SES v1.1 has a similar effect on growth compared to the original version. (c) Both constitutive GFP and pKI67-GFP were used in mouse primary cortical cultures that contain predominantly postmitotic neurons but also proliferating and postmitotic glial types. Immunofluorescence using the indicated antibodies shows that when directed by pKI67, expression of the transgene is only expressed in KI67-positive cells. + GFP was found in proliferating cells (red arrows and quantification) but never in MAP2 + As expected, constitutive GFP was observed in almost all cells.

[0243] Fig.11 .Generation of other ESFs.

[0244] (a) The construct generated based on the human SOX2 transcription factor fused to the epigenetic domains chromo shadow (CS) and V5 was named SES v2. The construct generated based on the human SOX2 transcription factor fused to the epigenetic domains YAF2-RYBP (YR) and V5 was named SES v3. (b) Infection efficiency of lentivirus carrying the indicated constructs in U251 cells (see inset). (c) Growth curves of U251 cells infected with the indicated constructs, showing that SES v2 and v3 were able to kill cells after 9 days of culture, ****p<0.0001; statistical comparisons were performed with two-way ANOVA. (d) The construct generated based on the human TEAD1 transcription factor and the epigenetic domains KRAB, DNMT3a (3A), DNMT3L (3L), and V5 was named TES, while the construct generated based on the human MYC transcription factor and the epigenetic domains KRAB, DNMT3a (3A), DNMT3L (3L), and V5 was named MES. (e) Infection efficiency of lentivirus carrying the indicated constructs in U251 cells (see inset, right). (e) Growth curves of U251 cells infected with the indicated constructs show that MES is able to kill cells after 9 days of culture, ****p<0.0001.

[0245] Fig.12 In vitro efficacy of SESv3 on patient-derived cancer stem cells

[0246] (a) Micrographs and (b) growth curves of patient-derived stem cells (CSCs) of classical GBM subtypes infected with GFP or SESv3. ****p<0.0001; ***p<0.001; statistical comparisons were performed with two-way ANOVA.

[0247] Fig.13 In vitro efficacy of TES and MES on patient-derived cancer stem cells

[0248] (ac) Micrographs (a), growth curves, and percentage of dead cells (bc) of patient-derived stem cells (CSCs) of classical (left) and mesenchymal (right) GBM subtypes that were not infected (NI) or infected with GFP or TES. *p<0.05; ***p<0.001; ****p<0.0001; ns=not significant; statistical comparisons were performed with two-way ANOVA. (de) Growth curves and percentage of dead cells of patient-derived stem cells (CSCs) of classical (left, d) and mesenchymal (right, e) GBM subtypes that were not infected (NI) or infected with GFP or TES. *p<0.05; ***p<0.001; ****p<0.0001; ns=not significant; statistical comparisons were performed with two-way ANOVA.

[0249] Fig.14.TES / MES functionality in vivo: heterotopic xenotransplantation

[0250] (a) Heterotopic xenografts were performed by subcutaneous injection of 3,000,000 classical CSCs in NSG mice pre-infected with mock (GFP) or TES or MES. Four weeks after injection, mock CSCs always generated giant masses, while smaller tumors emerged from TES (bc) or MES-infected cells (de).

[0251] Fig.15 .TES / MES in vivo functionality: orthotopic xenografts

[0252] (a) Orthotopic xenografting was performed in the brain of NSG mice pre-infected with mock (GFP) or TES or MES by injection of 300.000 classical CSCs in the striatum. (b) 5 weeks after injection (WPI), mock CSCs always generated giant tumors that were also able to invade the cortex, while TES cells formed smaller tumors in the brain. Evaluation by Nissl histological staining. (c) Estimation of tumor volume (n = 4 animals per group). (d) 3 weeks after injection (WPI), mock CSCs already showed important tumor masses, while MES cells formed smaller tumors. Evaluation by DAPI staining. (c) Estimation of tumor volume (n = 4 animals per group).

[0253] Fig.16 .TES functionality in vivo: processing of preformed orthotopic xenografts

[0254] (a) Orthotopic xenografts were generated by injecting 300,000 classical CSCs into the striatum of NSG mice; 7 days later, animals were reoperated to inject lentivirus carrying mock (GFP) or TES and evaluated 3 weeks after (WPT) (4 weeks total). (b) DAPI staining at the end of the protocol (post-treatment) shows that tumor growth was limited to the injection site in TES-treated animals, while large clumps were present in mock-treated mice. (c) Estimation of tumor volume 3 weeks from lentiviral injection (treatment) (n=3 animals per group).

[0255] Fig.17 .Epigenetic silencing factor constructs.

[0256] Schematic diagram of ESF constructs. (a) Constructs generated based on human TEAD1 transcription factor and epigenetic repressor domains ChromoShadow (CS) (from gene CBX5) and YAF2-RYBP (YR) (from gene RYBP), each variant has a V5 tag at the C-terminus. (b) Constructs generated based on human MYC transcription factor and epigenetic repressor domains Chromo Shadow (CS) (from gene CBX5) and YAF2-RYBP (YR) (from gene RYBP). Each variant has a 3' V5 tag. The subscripts indicate the amino acid range of each protein domain encoded by the construct.

[0257] Fig.18 .Use of miRNA-based de-targeting silencing factor constructs to enhance ESF specificity.

[0258] (a) Schematic diagram of both the control vector and the SES vector, both of which contain the Tmir box (four copies of each of the miRNA target sequences of miR124, miR338-3p and miR31) within the 3'UTR. The Tmir box allows the transgene to be expressed in cancer cells, but not in brain cells (neurons, oligodendrocytes and astrocytes). (b) Both GFP-Tmir and SES-Tmir are expressed in the U251 GBM cancer cell line as shown by GFP / V5 expression counterstained with Hoecst. SES-Tmir is able to reduce the proliferation of cancer cells in vitro. (c) SES-Tmir is not expressed (de-targeted) in mouse primary cortical cultures containing postmitotic neurons, postmitotic and proliferating astrocytes, and proliferating oligodendrocyte precursor cells (OPCs).

[0259] Fig.19 .AAV Function on Patient-Derived Cancer Stem Cells.

[0260] Recombinant AAV serotypes containing the Ef1a::GFP construct were used to infect patient-derived GBM cancer stem cells in vitro. Four days after infection, cells were fixed and subjected to immunofluorescence against GFP, and cells were stained with the nuclear marker Hoecst.

[0261] Fig. 20 .The efficacy of ESF on different cancer cell types.

[0262] (a) Growth curve of CT26 cells, derived from liver metastasis of colon adenocarcinoma in mice, treated with lentivirus carrying GFP or SES (Sox2 epigenetic silencer). ****p<0.0001. (b) Growth curve of BxPC-3 cells (human pancreatic ductal carcinoma cells) treated with lentivirus carrying GFP or TES (Tead epigenetic silencer). ****p<0.0001. (c) Growth curve of CFPAC-1 cells (human pancreatic ductal carcinoma cells) treated with lentivirus carrying GFP or TES (Tead epigenetic silencer). ****p<0.0001.

[0263] Fig.21 .Effects of SESv3 on SNB19 cells.

[0264] (a) Infection efficiency, micrographs and quantification of lentiviruses carrying the indicated constructs in SNB 19 cells. (b) Growth curves of SNB19 cells treated with lentiviruses carrying GFP or SES or SESv3; control means untreated cells. **p<0.01; ns p>0.05. (c) Percentage of cell death (cells positive for trypan blue solution) of SNB19 cells treated with lentiviruses carrying GFP or SES or SESv3; control means untreated cells. ***p<0.001; ns p>0.05.

[0265] Fig. 22 Comparative effects of TES in SNB19 cells.

[0266] (a) Growth curve of SNB19 cells treated with lentivirus carrying GFP or TES, TES mutated to affect DNA binding ability, and YAP / TAZ inhibitor verteporfin (concentration 2uM); control means untreated cells. **p<0.01. (b) Percentage of cell death (cells positive for trypan blue solution) of SNB19 cells treated with lentivirus carrying GFP or TES, TES mutated to affect DNA binding ability, and YAP / TAZ inhibitor verteporfin (concentration 2uM); control means untreated cells. ***p<0.001; *p<0.05.

[0267] Fig.23 .Transcriptional effects of TES in SNB19 cells.

[0268] (a) Volcano plot showing differentially expressed genes from RNAseq experiments comparing SNB19 cells treated with lentivirus carrying either GFP or TES. (b) Gene set enrichment analysis (GSEA) for a manually curated set of genes from a revision of the literature on experimentally validated YAP / TAZ targets. (c) Motif enrichment analysis of promoters (–1,000 + 100 bp from TSS) of genes downregulated in TES-treated SNB19 using Homer. (d) Summary of gene ontology analysis (biological processes) of genes upregulated or downregulated after TES treatment.

[0269] Fig.24 .TES impairs migration activity in SNB19 cells.

[0270] (a) Gene set enrichment analysis (GSEA) for the indicated gene sets associated with the migration capacity of different cell types. (b) Representative digital photos of wound healing assays taken 0, 24 and 48 hr after wounding performed 2 days after the start of the indicated treatments. Quantification of the results is shown on the right.

[0271] Fig.25 .SES impairs the growth of metastatic cells from a mouse model.

[0272] (a) Schematic diagram of Flex construct and function. (b) Stable line of MC38 cells (derived from liver metastasis of colorectal cancer-CRC mouse model) carrying Flex-SES construct has shown SES vulnerability after 6 days of CRE recombination-mediated SES expression. (c) Stable line of K8484 cells (derived from liver metastasis of pancreatic ductal adenocarcinoma-PDAC mouse model) carrying Flex-SES construct has shown SES vulnerability after 6 days of CRE recombination-mediated SES expression. DETAILED DESCRIPTION OF THE INVENTION

[0274] As used herein, the term "comprising" is synonymous with "including" or "containing", and is inclusive or open-ended and does not exclude additional, unrecited members, elements or steps. The term "comprising" also includes the term "consisting of".

[0275] It will be understood that when reference is made herein to a protein or polypeptide, the same applies to the polynucleotide encoding the same, and vice versa where relevant (ie when reference is made to a coding sequence within a polynucleotide).

[0276] It will be appreciated that any of the following aspects of the invention may be combined as appropriate in the practice of the invention.For example, a miRNA transgene cassette according to the invention may be used in conjunction with an ESF according to another aspect of the invention, or may be used independently, to deliver any suitable transgene.

[0277] Polynucleotides and transgene cassettes

[0278] Triplet miRNA cassette

[0279] Herein, the inventors provide a transgenic expression cassette comprising a target sequence recognized by a micro RNA (miRNA) to regulate transgenic expression. The expression cassette allows for regulation of transgenic expression so as to reduce or eliminate unwanted expression in a cell type comprising the miRNA, thereby improving safety and reducing off-target effects.

[0280] In one aspect, polynucleotides are provided that comprise at least one miR-124 target sequence, at least one miR-338-3p target sequence, and at least one miR-31 target sequence, wherein the miRNA target sequence is operably linked to a transgene.

[0281] In one embodiment, the number of copies of each miRNA target sequence is independently selected from the group consisting of: one, two, three, and four.

[0282] In one embodiment, the copy number of each miRNA target sequence is one.

[0283] In one embodiment, the copy number of each miRNA target sequence is two.

[0284] In one embodiment, the copy number of each miRNA target sequence is three.

[0285] In one embodiment, the copy number of each miRNA target sequence is four.

[0286] In one embodiment, the number of copies of each miRNA target sequence is greater than four, such as five, six, seven, eight, nine or ten.

[0287] Suitable miRNA target sequences for miR-124 are:

[0288] attgccttatttc[SEQ ID NO:1]

[0289] In one embodiment, the miRNA target sequence comprises the sequence of SEQ ID NO:1.

[0290] Suitable miRNA target sequences for miR-338-3p are:

[0291] caacaaaatcactgatgctgga[SEQ ID NO:2]

[0292] In one embodiment, the miRNA target sequence comprises the sequence of SEQ ID NO:2.

[0293] Suitable miRNA target sequences for miR-31 are:

[0294] cagctatgccagcatcttgcc[SEQ ID NO:3]

[0295] In one embodiment, the miRNA target sequence comprises the sequence of SEQ ID NO:3.

[0296] In one embodiment, the miRNA target sequence comprises SEQ ID NOs: 1, 2, and 3.

[0297] In one embodiment, the miRNA target sequence is located downstream, ie, 3', of the transgene.

[0298] In one embodiment, the miRNA target sequence is located within the 3'-UTR of the transgene.

[0299] In one embodiment, the miRNA target sequence or cluster of copies of the sequence is arranged from 5' to 3' in the following order: miR-124, miR-338-3p, and miR-31.

[0300] According to the aforementioned embodiment, the target sequences or clusters comprising one or more copies thereof are arranged from 5' to 3' such that they form groups according to their target specificity, i.e., 5'-[miR-124 target sequence]4-[miR-338-3p target sequence]4-[miR-31 target sequence]4-3'.

[0301] Both the single target sequence and the cluster of sequences can be adjacent to each other, separated by a spacer sequence, or any combination thereof.

[0302] Thus, in one embodiment, the miRNA target sequences are separated by a spacer sequence.

[0303] In embodiments where the polynucleotide comprises four target sequences for each of miR-124, miR-338-3p, and miR-31, the polynucleotide may comprise the sequence set forth in SEQ ID NO:4.

[0304] Triple miRNA target sequence (Tmir) (SEQ ID NO:4):

[0305]

[0306] miR124 target sequences

[0307] miR338-3p target sequence

[0308]

[0309] In one embodiment, the polynucleotide comprises the sequence shown in SEQ ID NO:4.

[0310] In one embodiment, the polynucleotide consists of the sequence shown in SEQ ID NO:4.

[0311] The polynucleotides and transgene cassettes described herein can be used with any transgene, such as the ESFs described herein.

[0312] miR-124 may, for example, also be referred to as miRNA-124 or miR124; miR-338-3p may also be referred to as miR-338-3p or miRNA338-3p; and miR-31 may also be referred to as miRNA-31 or miR31.

[0313] ESF-containing miRNA cassette

[0314] According to the present invention, miRNA-mediated regulation of transgene expression can be used to control ESF. Therefore, in one aspect, a polynucleotide is provided, which comprises an epigenetic silencing factor (ESF) sequence operably linked to at least one micro RNA (miRNA) target sequence, wherein the ESF comprises a transcription factor DNA binding domain operably linked to at least one epigenetic effector domain, and wherein the transcription factor is an oncogenic transcription factor or a cancer-associated transcription factor.

[0315] In one embodiment, the miRNA target sequence is located downstream, ie, 3', of the ESF coding sequence.

[0316] In one embodiment, the miRNA target sequence is located within the 3'-UTR of the transgene.

[0317] A miRNA target sequence may be represented by one or more copies of a sequence of a given identity. The number of copies of each target sequence may be selected independently, i.e., the number of copies of a given sequence does not necessarily depend on the number of copies of another different sequence.

[0318] Thus, in one embodiment, the one or more miRNA target sequences comprises more than one copy of said miRNA target sequence.

[0319] In one embodiment, the one or more miRNA target sequences comprise one copy of said miRNA target sequence.

[0320] In one embodiment, the one or more miRNA target sequences comprise two copies of said miRNA target sequence.

[0321] In one embodiment, the one or more miRNA target sequences comprises three copies of said miRNA target sequence.

[0322] In one embodiment, the one or more miRNA target sequences comprise four copies of said miRNA target sequence.

[0323] In one embodiment, the one or more miRNA target sequences comprise more than four copies, such as five, six, seven, eight, nine or ten copies of said miRNA target sequence.

[0324] The miRNA target sequence can be designed to be recognized by any suitable miRNA or selected from any suitable known miRNA target sequence, such as a miRNA expressed in a cell type in which transgene expression is undesirable.

[0325] In one embodiment, the miRNA target sequence is a target of a miRNA selected from the group consisting of miR-124, miR-338-3p, and miR-31.

[0326] In one embodiment, the miRNA target sequence is the target site of miR-124.

[0327] In one embodiment, the miRNA target sequence is the target site of miR-338-3p.

[0328] In one embodiment, the miRNA target sequence is the target site of miR-31.

[0329] In one embodiment, the one or more miRNA target sequences comprise a sequence selected from the group consisting of SEQ ID NOs: 1, 2, and 3.

[0330] In one embodiment, the miRNA target sequence comprises a sequence according to SEQ ID NO:1.

[0331] In one embodiment, the miRNA target sequence comprises a sequence according to SEQ ID NO:2.

[0332] In one embodiment, the miRNA target sequence comprises a sequence according to SEQ ID NO:3.

[0333] In one embodiment, the miRNA target sequence comprises target sites for miR-124, miR-338-3p, and miR-31.

[0334] In one embodiment, the miRNA target sequence comprises SEQ ID NOs: 1, 2, and 3.

[0335] In a preferred embodiment, the miRNA target sequence comprises four copies of each of the target sequences for miR-124, miR-338-3p, and miR-31.

[0336] The order of the miRNA target sequences may vary. In one embodiment, the miRNA target sequences or clusters of copies of sequences are arranged from 5' to 3' in the following order: miR-124, miR-338-3p and miR-31.

[0337] In another embodiment, the miRNA target sequence or cluster of copies of the sequence is arranged from 5' to 3' in the following order: miR-124, miR-31, and miR-338-3p.

[0338] In another embodiment, the miRNA target sequence or cluster of copies of sequences are arranged from 5' to 3' in the following order: miR-338-3p, miR-124, and miR-31.

[0339] In another embodiment, the miRNA target sequence or cluster of copies of the sequence is arranged from 5' to 3' in the following order: miR-338-3p, miR-31, and miR-124.

[0340] In another embodiment, the miRNA target sequence or cluster of copies of the sequence is arranged from 5' to 3' in the following order: miR-31, miR-124, and miR-338-3p.

[0341] In another embodiment, the miRNA target sequence or cluster of copies of the sequence is arranged from 5' to 3' in the following order: miR-31, miR-338-3p, and miR-124.

[0342] Both the single target sequence and the cluster of sequences can be adjacent to each other, separated by a spacer sequence, or any combination thereof.

[0343] Thus, in one embodiment, the miRNA target sequences are separated by a spacer sequence.

[0344] In one embodiment, the polynucleotide comprises the sequence according to SEQ ID NO:4.

[0345] Spacer

[0346] As used herein, a "spacer" can be a sequence (eg, a nucleotide or amino acid sequence) that can be used to separate other sequence elements within a larger polymer.

[0347] In one embodiment, one or more spacer sequences separate polynucleotide sequences (eg, miRNA target sequences).

[0348] A single miRNA target sequence or a group of miRNA target sequences can be separated by one or more spacer sequences. In one embodiment, the miRNA target sequences are separated by one or more spacer sequences. The spacer sequence can comprise, for example, at least one, at least two, at least three, at least four or at least five, at least ten, at least twenty or at least thirty nucleotide bases.

[0349] By way of non-limiting example, in the following triple miRNA target sequence, each of the miRNA target sequences is separated by a sequence that may not be considered part of the miRNA target sequence. Such sequences may be considered to be spacers separating functional sequence elements.

[0350] Triple miRNA target sequence (Tmir) (SEQ ID NO:4):

[0351]

[0352] The spacer sequences in SEQ ID NO: 4 include: (a) at; (b) cgatt; (c) gcatt; (d) tcact; (e) cgatcccggggtttaaaccgat (SEQ ID NO: 175); (f) cgat; NO: 176); (i) cgatcctgcaggagatct (SEQ ID NO: 177).

[0353] In one embodiment, the spacer is selected from the group consisting of: (a)-(i).

[0354] In one embodiment, the polynucleotide comprises one or more spacers selected from the group consisting of: (a)-(i).

[0355] In one embodiment, the spacer sequences separating clusters of miRNA target sequences are longer than the spacer sequences separating miRNA target sequences within a cluster.

[0356] genetically modified

[0357] The polynucleotide according to the invention may comprise any suitable transgene. A suitable transgene may be operably linked to a miRNA target sequence according to the invention such that expression of the transgene is dependent on the presence of the miRNA.

[0358] In one embodiment, the transgene is ESF.

[0359] Epigenetic Silencing Factor (ESF)

[0360] The present invention provides polynucleotides encoding polypeptide transcription factor DNA binding domains, which are operably connected to at least one epigenetic effector domain, preferably wherein the transcription factor is an oncogenic transcription factor or a cancer-associated transcription factor. The polypeptide can be used to reduce the transcription and / or expression of one or more target genes. The polypeptide or the transgenic encoding the polypeptide can be referred to as an epigenetic silencing factor (ESF). The polypeptide can be a multimeric polypeptide, for example including two, three or more polypeptide chains. For example, the polypeptide can be a dimer, such as a heterodimer. The polypeptide can include a single polypeptide chain. The polypeptide can be a fusion protein.

[0361] ESF is a reagent that can reduce the transcription and / or expression of one or more target genes (e.g., silence one or more target genes). The ESF of the present invention can include at least a portion of a transcription factor that is bound to DNA, wherein the portion is operably connected to an epigenetic effector domain. The effector domain can have transcription repression activity and can silence (e.g., permanently silence) one or more target genes of the transcription factor. Specifically, when the transcription factor is an oncogenic transcription factor or a cancer-associated transcription factor, the ESF can block the gene expression cascade involved in tumor growth.

[0362] ESF can be a chimeric protein or a fusion protein comprising a DNA binding domain operably linked to an effector domain (e.g., a KRAB domain, a Chromo Shadow domain, a YAF2-RYBP domain, a DNMT3A domain, and / or a DNMT3L domain). The effector domain can have a catalytic activity that suppresses the transcription and / or expression of one or more target genes. Alternatively or additionally, the effector domain can recruit additional agents within the cell to one or more target genes, which can suppress the transcription and / or expression of the target genes.

[0363] "Operably linked" is understood to mean that the individual components are linked together in a manner that enables them to perform their functions (e.g., bind DNA, catalyze a reaction, or recruit additional agents from within a cell) substantially unhindered. For example, a DNA binding domain may be conjugated to an effector domain, e.g., to form a fusion protein. Methods for conjugating polypeptides are known in the art, e.g., by providing a linker amino acid sequence (e.g., a linker comprising glycine and / or serine residues) that connects the polypeptides. Alternative methods for conjugating polypeptides known in the art include chemical and light-induced conjugation methods (e.g., using chemical cross-linking agents). Preferably, the DNA binding domain and the effector domain of the ESF form a fusion protein.

[0364] In some embodiments, the ESF is a fusion protein comprising a transcription factor DNA binding domain and at least one epigenetic effector domain.

[0365] ESF can be formed by separate polypeptide chains that bind together to form a complex, such as a heterodimeric complex. For example, binding can be achieved by an epitope (e.g., Suntag) contained on a first chain and an epitope-binding molecule (such as a single-chain variable fragment (scFv)) contained on a second chain.

[0366] In some embodiments, the ESF comprises two epigenetic effector domains, e.g., fused to the same DNA binding domain. In some embodiments, the ESF comprises three epigenetic effector domains, e.g., fused to the same DNA binding domain. The ESF may comprise four, five, six or more epigenetic effector domains, e.g., fused to the same DNA binding domain.

[0367] When the ESF comprises more than one epigenetic effector domain, the effector domains may be different.When the ESF comprises more than one epigenetic effector domain, the effector domains may be the same.

[0368] In preferred embodiments, the ESF comprises a KRAB domain, a DNMT3A domain and / or a DNMT3L domain.

[0369] In other preferred embodiments, the ESF comprises a Chromo Shadow domain, a YAF2-RYBP domain, a DNMT3A domain and / or a DNMT3L domain.

[0370] Epigenetic effector domain

[0371] The term "epigenetic effector domain" is understood to refer to a portion of an ESF that provides an epigenetic effect on a target gene, for example by catalyzing a reaction on DNA or chromatin (e.g. methylation of DNA, methylation or acetylation of histones, or demethylation or deacetylation of histones), or by recruiting additional agents, leading to repression of gene transcription.

[0372] "Domain" is understood herein to refer to a portion of an ESF that has a specific function. The domain can be a single domain isolated from a native protein (e.g., a catalytic domain), or it can be an entire, full-length native protein. In other words, the full-length protein or a functional fragment thereof can be used as an effector domain. Thus, for example, a "KRAB domain" can refer to a portion of an ESF that comprises an amino acid sequence that has the function of a KRAB domain.

[0373] Chromatin remodeling enzymes known to be involved in permanent epigenetic silencing of endogenous retroviruses (ERVs; Feschotte, C. et al. (2012) Nat. Rev. Genet. 13:283-96; Leung, DC et al. (2012) Trends Biochem. Sci. 37:127-33) may provide effector domains suitable for development in the present invention.

[0374] In some embodiments, the epigenetic effector domain represses transcription and / or expression of at least one target gene. In some embodiments, the epigenetic effector domain is a repressor domain.

[0375] In some embodiments, the epigenetic effector domain catalyzes chemical modification of chromatin and / or chromatin remodeling.

[0376] In some embodiments, the epigenetic effector domain catalyzes a DNA modification, such as DNA methylation. In some embodiments, the epigenetic effector domain is a DNA methyltransferase and / or is capable of recruiting a DNA methyltransferase.

[0377] In some embodiments, the epigenetic effector domain catalyzes histone modifications, such as histone methylation or histone acetylation. In some embodiments, the epigenetic effector domain is a histone methyltransferase or a histone acetyltransferase. In some embodiments, the epigenetic effector domain catalyzes histone demethylation or histone deacetylation. In some embodiments, the epigenetic effector domain is a histone methylase or a histone acetylase.

[0378] Krüppel-associated box (KRAB) domain

[0379] The Krüppel-associated box family containing zinc finger proteins (KRAB-ZFP; Huntley, S. et al. (2006) Genome Res. 16: 669-77) plays an important role in the silencing of endogenous retroviruses. These transcription factors bind to specific ERV sequences through their ZFP DNA binding domains, while recruiting KRAB-associated protein 1 (KAP1) using their conserved KRAB domains. KAP1 in turn binds to a large number of effectors that promote the local formation of repressive chromatin (Iyengar, S. et al. (2011) J. Biol. Chem. 286: 26267-76). For example, they may induce repressive chromatin modifications (e.g., H3K9me3) and / or remove active marks (e.g., H3K4ac).

[0380] In some embodiments, the ESF comprises a KRAB domain.

[0381] There are many known KRAB domains in the KRAB-ZFP protein family. For example, the ESF of the present invention may comprise the KRAB domain of human zinc finger protein 10 (ZNF10; Szulc, J. et al. (2006) Nat. Methods 3: 109-16). An exemplary sequence of the KRAB domain of human zinc finger protein 10 is:

[0382] ALSPQHSAVTQGSIIKNKEGMDAKSLTAWSRTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGYQLTKPDVILRLEKGEEPWLVEREIHQETHPDSETAFEIKSSV

[0383] (SEQ ID NO:5)

[0384] Further examples of KRAB domains suitable for use in the present invention include:

[0385] MNNSQGRVTFEDVTVNFTQGEWQRLNPEQRNLYRDVMLENYSNLVSVGQGETTKPDVILRLEQGKEPWLEEEEVLGSGRAEKNGDIGGQIWKPKDVKESL

[0386] (KRAB domain of ZIM3 protein; SEQ ID NO 6)

[0387] ITLEDVAVDFTWEEWQLLGAAQKDLYRDVMLENYSNLVAVGYQASKPDALFKLEQGEQLWTIEDGIHSGACS

[0388] (KRAB domain of ZNF350 protein; SEQ ID NO: 7)

[0389] VMFEEVSVCFTSEEWACLGPIQRALYWDVMLENYGNVTSLEWETMTENEEVTSKPSSSQRADSHKGTSKRLQG

[0390] (KRAB domain of ZNF197 protein; SEQ ID NO: 8)

[0391] VSFKDVAVDFTQEEWQQLDPDEKITYRDVMLENYSHLVSVGYDTTKPNVIIKLEQGEEPWIMGGEFPCQHSP

[0392] (KRAB domain of RBAK protein; SEQ ID NO: 9)

[0393] VKIEDMAVSLILEEWGCQNLARRNLSRDNRQENYGSAFPQGGENRNENEESTSKAETSEDSASRGETTGRSQKE

[0394] (KRAB domain of ZKSCAN1 protein; SEQ ID NO: 10)

[0395] LTFKDVFVDFTLEEWQQLDSAQKNLYRDVMLENYSHLVSVGYLVAKPDVIFRLGPGEESWMADGGTPVRTCA

[0396] (KRAB domain of KRBOX4 protein; SEQ ID NO: 11)

[0397] VTFEDVTLGFTPEEWGLLDLKQKSLYREVMLENYRNLVSVEHQLSKPDVVSQLEEAEDFWPVERGIPQDTIP

[0398] (KRAB domain of ZNF274 protein; SEQ ID NO: 12)

[0399] An exemplary nucleotide sequence encoding a KRAB domain is:

[0400]

[0401] (SEQ ID NO:13)

[0402] DNA methyltransferase (DNMT) domain

[0403] In some embodiments, the ESF comprises a DNA methyltransferase (DNMT) domain. In some embodiments, the ESF comprises a DNMT3A domain, a DNMT3B domain, and / or a DNMT1 domain. In some embodiments, the ESF comprises a DNMT3A domain.

[0404] The ESF of the present invention may, for example, comprise a domain, preferably a catalytic domain, of human DNA methyltransferase 3A (DNMT3A; Law, JA et al. (2010) Nat. Rev. Genet. 11:204-20). An exemplary DNMT3A sequence is:

[0405]

[0406]

[0407] (SEQ ID NO:14)

[0408] Similar to DNMT3A, DNA methyltransferase 3B and DNA methyltransferase 1 (DNMT3B and DNMT1) are also responsible for the deposition and maintenance of DNA methylation and can also be used in the ESF of the present invention. Example sequences are:

[0409]

[0410] (Catalytic domain of human DNMT3B; SEQ ID NO: 15)

[0411]

[0412]

[0413] (DNMT3B: SEQ ID NO: 16)

[0414]

[0415] (Catalytic domain of human DNMT1; SEQ ID NO: 17)

[0416] DNMT-like domain

[0417] In some embodiments, the ESF comprises a DNMT-like domain. A "DNMT-like" domain refers to a protein that is a member of the DNMT family but does not have DNA methylation activity. DNMT-like proteins typically activate or recruit other epigenetic effector domains.

[0418] For example, the ESF of the invention may comprise DNA (cytosine-5)-methyltransferase 3-like (DNMT3L), which is a catalytically inactive DNA methyltransferase that activates DNMT3A by binding to the catalytic domain of DNMT3A. An exemplary DNMT3L sequence is:

[0419]

[0420] (SEQ ID NO:18)

[0421] The DNMT3A and DNMT3L domains may be used together and are referred to herein as "DNMT3A / 3L" or "DNMT3a3L".

[0422] An exemplary nucleotide sequence encoding the DNMT3A and DNMT3L domains is:

[0423]

[0424]

[0425] (SEQ ID NO:19)

[0426] Chromo Shadow(CS) domain

[0427] In some embodiments, the ESF comprises a Chromo Shadow (CS) domain. The CS domain may be the CS domain of CBX5.

[0428] An example CS domain sequence is:

[0429] MLEPEKIIGATDSCGDLMFLMKWKDTDEADLVLAKEANVKCPQIVIAFYEERLTWHAYPE

[0430] (SEQ ID NO:20)

[0431] An exemplary nucleotide sequence encoding a CS domain is:

[0432]

[0433] (SEQ ID NO:21)

[0434] YAF2-RYBP (YR) domain

[0435] In some embodiments, the ESF comprises a YAF2-RYBP (YR) domain.

[0436] An example YR domain sequence is:

[0437] MRPRLKNVDRSTAQQLAVTVGNVTVIITDFKEKTRSSSSSSTVTSSAGSEQQNQ

[0438] (SEQ ID NO:22)

[0439] An exemplary nucleotide sequence encoding a YR domain is:

[0440]

[0441] (SEQ ID NO:23)

[0442] Other epigenetic effector domains

[0443] Other example sequences of suitable epigenetic effector domains are:

[0444]

[0445] Exemplary nucleotide sequences encoding other suitable epigenetic effector domains are:

[0446]

[0447]

[0448]

[0449] The ESF of the present invention may, for example, comprise an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 5-12, 14-18, 20, 22 or 24-27, preferably wherein the amino acid sequence substantially retains the native function of the protein represented by SEQ ID NOs: 5-12, 14-18, 20, 22 or 24-27, respectively.

[0450] The ESF of the present invention can, for example, be encoded by a polynucleotide comprising a nucleic acid sequence encoding a peptide having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid identity with any one of SEQ ID NOs: 5-12, 14-18, 20, 22 or 24-27, preferably wherein the amino acid sequence substantially retains the native function of the protein represented by SEQ ID NOs: 5-12, 14-18, 20, 22 or 24-27, respectively.

[0451] The polynucleotides of the present invention may include a nucleic acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with any one of SEQ ID NO: 13, 19, 21, 23 or 28-32, preferably wherein the encoded amino acid sequence substantially retains the native function of the protein encoded by SEQ ID NO: 13, 19, 21, 23 or 28-32, respectively.

[0452] Transcription Factors

[0453] Transcription factors (TFs) can control the transcription of DNA by binding to certain DNA sequences. Transcription factors generally function as regulators of genes to control transcription and / or expression, for example depending on cell type and time. Groups of transcription factors can function in a coordinated manner to direct cell division, cell growth, and cell death; cell migration and organization during embryonic development; and in response to extracellular signals.

[0454] Transcription factors contain at least one DNA binding domain, which can target the transcription factor to certain sequences to direct its regulatory function.

[0455] The ESF of the present invention comprises at least one transcription factor DNA binding domain. A skilled person can easily identify a DNA binding domain from a transcription factor using well-known methods, for example using sequence comparison tools and / or databases.

[0456] The polynucleotides, polypeptides and ESFs of the present invention may contain minimal transcription factor sequences that retain their function of binding to DNA. However, it is preferred that as much transcription factor sequence as possible is retained in the polypeptides and ESFs of the present invention without adversely affecting the function of the ESF to reduce transcription and / or expression. Without wishing to be bound by theory, transcription factor sequences other than the DNA binding domain may enable the recruitment of additional factors within the cell.

[0457] Unmodified transcription factors may contain an activation domain (AD; also called a transactivation domain), which may function to activate gene transcription and / or expression. The polypeptides and ESFs of the present invention preferably do not contain a functional activation domain. The transcription factor sequence may be modified (e.g., mutated or truncated) to disrupt activation domain function. The transcription factor sequence incorporated into a polypeptide or ESF may lack an activation domain.

[0458] In a preferred embodiment, the ESF does not comprise a functional transcription factor activation domain. For example, the ESF may not comprise a transcription factor activation domain. The ESF may, for example, comprise a transcription factor fragment lacking a functional transcription activation domain and comprising a functional DNA binding domain.

[0459] In a preferred embodiment, the transcription factor is an oncogenic transcription factor.

[0460] As used herein, the term "oncogenic transcription factor" refers to a transcription factor that can transform healthy cells into cancer cells, for example by causing inappropriate gene expression patterns, which can, for example, promote tumor initiation and progression.

[0461] In some embodiments, the transcription factor is a cancer-associated transcription factor.

[0462] As used herein, the term "cancer-associated transcription factor" refers to a transcription factor that can induce tumorigenic properties but cannot transform healthy cells into cancer cells.

[0463] In some embodiments, the transcription factor is selected from the group consisting of SOX2, MYC, MYCN, TEAD1, TEAD2, TEAD3, TEAD4, FOXA1, FOXA2, ELK1, ELK3, ELK4, SRF, FOXM1, FOXC1, FOXC2, TWIST1, SALL4, ELF1, HIF1A, SOX9, SOX12, SOX18, ETS1, PAX3, PAX8, GLI1, GLI2, GLI3, ETV1, ETV2, ETV3, RUNX1, RUNX2, RUNX3, MAFB, TFAP2C, and E2F1.

[0464] In some embodiments, the transcription factor is SOX2. The ESF may comprise a fragment of human SOX2 consisting of amino acids 1-179.

[0465] In some embodiments, the transcription factor is MYC. The ESF may comprise a fragment of human MYC consisting of amino acids 144-454.

[0466] In some embodiments, the transcription factor is TEAD1. The ESF may comprise a fragment of human TEAD1 consisting of amino acids 1-166.

[0467] An exemplary sequence comprising a suitable transcription factor DNA binding domain is:

[0468]

[0469]

[0470]

[0471]

[0472]

[0473]

[0474]

[0475]

[0476]

[0477]

[0478]

[0479]

[0480]

[0481]

[0482] An exemplary nucleotide sequence encoding a polypeptide comprising a transcription factor DNA binding domain is:

[0483]

[0484]

[0485]

[0486]

[0487]

[0488]

[0489]

[0490]

[0491]

[0492]

[0493]

[0494]

[0495]

[0496]

[0497]

[0498]

[0499]

[0500]

[0501]

[0502]

[0503]

[0504]

[0505]

[0506]

[0507]

[0508]

[0509]

[0510]

[0511]

[0512]

[0513]

[0514]

[0515]

[0516]

[0517]

[0518]

[0519]

[0520]

[0521]

[0522]

[0523]

[0524]

[0525]

[0526]

[0527]

[0528]

[0529]

[0530]

[0531]

[0532]

[0533]

[0534]

[0535]

[0536]

[0537]

[0538]

[0539]

[0540]

[0541]

[0542]

[0543]

[0544] The transcription factor DNA binding domain may, for example, comprise an amino acid sequence that is at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of SEQ ID NOs: 33-70, preferably wherein the amino acid sequence substantially retains the native function of the protein represented by SEQ ID NOs: 33-70, respectively.

[0545] The transcription factor DNA binding domain can, for example, be encoded by a polynucleotide comprising a nucleic acid sequence encoding a peptide having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid identity with any one of SEQ ID NOs: 33-70, preferably wherein the amino acid sequence substantially retains the native function of the protein represented by SEQ ID NOs: 33-70, respectively.

[0546] The transcription factor DNA binding domain can, for example, be encoded by a polynucleotide comprising a nucleic acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOs:71-108, preferably wherein the encoded amino acid sequence substantially retains the native function of the protein encoded by SEQ ID NOs:71-108, respectively.

[0547] Exemplary ESF

[0548] In some embodiments, the ESF comprises or consists of a KRAB domain, a SOX2 DNA binding domain, a DNMT3A domain, and a DNMT3L domain.

[0549] In some embodiments, the ESF comprises or consists of a CS domain and a SOX2 DNA binding domain.

[0550] In some embodiments, the ESF comprises or consists of a SOX2 DNA binding domain and a YR domain.

[0551] In some embodiments, the ESF comprises or consists of a CS domain, a SOX2 DNA binding domain, and a YR domain.

[0552] In some embodiments, the ESF comprises or consists of a KRAB domain, a TEAD1 DNA binding domain, a DNMT3A domain, and a DNMT3L domain.

[0553] In some embodiments, the ESF comprises or consists of a KRAB domain, a DNMT3A domain, a DNMT3L domain, and a MYC DNA binding domain.

[0554] An exemplary sequence of an ESF of the present invention is:

[0555] KRAB-hSOX2 1-179 -DNMT3a3L

[0556]

[0557]

[0558] (SEQ ID NO: 109)

[0559] KRAB

[0560] hSOX2 1-179

[0561] DNMT3a3L CS-hSOX2 1-179

[0562]

[0563] (SEQ ID NO:110)

[0564] CS

[0565] hSOX2 1-179 SOX2 1-179 -YR

[0566]

[0567] (SEQ ID NO:111)

[0568] hSOX2 1-179

[0569] Y

[0570] KRAB-hTEAD 1-166 -DNMT3a3L

[0571]

[0572] (SEQ ID NO:112)

[0573] KRAB

[0574] hTEAD1 1-166

[0575] DNMT3a3L

[0576] KRAB-DNMT3a3L-hMYC 144-454

[0577]

[0578] (SEQ ID NO:113)

[0579] KRAB

[0580] DNMT3a3L

[0581] hMYC 144-454

[0582] An exemplary nucleotide sequence encoding an ESF of the present invention is

[0583] KRAB-hSOX2 1-179 -DNMT3a3L

[0584]

[0585]

[0586] (SEQ ID NO:114)

[0587] KRAB

[0588] hSOX2 1-179

[0589] DNMT3a3L

[0590] KRAB-hSOX2 1-179 -DNMT3a3L

[0591]

[0592]

[0593]

[0594] (SEQ ID NO:115)

[0595] KRAB

[0596] hSOX2 1-179

[0597] <h2 style=";text-align:left;direction:ltr"> DNMT3a3L <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0598] <h2 style=";text-align:left;direction:ltr"> CS-hSOX2<h2 style=";text-align:left;direction:ltr"> 1-179 <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0599] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0600] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0601] <h2 style=";text-align:left;direction:ltr"> (SEQ ID NO:116)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0602] <h2 style=";text-align:left;direction:ltr"> CS <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0603] <h2 style=";text-align:left;direction:ltr"> hSOX2<h2 style=";text-align:left;direction:ltr"> 1-179 <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0604] <h2 style=";text-align:left;direction:ltr"> SOX2<h2 style=";text-align:left;direction:ltr"> 1-179 <h2 style=";text-align:left;direction:ltr"> -YR<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0605] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0606] <h2 style=";text-align:left;direction:ltr"> (SEQ ID NO:117)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0607] <h2 style=";text-align:left;direction:ltr"> hSOX2<h2 style=";text-align:left;direction:ltr"> 1-179 <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0608] <h2 style=";text-align:left;direction:ltr"> Y <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0609] <h2 style=";text-align:left;direction:ltr"> KRAB-hTEAD<h2 style=";text-align:left;direction:ltr"> 1-166 <h2 style=";text-align:left;direction:ltr"> -DNMT3a3L<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0610] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0611] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0612] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0613] <h2 style=";text-align:left;direction:ltr"> (SEQ ID NO:118)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0614] <h2 style=";text-align:left;direction:ltr"> KRAB <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0615] <h2 style=";text-align:left;direction:ltr"> hTEAD<h2 style=";text-align:left;direction:ltr"> 1-166 <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0616] <h2 style=";text-align:left;direction:ltr"> DNMT3a3L <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0617] <h2 style=";text-align:left;direction:ltr"> KRAB-DNMT3a3L-hMYC<h2 style=";text-align:left;direction:ltr"> 144-454 <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0618] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0619] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0620] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0621] (SEQ ID NO:119)

[0622] KRAB

[0623] DNMT3a3L HkDJ 144-454

[0624] The ESF may, for example, comprise or consist of an amino acid sequence that is at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of SEQ ID NOs: 109-113, preferably, wherein the amino acid sequence substantially retains the native function of the protein represented by SEQ ID NOs: 109-113, respectively.

[0625] The ESF may, for example, be encoded by a polynucleotide comprising or consisting of a nucleic acid sequence encoding a protein having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid identity with any one of SEQ ID NOs: 109-113, preferably, wherein the amino acid sequence substantially retains the native function of the protein represented by SEQ ID NOs: 109-113, respectively.

[0626] The ESF may, for example, be encoded by a polynucleotide comprising a nucleic acid sequence or a fragment thereof having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide identity to any one of SEQ ID NOs: 114-119, preferably, wherein the encoded amino acid sequence substantially retains the native function of the protein encoded by SEQ ID NOs: 114-119, respectively.

[0627] A further example sequence of an ESF of the invention is:

[0628] CS-hTEAD 1-166 [TESv2]

[0629]

[0630] (SEQ ID NO: 120)

[0631] CS

[0632] hTEAD 1-166

[0633] hTEAD 1-166 -YR[TESv3]

[0634]

[0635] (SEQ ID NO:121)

[0636] hTEAD 1-166

[0637] Y-R

[0638] CS-hTEAD 1-166 –Y-R[TESv4]

[0639]

[0640] (SEQ ID NO:122)

[0641] CS

[0642] hTEAD 1-166

[0643] y-r

[0644] CS-hMYC 144-454 [MESv2]

[0645]

[0646] (SEQ ID NO:123)

[0647] CS

[0648] hMYC 144-454

[0649] YR-hMYC 144-454 [MESv3]

[0650]

[0651] (SEQ ID NO:124)

[0652] Y

[0653] hMYC 144-454

[0654] CS-YR-hMYC 144-454 [MESv4]

[0655]

[0656]

[0657] (SEQ ID NO:125)

[0658] CS

[0659] yr

[0660] HkDJ 144-454

[0661] Other exemplary nucleotide sequences encoding ESF of the present invention are:

[0662] CS-hTEAD 1-166

[0663]

[0664] (SEQ ID NO:126)

[0665] CS

[0666] hTEAD 1-166

[0667] hTEAD 1-166 -YR

[0668]

[0669]

[0670] (SEQ ID NO: 127)

[0671] hTEAD 1-166

[0672] Y

[0673] CS-hTEAD 1-166 –YR

[0674]

[0675] (SEQ ID NO:128)

[0676] CS

[0677] hTEAD 1-166

[0678] yr

[0679] CS-hMYC 144-454

[0680]

[0681] (SEQ ID NO:129)

[0682] CS

[0683] HkDJ 144-454

[0684] YR-hMYC 144-454

[0685]

[0686] (SEQ ID NO:130) Y HkDJ 144-454 CS-YR-hMYC 144-454

[0687]

[0688]

[0689] (SEQ ID NO:131)

[0690] CS

[0691] yr

[0692] HkDJ 144-454

[0693] The ESF may, for example, comprise or consist of an amino acid sequence that is at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of SEQ ID NOs: 120-125, preferably, wherein the amino acid sequence substantially retains the native function of the protein represented by SEQ ID NOs: 120-125, respectively.

[0694] The ESF may, for example, be encoded by a polynucleotide comprising or consisting of a nucleic acid sequence encoding a peptide having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid identity with any one of SEQ ID NOs: 120-125, preferably wherein the amino acid sequence substantially retains the native function of the protein represented by SEQ ID NOs: 120-125, respectively.

[0695] ESF can be encoded, for example, by a polynucleotide comprising a nucleic acid sequence or a fragment thereof having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide identity to any one of SEQ ID NOs: 126-131, preferably, wherein the encoded amino acid sequence substantially retains the native function of the protein encoded by SEQ ID NOs: 126-131, respectively.

[0696] Label

[0697] The polynucleotides of the invention may encode a tag, such as a polypeptide tag. The tag may be selected depending on the intended purpose of the polynucleotide in question.

[0698] In one embodiment, the polynucleotide according to the present invention further comprises a tag.

[0699] In one embodiment, the tag is a V5 tag.

[0700] The ESF of the present invention may have a polypeptide tag.

[0701] In one embodiment, the polypeptide tag is a V5 tag.

[0702] Example V5 tag (SEQ ID NO: 132): GKPIPNPLLGLDST

[0703] In one embodiment, the polypeptide tag comprises or consists of SEQ ID NO:132.

[0704] The polypeptide tag can be separated from the ESF sequence by a spacer.

[0705] In one embodiment, the polypeptide tag is a C-terminal tag.

[0706] In one embodiment, the polypeptide tag has an N-terminal spacer sequence between the tag and the ESF.

[0707] Example V5 tag and spacer (SEQ ID NO: 133): gap GKPIPNPLLGLDST

[0708] It will be appreciated that such tags may not be advantageous in certain contexts, but may be advantageous in other contexts. For example, a tag may be advantageous for in vitro testing, but may not be desirable in a polynucleotide being considered for medical use.

[0709] In one embodiment, the ESF of the invention does not comprise a polypeptide tag, such as a V5 tag. In one embodiment, the invention provides a sequence obtained from any sequence described herein comprising a V5 tag by deleting the V5 tag (and optionally any spacer sequence between the V5 tag and the ESF).

[0710] The aforementioned ESF can be advantageously used in combination with any of the miRNA target sequence cassettes of the present invention. In addition, the aforementioned ESF, whether or not comprised in a polynucleotide or cassette according to the present invention, can be advantageously used in the treatment of cancer according to the present invention.

[0711] Expression control sequences

[0712] The polynucleotides of the invention may comprise one or more expression control sequences. Suitably, the nucleic acid sequence encoding the ESF or transgene is operably linked to one or more expression control sequences.

[0713] As used herein, an "expression control sequence" is any nucleotide sequence that controls the expression of a transgene, eg, promotes and / or increases expression in certain cell types and / or decreases expression in other cell types.

[0714] The expression control sequence and the transgene (eg, a nucleic acid sequence encoding ESF) can be in any suitable arrangement in the polynucleotide, so long as the expression control sequence is operably linked to the transgene (eg, a nucleic acid sequence encoding ESF).

[0715] Promoter

[0716] In some embodiments, the expression control sequence is a promoter.

[0717] Any suitable promoter can be used, and the skilled person can easily select. The promoter sequence can be constitutively active (i.e., running in any host cell background), or can be active only in a specific host cell environment, thereby allowing the targeted expression of the nucleotide of interest (e.g., ESF) in a specific cell type (e.g., tissue-specific promoter). The promoter can show inducible expression in response to the presence of another factor (e.g., a factor present in the host cell). In any case, in the case of administering the vector for therapy, it is preferred that the promoter should function in the target cell (e.g., cancer cell) background.

[0718] In some embodiments, the polynucleotide further comprises a promoter operably linked to the transgene. In some embodiments, the polynucleotide further comprises a promoter operably linked to the nucleic acid sequence encoding ESF.

[0719] In some embodiments, the promoter is a constitutive promoter. In some embodiments, the constitutive promoter is the Ef1a promoter.

[0720] An example sequence for the Ef1a promoter is:

[0721] CGATAAGCTTTGCAAAGATGGATAAAGTTTTAAACAGAGAGGAA

[0722] TCTTTGCAGCTAATGGACCTTCTAGGTCTTGAAAGGAGTGGGAAT

[0723] TGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAG

[0724] TCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCC

[0725] TAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTA

[0726] CTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAA

[0727] GTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCG

[0728] CCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGC

[0729] CTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACTG

[0730] GCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGG

[0731] GTGGGAGAGTTCGAGGCCTTGCCGCTTAAGGAGCCCCTTCGCCTC

[0732]

[0733] (SEQ ID NO:134)

[0734] In some embodiments, the promoter is a tissue-specific promoter, preferably a cancer cell-specific promoter.

[0735] In some embodiments, the promoter is a proliferating cell-specific promoter.

[0736] In some embodiments, the promoter is selected from the group consisting of Mki67 promoter, Ccnd1 promoter, Ccnb2 promoter, Ccna2 promoter, Cdc25c promoter, Cdc2 promoter, Cks1 promoter, PCNA promoter, CDC6 promoter, POLD1 promoter, CSK1B promoter, MCM2 promoter and PLK1 promoter.

[0737] In some embodiments, the promoter is the Mki67 promoter.

[0738] An example sequence for the Mki67 promoter is:

[0739]

[0740] (SEQ ID NO:135)

[0741] The promoter may, for example, comprise or consist of a nucleic acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NO: 134 or 135; preferably wherein the promoter substantially retains the native function of the promoter of SEQ ID NO: 134 or 135, respectively.

[0742] In some embodiments, the promoter is the Ef1a promoter. An exemplary polynucleotide sequence comprising the EF1a promoter operably linked to a transgene encoding ESF is as follows:

[0743] Ef1a::CS-hTEAD 1-166 (SEQ ID NO:158)

[0744]

[0745]

[0746]

[0747] Ef1a promoter

[0748] CS

[0749] hTEAD 1-166

[0750] Ef1a::hTEAD 1-166 -YR (SEQ ID NO: 159)

[0751]

[0752]

[0753] Ef1a promoter

[0754] hTEAD 1-166

[0755] Y

[0756] Ef1a::CS-hTEAD 1-166 –YR (SEQ ID NO: 160)

[0757]

[0758]

[0759]

[0760] Ef1a promoter

[0761] CS

[0762] hTEAD 1-166

[0763] yr

[0764] Ef1a::CS-hMYC 144-454 (SEQ ID NO: 161)

[0765]

[0766]

[0767]

[0768] Ef1a promoter

[0769] CS

[0770] HkDJ 144-454

[0771] Ef1a::YR-hMYC 144-454 (SEQ ID NO: 162)

[0772]

[0773]

[0774] Ef1a promoter

[0775] Y

[0776] HkDJ 144-454

[0777] Ef1a::CS-YR-hMYC 144-454 (SEQ ID NO: 163)

[0778]

[0779]

[0780] Ef1a promoter

[0781] CS

[0782] yr

[0783] HkDJ 144-454

[0784] Ef1a::KRAB-hTEAD 1-166 – DNMT3A / L (SEQ ID NO: 178)

[0785]

[0786]

[0787]

[0788] Ef1a promoter

[0789] KRAB

[0790] hTEAD 1-166

[0791] DNMT3a3L

[0792] Ef1a::KRAB-DNMT3A / L-hMYC 144-454 (SEQ ID NO: 179)

[0793]

[0794]

[0795]

[0796]

[0797] Ef1a promoter

[0798] KRAB

[0799] DNMT3a3L

[0800] HkDJ 144-454

[0801] Ef1a::KRAB-hSOX2 1-179 – DNMT3A / L (SEQ ID NO: 180)

[0802]

[0803]

[0804]

[0805]

[0806] Ef1a promoter

[0807] KRAB

[0808] hSOX2 1-179

[0809] DNMT3a3L

[0810] Ef1a::CS-hSOX2 1-179 (SEQ ID NO: 181)

[0811]

[0812]

[0813]

[0814] Ef1a promoter

[0815] CS

[0816] hSOX2 1-179

[0817] Ef1a::SOX2 1-179 -YR (SEQ ID NO: 182)

[0818]

[0819]

[0820] Ef1a promoter

[0821] hSOX2 1-179

[0822] Y

[0823] In one embodiment, the polynucleotide comprises a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 158-163, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

[0824] In one embodiment, the polynucleotide comprises a sequence according to any one of SEQ ID NOs: 158-163.

[0825] In one embodiment, the polynucleotide comprises a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 178-182, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

[0826] In one embodiment, the polynucleotide comprises a sequence according to any one of SEQ ID NOs: 178-182.

[0827] miRNA target sequences

[0828] In some embodiments, the polynucleotide further comprises one or more miRNA target sequences. Suitably, the nucleic acid sequence encoding the transgene (eg, ESF) is operably linked to one or more miRNA target sequences.

[0829] MicroRNA (miRNA) genes are scattered on all human chromosomes except the Y chromosome. They can be located in non-coding regions of the genome or in introns of protein-coding genes. About 50% of miRNAs appear in clusters and are transcribed as polycistronic primary transcripts. Similar to protein-coding genes, miRNAs are usually transcribed by polymerase II promoters to generate so-called primary miRNA transcripts (pri-miRNAs). The pri-miRNA is then processed through a series of endonucleolytic cleavage steps, which are performed by two enzymes Drosha and Dicer belonging to the RNAse type III family. From the pri-miRNA, a stem loop of about 60 nucleotides in length, called the miRNA precursor (pre-miRNA), is removed by a specific nuclear complex composed of Drosha and DiGeorge syndrome critical region gene (syndrome critical region gene) (DGCR8), which cuts the two chains close to the base of the primary stem loop and leaves a 5' phosphate and a 2bp long 3' overhang. The pre-miRNA is then actively transported from the nucleus to the cytoplasm by RAN-GTP and nuclear export protein (Exportin). Dicer then performs double-stranded cleavage at the undefined stem-loop end of Drosha cleavage to generate a 19-24bp duplex consisting of a mature miRNA and the opposite strand of the duplex (called miRNA*). Consistent with the thermodynamic asymmetry rule, only one strand of the duplex is selectively loaded into the RNA-induced silencing complex (RISC) and accumulated as a mature microRNA. This strand is usually the strand whose 5' end is not tightly paired with its complementary strand, as evidenced by the single nucleotide mismatch introduced at the 5' end of each strand of the siRNA duplex. However, some miRNAs support the accumulation of both duplexes to a similar extent.

[0830] MicroRNAs trigger RNAi, much like small interfering RNAs (siRNAs) widely used for experimental gene knockdown. The main difference between miRNAs and siRNAs is their biogenesis. Once loaded into RISC, the guide strand of the small RNA molecule interacts with the mRNA target sequence that is preferentially found in the 3' untranslated region (3'UTR) of protein-coding genes. The 2nd to 8th nucleotides from the 5' end of the miRNA, the so-called seed sequence, have been shown to be essential for triggering RNAi. If the entire guide strand sequence is fully complementary to the mRNA target, which is usually the case for siRNAs and plant miRNAs, the mRNA is endonucleolytically cleaved into the RNA-induced silencing complex (RISC) by the involvement of Argonaute (Ago) proteins, also known as "slicers" of small RNA duplexes. DGCR (DiGeorge syndrome critical region gene 8) and TRBP (TAR (HIV) RNA binding protein 2) are double-stranded RNA binding proteins that promote the biogenesis of mature miRNAs through the Drosha and Dicer RNase III enzymes, respectively. The guide strand of the miRNA duplex is incorporated into the effector complex RISC, which recognizes specific targets through imperfect base pairing and induces posttranscriptional gene silencing. Several mechanisms have been proposed for this mode of regulation: miRNAs can induce repression of translation initiation by marking target mRNAs for degradation through deadenylation or by sequestering targets into cytoplasmic P bodies.

[0831] On the other hand, if only the seed is fully complementary to the target mRNA, but the remaining bases show incomplete pairing, RNAi can act through multiple mechanisms, leading to translational repression. Eukaryotic mRNA degradation occurs primarily through shortening of the polyA tail at the 3' end of the mRNA, as well as decapping at the 5' end, followed by 5'-3' exonuclease digestion and accumulation of miRNAs in discrete cytoplasmic regions, so-called P bodies, which are enriched among the components of the mRNA decay pathway.

[0832] The expression of a nucleic acid sequence encoding a transgene (e.g., ESF) can be regulated by one or more endogenous miRNAs using one or more corresponding miRNA target sequences. Using this method, one or more miRNAs endogenously expressed by a cell prevent or reduce transgene expression in the cell by binding to its corresponding miRNA target sequence located in a polynucleotide or vector.

[0833] The target sequence can be completely or partially complementary to the miRNA. As used herein, the term "completely complementary" can refer to a target sequence having a nucleic acid sequence that is 100% complementary to the sequence of the miRNA that recognizes it. As used herein, the term "partially complementary" can refer to a target sequence that is only partially complementary to the sequence of the miRNA that recognizes it, whereby the partially complementary sequence is still recognized by the miRNA. In other words, the partially complementary target sequence in the context of the present invention can effectively recognize the corresponding miRNA and effectively prevent or reduce transgenic expression in cells expressing the miRNA. Suitably, a partially complementary miRNA target sequence can be completely complementary to a miRNA seed sequence.

[0834] The miRNA target sequence comprising more than one copy can increase the effectiveness of the system. In addition, different miRNA target sequences can be included. For example, a protein coding (e.g., ESF coding) sequence can be operably linked to more than one miRNA target sequence, and the miRNA target sequences can be different, or can be identical. The miRNA target sequence can be tandem, but other arrangements are envisioned. The polynucleotide can, for example, comprise 1, 2, 3, 4, 5, 6, 7 or 8 copies of the same or different miRNA target sequence. Suitably, the polynucleotide comprises 4 copies of every kind of miRNA target sequence.

[0835] The copies of the miRNA target sequence can be separated by a spacer sequence. The spacer sequence can comprise, for example, at least 1, at least 2, at least 3, at least 4, or at least 5 nucleotide bases.

[0836] One or more miRNA target sequences can, for example, inhibit the expression of a transgene (e.g., a nucleic acid sequence encoding ESF) in non-cancerous cells. For example, this can improve the safety of a therapy using ESF. The expression of a transgene (e.g., a nucleic acid sequence encoding ESF) in cancer cells may, for example, not be inhibited by one or more miRNA target sequences.

[0837] One or more miRNA target sequences can inhibit transgene expression in one or more cells other than cancer cells (e.g., neurons, astrocytes, and / or oligodendrocytes). In one embodiment, one or more miRNA target sequences inhibit transgene expression in neurons. In one embodiment, one or more miRNA target sequences inhibit transgene expression in astrocytes. In one embodiment, one or more miRNA target sequences inhibit transgene expression in oligodendrocytes.

[0838] As used herein, the term "inhibit expression" can refer to a reduction in expression of a transgene operably linked to one or more miRNA target sequences in a relevant cell type compared to transgene expression in the absence of one or more miRNA target sequences but under otherwise substantially identical conditions. In some embodiments, transgene expression is inhibited by at least 50%. In some embodiments, transgene expression is inhibited by at least 60%, 70%, 80%, 90%, or 95%. In some embodiments, transgene expression is substantially prevented.

[0839] In some embodiments, the transgene encoding ESF is operably linked to the Ef1a promoter and one or more miRNA target sequences. An exemplary polynucleotide sequence comprising a transgene encoding ESF operably linked to the Ef1a promoter and one or more miRNA target sequences is as follows:

[0840] Ef1a::KRAB-hSOX2 1-179 -DNMT3a3L-Tmir[SES-Tmir](SEQ ID NO:164)

[0841]

[0842]

[0843]

[0844]

[0845] Ef1a promoter

[0846] KRAB

[0847] hSOX2 1-179

[0848] DNMT3a3L

[0849] miR124 target sequences

[0850] miR338-3p target sequence

[0851]

[0852] Ef1a::CS-hSOX2 1-179 –Tmir[SESv2-Tmir](SEQ ID NO:165)

[0853]

[0854]

[0855]

[0856] Ef1a promoter

[0857] CS

[0858] hSOX2 1-179

[0859] miR124 target sequences

[0860] miR338-3p target sequence

[0861]

[0862] Ef1a::SOX2 1-179 -Y-R-Tmir[SESv3-Tmir](SEQ ID NO:166)

[0863]

[0864]

[0865] Ef1a promoter

[0866] hSOX2 1-179

[0867] Y

[0868] miR124 target sequences

[0869] miR338-3p target sequence

[0870]

[0871] Ef1a::KRAB-hTEAD 1-166 -DNMT3a3L-Tmir[TES-Tmir](SEQ ID NO:167)

[0872]

[0873]

[0874]

[0875] Ef1a promoter

[0876] KRAB

[0877] hTEAD 1-166

[0878] DNMT3a3L

[0879] miR124 target sequences

[0880] miR338-3p target sequence

[0881]

[0882] Ef1a::CS-hTEAD 1-166 -Tmir[TESv2-Tmir](SEQ ID NO:168)

[0883]

[0884]

[0885] Ef1a promoter

[0886] CS

[0887] hTEAD 1-166

[0888] miR124 target sequences

[0889] miR338-3p target sequence

[0890]

[0891] Ef1a::hTEAD 1-166 -YR–Tmir[TESv3-Tmir](SEQ ID NO:169)

[0892]

[0893]

[0894]

[0895] Ef1a promoter

[0896] hTEAD 1-166

[0897] Y-R

[0898] miR124 target sequences

[0899] miR338-3p target sequence

[0900]

[0901] Ef1a::CS-hTEAD 1-166 –Y-R-Tmir[TESv4-Tmir](SEQ ID NO:170)

[0902]

[0903]

[0904]

[0905] Ef1a promoter

[0906] CS

[0907] hTEAD 1-166

[0908] y-r

[0909] miR124 target sequences

[0910] miR338-3p target sequence

[0911]

[0912] Ef1a::CS-hMYC 144-454 -Tmir[MESv2-Tmir](SEQ ID NO:171)

[0913]

[0914]

[0915]

[0916] Ef1a promoter

[0917] CS

[0918] hMYC 144-454

[0919] miR124 target sequences

[0920] miR338-3p target sequence

[0921]

[0922] Ef1a::YR-hMYC 144-454 -Tmir[MESv3-Tmir](SEQ ID NO:172)

[0923]

[0924]

[0925]

[0926] Ef1a promoter

[0927] Y

[0928] hMYC 144-454

[0929] miR124 target sequences

[0930] miR338-3p target sequences

[0931]

[0932] Ef1a::CS-YR-hMYC 144-454 -Tmir[MESv4-Tmir](SEQ ID NO:173)

[0933]

[0934]

[0935]

[0936] Ef1a promoter

[0937] CS

[0938] yr

[0939] hMYC 144-454

[0940] V5

[0941] miR124 target sequences

[0942] miR338-3p target sequence

[0943]

[0944] Ef1a::KRAB-DNMT3a3L-hMYC 144-454 -Tmir[MES-Tmir](SEQ ID NO:174)

[0945]

[0946]

[0947]

[0948]

[0949] Ef1a promoter

[0950] KRAB

[0951] DNMT3a3L

[0952] HkDJ 144-454

[0953] miR124 target sequences

[0954] miR338-3p target sequence

[0955]

[0956] In one embodiment, the polynucleotide comprises a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 164-174, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

[0957] In one embodiment, the polynucleotide comprises a sequence according to any one of SEQ ID NOs: 164-174.

[0958] In one embodiment, the polynucleotide comprises a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 168-173, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

[0959] In one embodiment, the polynucleotide comprises a sequence according to any one of SEQ ID NOs: 168-173.

[0960] Exemplary constructs

[0961] The polynucleotide may, for example, comprise or consist of a nucleic acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOs: 114-119, 126-131 and 136-174, or a fragment thereof; preferably, wherein the polynucleotide substantially retains the natural function of the polynucleotide of SEQ ID NOs: 114-119, 126-131 or 136-174, respectively.

[0962] Target gene transcription and expression

[0963] The ESF of the invention may be used in a method of repressing transcription and / or expression of at least one target gene. Suitably, the target gene is an endogenous gene.

[0964] At least one target gene transcription and / or expression can be repressed by epigenetic editing.

[0965] The transcription or expression level of the target gene can be reduced, for example, by at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% compared to the transcription or expression level in the absence of ESF.

[0966] At least one target gene can be silenced. So-called "silencing target gene" should be understood as reducing the transcription and / or expression of the target gene to a degree sufficient to achieve the desired effect. The reduced expression may be sufficient to achieve the effect associated with treatment, such as preventing or treating a disease (such as cancer). For example, the target gene is preferably repressed to the point where the target gene is not transcribed and / or not expressed, or the residual level of the target gene transcription and / or expression is sufficiently low to improve or prevent the degree of a disease state.

[0967] Methods for analyzing gene transcription or expression are well known in the art. Methods for determining gene transcription, including reverse transcription PCR and Northern blot-based methods are known in the art. Methods for determining gene expression, including Western blot-based methods or flow cytometry are known in the art.

[0968] Preferably, repression of the target gene occurs following transient delivery of the ESF of the invention to the cell or expression within the cell.

[0969] By "transient expression" it is understood that the expression of ESF is not stable for a long period of time. Preferably, the polynucleotide encoding ESF is not integrated into the host genome. More specifically, transient expression can be expression that is substantially lost within 20 weeks after the polynucleotide encoding ESF is introduced into the cell. Preferably, the expression is substantially lost within 12, 6, 4 or 2 weeks after the polynucleotide encoding ESF is introduced into the cell.

[0970] Similarly, "transient delivery" is understood to mean that the ESF is not substantially retained in the cells (i.e., substantially lost by the cells) for a long period of time. More specifically, transient delivery may result in the ESF being substantially lost by the cells within 20 weeks after the ESF is introduced into the cells. Preferably, the ESF is substantially lost within 12, 6, 4, or 2 weeks after the ESF is introduced into the cells.

[0971] In some embodiments, ESF is delivered transiently. Transient delivery may result in permanent changes

[0972] Preferably, at least one target gene is permanently repressed or silenced. The so-called "permanent repression" or "permanent silencing" of a target gene is understood to mean that the transcription or expression of the target gene is reduced (e.g., reduced by at least 60%, at least 70%, at least 80%, at least 90% or 100%) for at least 2 months, 6 months, 1 year, 2 years or the entire life cycle of the cell / organism compared to the transcription or expression level in the absence of ESF. Preferably, the permanently repressed or silenced target gene remains repressed or silenced for the remaining life of the cell.

[0973] In some embodiments, the ESF is stably expressed.

[0974] protein

[0975] As used herein, the term "protein" includes single-chain polypeptide molecules and multiple polypeptide complexes in which the individual constituent polypeptides are linked by covalent or non-covalent means. As used herein, the terms "polypeptide" and "peptide" refer to polymers in which the monomers are amino acids and are linked together by peptide bonds or disulfide bonds.

[0976] Protein transduction

[0977] As an alternative to delivering polynucleotides to cells, the ESFs of the invention may be delivered to cells by protein transduction.

[0978] Protein transduction can be performed by vector delivery (Cai et al. (2014) Elife 3:e01911; Maetziget al. (2012) Curr. Gene Ther. 12:389-409). Vector delivery involves engineering of viral particles (e.g., lentiviral particles) to contain the protein to be delivered to the cell. Thus, when the engineered viral particles enter the cell as part of their natural life cycle, the protein contained in the particles is carried into the cell.

[0979] Protein transduction can be performed by protein delivery (Gaj et al. (2012) Nat. Methods 9: 805-7). Protein delivery can be achieved, for example, by using a vehicle (e.g., nanoparticles such as liposomes) or by directly administering the protein itself to the cell.

[0980] In some embodiments, the ESF is contained in a nanoparticle. In some embodiments, the nanoparticle is a polymer nanoparticle, an inorganic nanoparticle, or a lipid nanoparticle. In some embodiments, the nanoparticle is a liposome.

[0981] Nanoparticles can be targeted to specific cell types (eg, cancer cells) using one or more ligands displayed on their surface.

[0982] Polynucleotide

[0983] Polynucleotides of the present invention may comprise DNA or RNA. They may be single-stranded or double-stranded. It will be appreciated by the skilled person that, due to the degeneracy of the genetic code, many different polynucleotides may encode the same polypeptide. In addition, it will be appreciated that the skilled person may use conventional techniques to replace nucleotides that do not affect the polypeptide sequence encoded by the polynucleotides of the present invention, to reflect the codon selection of any specific host organism that will express the polypeptide of the present invention.

[0984] The transgenes and coding sequences of the invention (such as those disclosed herein) may also include a stop codon, eg, TGA, at the 3' end of the transgene or coding sequence.

[0985] The polynucleotides may be modified by any method available in the art. Such modifications may be performed to increase the in vivo activity or lifespan of the polynucleotides of the invention.

[0986] Polynucleotides such as DNA polynucleotides can be produced recombinantly, synthetically or by any means available to those skilled in the art. They can also be cloned by standard techniques.

[0987] Longer polynucleotides are usually produced using recombinant methods, such as using polymerase chain reaction (PCR) cloning techniques. This may involve making a pair of primers (e.g., about 15 to 30 nucleotides) flanking the target sequence desired to be cloned, contacting the primers with mRNA or cDNA obtained from animal or human cells, performing a polymerase chain reaction under conditions that result in amplification of the desired region, isolating the amplified fragments (e.g., by purifying the reaction mixture with an agarose gel) and recovering the amplified DNA. The primers may be designed to contain suitable restriction enzyme recognition sites so that the amplified DNA can be cloned into a suitable vector.

[0988] Carrier

[0989] A vector is a tool that allows or promotes the transfer of an entity from one environment to another. According to the present invention, for example, some vectors used in recombinant nucleic acid technology allow entities, such as segments of nucleic acids (e.g., heterologous DNA segments, such as heterologous cDNA segments) to be transferred to target cells. Vectors can be used to maintain heterologous nucleic acids (DNA or RNA) in cells, promote the replication of vectors containing nucleic acid segments, or promote the expression of proteins encoded by nucleic acid segments. Vectors can be non-viral or viral. Examples of vectors used in recombinant nucleic acid technology include, but are not limited to, plasmids, mRNA molecules (e.g., in vitro transcribed mRNA), chromosomes, artificial chromosomes, and viruses. Vectors can also be, for example, naked nucleic acids (e.g., DNA). In its simplest form, the vector itself can be a nucleotide of interest.

[0990] The vector used in the present invention may be, for example, a plasmid, mRNA or viral vector, and may include a promoter for expressing the polynucleotide and optionally a regulator of the promoter.

[0991] A variety of techniques known in the art, such as transfection, transformation and transduction, can be used to introduce vectors comprising polynucleotides used in the present invention into cells. Several such techniques are known in the art, such as infection with recombinant viral vectors, such as retrovirus, lentivirus (e.g., integration-defective lentivirus), adenovirus, adeno-associated virus, baculovirus and herpes simplex virus vectors, direct injection of nucleic acids and biolistic transformation.

[0992] Non-viral delivery systems include, but are not limited to, DNA transfection methods. Here, transfection includes the process of using a non-viral vector to deliver a gene to a target cell. Typical transfection methods include electroporation, DNA bio-ballistics, lipid-mediated transfection, compressed DNA-mediated transfection, liposomes, immunoliposomes, lipofection, cationic agent-mediated transfection, cationic surface amphiphiles (CFA) (Nat. Biotechnol. (1996) 14: 556) and combinations thereof.

[0993] For example, transfection of cells with mRNA vectors can be achieved using nanoparticles such as liposomes.

[0994] In some embodiments, the carrier (e.g., mRNA carrier) is contained in a nanoparticle. In some embodiments, the nanoparticle is a polymer nanoparticle, an inorganic nanoparticle, or a lipid nanoparticle. In some embodiments, the nanoparticle is a liposome.

[0995] Nanoparticles can be targeted to specific cell types (eg, cancer cells) using one or more ligands displayed on their surface.

[0996] Viral vectors

[0997] In a preferred embodiment, the vector is a viral vector. The viral vector may be in the form of a viral vector particle.

[0998] The viral vector can be, for example, a retroviral, lentiviral, adeno-associated viral (AAV) or adenoviral vector.

[0999] In some embodiments, the vector is a lentiviral vector. In some embodiments, the vector is an AAV vector. In some embodiments, the vector is an AAV vector particle.

[1000] Retroviral and lentiviral vectors

[1001] Retroviral vectors can be derived from or can be derived from any suitable retrovirus. A large number of different retroviruses have been identified. Examples include murine leukemia virus (MLV), human T-cell leukemia virus (HTLV), mouse mammary tumor virus (MMTV), Rous sarcoma virus (RSV), Fujinami sarcoma virus (FuSV), Moloney murine leukemia virus (Mo-MLV), FBR murine osteosarcoma virus (FBR MSV), Moloney murine sarcoma virus (Mo-MSV), Abelson murine leukemia virus (A-MLV), avian myelocytoma virus 29 (MC29) and avian erythroblastosis virus (AEV). A detailed list of retroviruses can be found in Coffin et al. (1997) Retroviruses, Cold Spring Harbour Laboratory Press, 758-63.

[1002] Retroviruses can be roughly divided into two categories: "simple" and "complex". Retroviruses can be further divided into seven groups. Five of these groups represent retroviruses with oncogenic potential. The remaining two groups are lentiviruses and spumaviruses. These retroviruses are reviewed by Coffin et al. (1997) Retroviruses, Cold Spring Harbour Laboratory Press, 758-63.

[1003] The basic structure of retroviral and lentiviral genomes has many common features, such as 5'LTR and 3'LTR. Between or within them are packaging signals that enable the genome to be packaged, primer binding sites, integration sites that enable integration into the host cell genome, and gag, pol, and env genes that encode packaging components - polypeptides required for the assembly of viral particles. Lentiviruses have other features, such as the rev and RRE sequences in HIV, which enable efficient export of the RNA transcripts of the integrated provirus from the nucleus to the cytoplasm of the infected target cell.

[1004] In the provirus, these genes are flanked at both ends by regions called long terminal repeats (LTRs). LTRs are responsible for proviral integration and transcription. LTRs also act as enhancer-promoter sequences and can control the expression of viral genes.

[1005] The LTR itself is an identical sequence that can be divided into three elements: U3, R, and U5. U3 is derived from a sequence unique to the 3' end of the RNA. R is derived from a sequence repeated at both ends of the RNA. U5 is derived from a sequence unique to the 5' end of the RNA. The sizes of these three elements can vary greatly between different retroviruses.

[1006] In defective retroviral vector genomes, gag, pol, and env may be absent or inoperative.

[1007] In a typical retroviral vector, at least a portion of one or more protein coding regions essential for replication may be removed from the virus. This renders the viral vector replication defective.

[1008] Lentiviral vectors are part of a larger group of retroviral vectors. A detailed list of lentiviruses can be found in Coffin et al. (1997) Retroviruses, Cold Spring Harbour Laboratory Press, 758-63. Lentiviruses can be divided into primate and non-primate groups. Examples of primate lentiviruses include, but are not limited to, human immunodeficiency virus (HIV), the pathogen of human acquired immunodeficiency syndrome (AIDS); and simian immunodeficiency virus (SIV). Examples of non-primate lentiviruses include the prototype "lentivirus" Vesna / Medi virus (VMV), as well as the related caprine arthritis encephalitis virus (CAEV), equine infectious anemia virus (EIAV), and the recently described feline immunodeficiency virus (FIV) and bovine immunodeficiency virus (BIV).

[1009] The lentivirus family differs from retroviruses in that lentiviruses have the ability to infect both dividing and non-dividing cells (Lewis et al. (1992) EMBO J. 11: 3053-8; Lewis et al. (1994) J. Virol. 68: 510-6). In contrast, other retroviruses, such as MLV, are unable to infect non-dividing or slowly dividing cells, such as those that make up muscle, brain, lung, and liver tissue.

[1010] As used herein, a lentiviral vector is a vector comprising at least one component that is derivable from a lentivirus. Preferably, the component is involved in the biological machinery by which the vector infects cells, expresses genes or replicates.

[1011] The lentiviral vector may be a "primate" vector. The lentiviral vector may be a "non-primate" vector (i.e., derived from a virus that does not primarily infect primates, especially humans). Examples of non-primate lentiviruses may be any member of the Lentiviridae family that does not naturally infect primates.

[1012] Preferably, the viral vector used in the present invention has a minimal viral genome.

[1013] "Minimal viral genome" is understood to be a viral vector that has been manipulated to remove non-essential elements and retain essential elements in order to provide the functions required for infection, transduction and delivery of the nucleotide sequence of interest to the target host cell. Further details of this strategy can be found in WO1998 / 017815.

[1014] Preferably, the plasmid vector used to produce the viral genome in the host cell / packaging cell will have sufficient lentiviral genetic information to allow the RNA genome to be packaged into viral particles capable of infecting the target cell in the presence of packaging components, but will not be able to replicate independently to produce infectious viral particles in the final target cell. Preferably, the vector lacks functional gag-pol and / or env genes and / or other genes essential for replication.

[1015] However, the plasmid vector used to produce the viral genome in the host cell / packaging cell will also include transcriptional regulatory control sequences operably linked to the lentiviral genome to direct the transcription of the genome in the host cell / packaging cell. These regulatory sequences can be natural sequences associated with the transcribed viral sequence (i.e., 5' U3 region), or they can be a heterologous promoter, such as another viral promoter (e.g., CMV promoter).

[1016] The vector can be a self-inactivating (SIN) vector in which the viral enhancer and promoter sequences have been deleted. SIN vectors can be generated in vivo and transduce non-dividing cells with similar efficacy to wild-type vectors. Transcriptional inactivation of the long terminal repeats (LTRs) in the SIN provirus should prevent the mobilization of replication-competent viruses. This should also be able to regulate gene expression from internal promoters by eliminating any cis-acting effects of the LTRs.

[1017] The vector may be integration-defective. For example, an integration-defective lentiviral vector (IDLV) can be produced by packaging the vector with a catalytically inactive integrase (such as HIV integrase with a D64V mutation in the catalytic site), or by modifying or deleting essential att sequences in the vector LTR, or a combination of the above.

[1018] Adeno-associated virus (AAV) vector

[1019] Adeno-associated virus (AAV) is an attractive vector system for the present invention because it has a high integration frequency. In addition, due to its small size and reduced binding to cell membranes, AAV can diffuse through brain tissue.

[1020] AAV has a broad host range of infection. Details on the production and use of AAV vectors are described in U.S. Pat. No. 5,139,941 and U.S. Pat. No. 4,797,368.

[1021] Recombinant AAV vectors have been successfully used for the transduction of marker genes and human disease-related genes in vitro and in vivo.

[1022] In some embodiments, the vector is an AAV2 vector. In some embodiments, the vector is an AAV5 vector.

[1023] In some embodiments, the vector is an AAV9 vector.

[1024] The viral vector may be a modified or mutated viral vector.

[1025] Such vectors may be vectors modified to have certain desired properties. For example, the AAV2 vector HBKO (or AAV2-HBKO) is a modified AAV2 that is unable to bind to the heparan sulfate proteoglycan receptor (Naidoo et al. (2018) Mol Ther 26:2418-2430).

[1026] In one embodiment, the vector is a modified AAV2 vector.

[1027] In one embodiment, the vector is an AAV2-HBKO vector.

[1028] Modified viral vectors may comprise proteins that confer, for example, altered cellular tropism, and have been described, for example, in WO2021155137 and WO2015168666.

[1029] In one embodiment, the vector is a modified AAV5 vector.

[1030] In one embodiment, the vector comprises a capsid having one or more mutations in one or more capsid proteins.

[1031] In one embodiment, the vector comprises a capsid having one or more mutations in VP1.

[1032] In one embodiment, the vector is an AAV vector, preferably an AAV5 vector, which comprises a capsid protein (e.g., a VP1 protein) comprising: (a) a G at a position corresponding to amino acid 194; (b) an R at a position corresponding to amino acid 474; (c) an R at a position corresponding to amino acid 564; and / or (d) an R at a position corresponding to amino acid 573, wherein the amino acids are numbered with reference to VP1 of AAV5.

[1033] Variants, derivatives, analogs, homologs and fragments

[1034] In addition to the specific proteins and polynucleotides mentioned herein, the present invention also encompasses the use of variants, derivatives, analogs, homologs and fragments thereof.

[1035] In the context of the present invention, a variant of any given sequence is one in which the specific sequence of residues (whether amino acid residues or nucleic acid residues) has been modified in such a way that the polypeptide or polynucleotide in question substantially retains at least one of its endogenous functions. Variant sequences can be obtained by addition, deletion, substitution, modification, replacement and / or variation of at least one residue present in a naturally occurring protein.

[1036] As used herein, the term "derivative" in relation to a protein or polypeptide of the invention includes any substitution, variation, modification, replacement, deletion and / or addition from or to one or more amino acid residues of the sequence, provided that the resulting protein or polypeptide substantially retains at least one of its endogenous functions.

[1037] As used herein, the term "analog" in relation to a polypeptide or polynucleotide includes any mimetic, ie, a compound that has at least one endogenous function of the polypeptide or polynucleotide that it mimics.

[1038] Typically, amino acid substitutions, for example 1, 2 or 3 to 10 or 20 amino acid substitutions, may be made, provided that the modified sequence substantially retains the desired activity or ability.Amino acid substitutions may include the use of non-naturally occurring analogs.

[1039] The proteins used in the present invention may also have deletions, insertions or substitutions of amino acid residues that produce silent changes and produce functionally equivalent proteins. Deliberate amino acid substitutions may be made based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic properties of the residues, as long as endogenous function is retained. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values ​​include asparagine, glutamine, serine, threonine, and tyrosine.

[1040] For example, conservative substitutions can be made according to the following table. Amino acids in the same block in the second column, and preferably in the same row in the third column, can replace each other:

[1041]

[1042] As used herein, the term "homologue" refers to an entity having a certain homology with a wild-type amino acid sequence or a wild-type nucleotide sequence. The term "homology" can be equivalent to "identity".

[1043] Homologous sequences may include amino acid sequences that are at least 50%, 55%, 65%, 75%, 85% or 90% identical, preferably at least 95% or 97% or 99% identical to the subject sequence. Typically, homologs will contain the same active sites, etc. as the subject amino acid sequence. Although homology can also be considered in terms of similarity (i.e., amino acid residues with similar chemical properties / functions), in the context of the present invention, it is preferred to express homology in terms of sequence identity.

[1044] Homologous sequences can include nucleotide sequences that are at least 50%, 55%, 65%, 75%, 85% or 90% identical, preferably at least 95% or 97% or 99% identical to the subject sequence. Although homology can also be considered from the perspective of similarity, in the context of the present invention, homology is preferably represented from the perspective of sequence identity.

[1045] Preferably, reference to a sequence having a percentage identity to any SEQ ID NO disclosed herein refers to the sequence having said percentage identity over the entire length of the SEQ ID NO in question.

[1046] Homology comparisons can be performed by eye, or more usually with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate the percent homology or identity between two or more sequences.

[1047] The percent homology of consecutive sequences can be calculated by aligning one sequence with the other and directly comparing each amino acid in one sequence with the corresponding amino acid in the other sequence, one residue at a time. This is called a "ungapped" alignment. Typically, such ungapped alignments are performed only over a relatively small number of residues.

[1048] Although this is a very simple and consistent approach, it does not take into account that, for example, a single insertion or deletion in a nucleotide sequence in a pair of otherwise identical sequences may cause subsequent codons to be out of alignment, thus potentially resulting in a large reduction in the homology percentage when a global alignment is performed. Therefore, most sequence comparison methods aim to produce an optimal alignment, taking into account possible insertions and deletions without overly penalizing the overall homology score. This is achieved by inserting "gaps" in the sequence alignment in an attempt to maximize local homology.

[1049] However, these more complex methods assign a "gap penalty" to each gap that occurs in the alignment, so that for the same number of identical amino acids, the sequence alignment has as few gaps as possible, reflecting a higher correlation between the two compared sequences, and will receive a higher score than a sequence with many gaps. An "affine gap cost" is usually used, which charges a relatively high cost for the existence of a gap and a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. A high gap penalty will of course produce an optimized alignment with fewer gaps. Most alignment programs allow the gap penalty to be modified. However, when using such software for sequence comparison, it is preferred to use the default values. For example, when using the GCG WisconsinBestfit software package, the default gap penalty for amino acid sequences is -12 for a gap and -4 for each extension.

[1050] Therefore, calculating the maximum percentage homology first requires producing an optimal alignment while taking into account gap penalties. A suitable computer program for performing such an alignment is the GCG Wisconsin Bestfit software package (University of Wisconsin, USA; Devereux et al. (1984) Nucleic Acids Res. 12: 387). Examples of other software that can perform sequence comparisons include, but are not limited to, the BLAST package, FASTA (Atschul et al. (1990) J. Mol. Biol. 403-410) and the GENEWORKS comparison tool suite. Both BLAST and FASTA can be used for offline and online searches. However, for some applications, it is best to use the GCG Bestfit program. Another tool called BLAST 2 Sequences can also be used to compare protein and nucleotide sequences (see FEMS Microbiol. Lett. (1999) 174: 247-50; FEMS Microbiol. Lett. (1999) 177: 187-8).

[1051] Although the final homology percentage can be measured according to identity, the alignment process itself is usually not based on an all-or-nothing paired comparison. On the contrary, a hierarchical similarity scoring matrix that assigns each paired comparison score based on chemical similarity or evolutionary distance is usually used. An example of such a matrix commonly used is the BLOSUM62 matrix--the default matrix of the BLAST program suite. The GCG Wisconsin program usually uses a public default value or a custom symbol comparison table (if provided) (see user manual for further details). For some applications, it is preferred to use the public default values ​​of the GCG package, or in the case of other software, a default matrix, such as BLOSUM62, is used.

[1052] Once the software has produced an optimal alignment, the percent homology, preferably the percent sequence identity, can be calculated. The software typically does this as part of the sequence comparison and generates a numerical result.

[1053] A "fragment" is also a variant, and the term generally refers to a selected region of a polypeptide or polynucleotide that is of interest functionally or, for example, in an assay. Thus, a "fragment" refers to an amino acid or nucleic acid sequence that is a portion of a full-length polypeptide or polynucleotide.

[1054] Such variants can be prepared using standard recombinant DNA techniques such as site-directed mutagenesis. In the case of an insertion, a synthetic DNA encoding the insertion and 5' and 3' flanking regions corresponding to the naturally occurring sequence on either side of the insertion site can be prepared. The flanking regions will contain convenient restriction sites corresponding to the sites in the naturally occurring sequence so that the sequence can be cut with an appropriate enzyme and the synthetic DNA can be connected to the nick. The DNA is then expressed according to the present invention to produce the encoded protein. These methods are only examples of various standard techniques known in the art for manipulating DNA sequences, and other known techniques may also be used.

[1055] Codon optimization

[1056] The polynucleotide used in the present invention can be codon optimized. Codon optimization has been previously described in WO1999 / 41397 and WO2001 / 79518. Different cells have different uses for specific codons. This codon bias corresponds to the bias of specific tRNA relative abundance in the cell type. By changing the codons in the sequence so that it is customized to match the relative abundance of the corresponding tRNA, expression can be increased. For the same reason, expression can be reduced by intentionally selecting codons that are rare in a specific cell type known to the corresponding tRNA. Therefore, an additional degree of translational control can be obtained.

[1057] Composition

[1058] The polynucleotides, proteins, vectors, nanoparticles and cells of the present invention can be formulated with a pharmaceutically acceptable carrier, diluent or excipient for administration to a subject. Suitable carriers and diluents include isotonic saline solutions, such as phosphate buffered saline, and may contain human serum albumin.

[1059] Materials used to formulate pharmaceutical compositions should be non-toxic and should not interfere with the efficacy of the active ingredients. The precise nature of the carrier or other material can be determined by the skilled person depending on the route of administration.

[1060] Pharmaceutical compositions are generally in liquid form. Liquid pharmaceutical compositions generally include liquid carriers, such as water, petroleum, animal or vegetable oils, mineral oils or synthetic oils. Physiological saline solutions, magnesium chloride, glucose or other sugar solutions, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included. In some cases, surfactants may be used, such as 0.001% pluronic acid (PF68). In some cases, serum albumin may be used in the compositions.

[1061] For injection, the active ingredient can be in the form of a pyrogen-free aqueous solution and have suitable pH, isotonicity and stability. The technician is fully capable of using, for example, isotonic vehicles such as sodium chloride injection, Ringer's injection or lactated Ringer's injection to prepare suitable solutions. Preservatives, stabilizers, buffers, antioxidants and / or other additives may be included as needed.

[1062] For delayed release, the drug may be contained in a pharmaceutical composition formulated for slow release, such as in microcapsules formed from biocompatible polymers or in a liposome carrier system according to methods known in the art.

[1063] Processing of cell therapy products is preferably performed in accordance with the FACT-JACIE international standards for cell therapy.

[1064] Treatment

[1065] In one aspect, the invention provides a polynucleotide, vector, protein, nanoparticle, cell, composition or pharmaceutical composition of the invention for use in therapy.

[1066] In another aspect, the present invention provides an ESF, a polynucleotide, a vector, a cell or a composition of the present invention for use in the treatment of cancer.

[1067] In some embodiments, the cancer is a glioma, glioblastoma, medulloblastoma, astrocytoma, neuroblastoma, ependymoma, meningioma, retinoblastoma, rhabdomyosarcoma, lung cancer, prostate cancer, breast cancer, liver cancer, pancreatic cancer, bladder cancer, oropharyngeal cancer, or kidney cancer. In some embodiments, the cancer is a brain tumor. In some embodiments, the cancer is glioblastoma multiforme.

[1068] In some embodiments, treatment reduces tumor size.

[1069] In some embodiments, treatment is as adjuvant therapy, optionally in combination with surgery.

[1070] All treatments mentioned herein include curative, palliative and preventive treatments. Treatment of mammals, particularly humans, is preferred. Both human and veterinary treatments are within the scope of the present invention.

[1071] In some embodiments, the method of treatment provides a polynucleotide, vector, ESF, protein, nanoparticle, cell, composition or pharmaceutical composition of the invention to a tumor.

[1072] In some embodiments, the method of treatment provides a polynucleotide, vector, ESF, protein, nanoparticle, cell, composition, or pharmaceutical composition of the invention to the brain of a subject.

[1073] Application

[1074] In some embodiments, the polynucleotide, vector, ESF, protein, nanoparticle, cell, composition, or pharmaceutical composition is administered topically to a subject.

[1075] In some embodiments, the polynucleotide, vector, ESF, protein, nanoparticle, cell, composition, or pharmaceutical composition is administered to the brain of a subject.

[1076] In preferred embodiments, the polynucleotide, vector, ESF, protein, nanoparticle, cell, composition or pharmaceutical composition is administered to a tumor.

[1077] In some embodiments, the polynucleotide, vector, ESF, protein, nanoparticle, cell, composition, or pharmaceutical composition is administered to a subject systemically, e.g., intravenously.

[1078] In some embodiments, the polynucleotide, vector, ESF, protein, nanoparticle, cell, composition, or pharmaceutical composition is administered topically to a subject.

[1079] As used herein, the term "systemic delivery" or "systemic administration" means administering an agent of the invention into the circulatory system, e.g., to achieve widespread distribution of the agent. In contrast, topical or local administration limits the delivery of an agent to a local area, e.g., a tumor.

[1080] dose

[1081] The technician can easily determine the appropriate dose of the medicament of the present invention applied to the subject. Usually, the doctor will determine the actual dose that is most suitable for the individual patient, which will depend on multiple factors, including the activity of the specific compound used, the metabolic stability and action time of the compound, age, body weight, general health, sex, diet, mode of administration and time, excretion rate, drug combination, severity of a particular condition and the individual undergoing therapy. Of course, there may be individual cases that require higher or lower dosage ranges, and this is within the scope of the present invention.

[1082] Subjects

[1083] As used herein, the term "subject" refers to a human or non-human animal.

[1084] Examples of non-human animals include vertebrates, e.g., mammals, such as non-human primates (particularly higher primates), dogs, rodents (e.g., mice, rats, or guinea pigs), pigs, and cats. The non-human animal may be a companion animal.

[1085] Preferably, the subject is a human.

[1086] The skilled person will understand that they can combine all features of the present invention disclosed herein without departing from the scope of the invention disclosed.

[1087] Preferred features and embodiments of the invention will now be described by way of non-limiting examples.

[1088] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of chemistry, biochemistry, molecular biology, microbiology and immunology, which are within the capabilities of one of ordinary skill in the art and are explained in the literature. See, for example, Sambrook, J., Fritsch, EF and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press; Ausubel, FMet al. (1995 and periodic supplements) Current Protocols in Molecular Biology, Ch. 9, 13 and 16, John Wiley & Sons; Roe, B., Crabtree, J. and Kahn, A. (1996) DNA Isolation and Sequencing: Essential Techniques, John Wiley & Sons; Polak, JM and McGee, J.O'D. (1990) In Situ Hybridization: Principles and Practice, Oxford University Press; Gait, MJ (1984) Oligonucleotide Synthesis: APractical Approach, IRL Press; and Lilley, DMand Dahlberg,JE(1992)Methods in Enzymology: DNA Structures Part A: Synthesis and Physical Analysis of DNA, Academic Press. Each of these general texts is incorporated herein by reference.

[1089] All publications mentioned in the above description are incorporated herein by reference. Without departing from the scope and spirit of the present invention, various modifications and variations of the polypeptides, polynucleotides, vectors, cells, compositions, uses and methods disclosed in the present invention are obvious to the technician. Although the present invention has been disclosed in conjunction with specific preferred embodiments, it should be understood that the claimed invention should not be unduly limited to these specific embodiments. In fact, various modifications of the disclosed modes for implementing the present invention that are obvious to those skilled in the art are intended to fall within the scope of the appended claims. Example

[1090] Example A

[1091] result

[1092] We sought to construct a panel of epigenetic silencing factors by adding the Kruppel-associated box (KRAB) domain and / or the catalytic domain of the DNA methyltransferase DNMT3A and its cofactor DNMT3L to full-length Sox2 or Sox2 lacking the C-terminal transcriptional activation domain. Figure 1 a).

[1093] The KRAB domain (from the zinc finger protein ZNF10) recruits different epigenetic complexes that are able to induce repressive chromatin modifications (such as H3K9me3) and remove active marks (such as H3K4ac), while the DNMT domain coordinates de novo DNA methylation, thereby completely preventing gene transcription (Amabile et al. (2016) Cell 167:219-232.e14). Only the factor composed of the N-terminal and DNA binding regions of SOX2 plus the KRAB and DNMT domains located at the 5' and 3' ends, respectively (called SES), rapidly kills glioma cell lines (such as U87, U251 and SNB19) in vitro ( Figure 1 bf). Thus, genes associated with cell proliferation and bona fide SOX2 targets (CDK1, CDC6, CCND1, etc.) were strongly repressed in SES-treated cancer cells ( Figure 1 g). Therefore, removal of the SOX2 transcriptional activation domain is essential for obtaining synthetic factors capable of suppressing cancer cell proliferation. Interestingly, proliferation of cell lines from prostate, liver, and pancreatic cancers was largely unaffected by SES treatment, suggesting that SES has cancer-specific efficacy ( Figure 1 h). We extended the in vitro analysis to patient-derived glioblastoma multiforme cancer stem cells (GBM CSCs) of both classical and mesenchymal subtypes that better retain the characteristics of the primary tumor. Following SES lentiviral (LV) transduction, both GBM CSC lines exhibited a robust loss of proliferation and high cell death ( Figure 2a). To determine SES specificity, we mutated two residues in the HMG box domain (arginine at position 74 and leucine at position 97 replaced by two prolines) that have been described to be important for Sox2 binding to DNA ( Figure 1 e). Expression of SES(R74P / L97P) failed to prevent CSC growth, indicating that SES activity is dependent on its DNA binding activity ( Figure 2 a). In addition, clonogenic analysis showed that SES-treated CSCs exhibited diminished self-renewal capacity, as well as impaired ability to form tumor spheres and sustain their growth ( Figure 2 b).

[1094] We next set out to assess the overall SES transcriptional output and its genome-wide occupancy. SES-treated GBM CSCs exhibited dramatic transcriptional changes in at least two different glioma cell lines, with widespread upregulation of apoptosis-related genes and silencing of genes encoding proliferation and tumor-promoting factors ( Figure 3 ac). Interestingly, using a list of putative SOX2 targets inferred by computational methods (Janky (2014) et al. PLoS Comput Biol. 10:e1003731), we found that they were largely downregulated in cells expressing SES ( Figure 3 d). Comparative ChIP-seq experiments to determine genome-wide binding of SOX2 and SES on CSCs showed that SES maintained the ability to bind to the majority of SOX2 genomic sites ( Figure 3 e). Given that SES contains the catalytic domain of the DNMT3A / L de novo DNA methyltransferase, we used MeDIP-seq to profile methylated DNA. Notably, the methylation status of regions bound by SOX2 was significantly increased in SES-transduced cells ( Figure 2 f).

[1095] We then asked whether SES expression in vivo could exert any antitumor activity. Initially, we performed subcutaneous xenografts in NSG immunodeficient mice using CSCs previously transduced with lentivirus expressing either GFP (mock) or SES. Importantly, xenografts were grown exclusively by transplanted mock cells, whereas SES-transduced cells were unable to sustain tumor growth ( Figure 4 ac), suggesting that the factor is toxic to glioma cells and prevents tumor formation. Even though some scar-like epithelial masses could be isolated within the injection site, no SES + cell( Figure 4 c). Similar results were obtained by inducing subcutaneous tumor growth using glioma cell lines (U87, SNB19, U251) ( Figure 4d) Next, orthotopic intracranial xenografts were performed with U87 cancer cells expressing GFP or SES. + The transplanted cells generated large tumor masses that extended through the striatum and into the cerebral cortex, which is rich in proliferating cells ( Figure 5 Therefore, all mice transplanted with simulated U87 cells died within 30 days after transplantation ( Figure 5 f) In contrast, transplanted SES + U87 cells completely lost their tumor initiating ability and no tumor mass could be recovered from transplanted mice showing an unchanged survival curve ( Figure 5 bg). Similar results were obtained with GBM CSCs treated with SES, where these cells were able to generate very small tumor masses 6 weeks after brain transplantation ( Figure 5 h). To test GBM CSC tumorigenicity in a fully humanized model system, we developed brain organoids by 3D differentiation of human iPSCs (Lancaster et al. (2013) Nature 501:373-9) and assembled them with GBM CSC-derived spheroids using Matrigel embedding (Linkous et al. (2019) Cell Rep. 26:3203-3211.e5; Goranci-Buzhala et al. (2020) Cell Rep. 31:107738). In these assembloids (GBM-cortical organoids), GFP was mimicked. + CSCs successfully infiltrate and spread within the cortical tissue over time ( Figure 6 a). In contrast, in SES-treated organoids, the GBM fraction remained very small, failed to grow over time, and stratified cells within the organoid regions ( Figure 6 b). These results strongly suggest that SES expression in GBM cancer cells inhibits their growth and survival, thereby abolishing their tumor-initiating capacity in vivo. Based on these findings based on the validation of SES antitumor activity based on in vitro transduction of SES in cancer cells prior to transplantation, we turned to an in vivo SES viral transduction approach to inhibit the development of already growing GBM masses in the brain parenchyma. Intracranial transplanted U87 cancer cells were allowed to proliferate and form tumor masses for 4 days ( Figure 7 a, b). Four weeks after lentiviral gene transfer, mock-transduced tumors developed large masses that spread throughout the striatum ( Figure 7 b, c). In contrast, in situ SES expression was sufficient to strongly reduce tumor mass and significantly prolong the survival of affected animals ( Figure 7bd). Notably, V5 immunohistochemistry in 4-week transplanted brains revealed that SES+ cells were not present in the tumor mass but only in the surrounding brain parenchyma ( Figure 7 e) This finding indicates that cancer cells transduced with the SES lentivirus disappeared over time and that the remaining tumor tissue at 4 weeks after transplantation was completely dominated by the SES - Cell composition.

[1096] We then challenged our approach by treating tumors generated from patient-derived CSCs. Lentivirus expressing mock (GFP) or SES was injected orthotopically (7 days after CSC transplantation), and MRI T1 scans were performed weekly to follow tumor growth until histological analysis 6 weeks later ( Figure 7 f). SES-treated tumors were smaller compared with sham-treated groups at both MRI screening and endpoint ( Figure 7 gj). These data suggest that in situ viral treatment with SES is able to reduce the mass of patient-derived GBM in mice.

[1097] SOX2 is a key factor in stem cells and neural progenitors, but is strongly downregulated during neuronal differentiation. Therefore, the effects of SES on mature brain neurons in adulthood should be less. To determine the effects of SES on brain cells, primary mouse cortical neuron cultures were treated with SES LV and survival, morphology, and gene expression were assessed two weeks later. Neurons expressing mock and SES showed similar morphology, no signs of cell damage, and PI + The number of dead cells is quite low ( Figure 8 a). Notably, the transcriptomes of mock and SES-transduced cultures were largely comparable with only Sox2 and 15 other genes differentially expressed between the two neuronal populations ( Figure 8 b) Similar results were collected using human iPSC-derived neuronal cultures, where SES treatment did not alter MAP2 + Neuronal survival and morphology ( Figure 8 c, d). Given the short survival time of primary neurons in vitro, this analysis could only last for no more than two weeks. We therefore turned to in vivo and transduced SES in the hippocampus of adult C57BL / 6 mice and assessed long-term behavioral manifestations ( Fig. 9 a, b). Four weeks after stereotaxic injection of mock (GFP) and SES LV, both mock and SES viral vectors were present in the hippocampus after death ( Fig. 9 c). Similar results were obtained by evaluating the mRNA of the transgene ( Fig. 9 c). Notably, similar levels of cell death were observed after both GFP and SES injections ( Fig. 9d). Before sacrifice, mice were evaluated in spontaneous alternation, radial maze, and Morris water maze tests to assess their exploratory behavior and cognitive functions related to spatial learning and memory ( Fig. 9 Both groups of animals performed equally well on these tasks, indicating that SES expression does not induce significant functional changes in hippocampal neurons.

[1098] We then devised a strategy to restrict SES expression to cancer cells after brain viral inoculation. We isolated the KI67 promoter (Zambon (2010) Cytometry A 77:564-70) and inserted it into the LV cassette (SES v1.1) to drive SES expression only in proliferating cells ( Fig.10 a). First, we confirmed that our strategy did not affect GFP (when placed downstream of the KI67 promoter) or SES expression levels in cancer cells and, in the case of SES, triggered significant cell loss in transduced cancer cells in vitro ( Fig.10 b). Next, mouse primary neuronal cultures composed of neurons and glial cells were transfected with EF1a-GFP (constitutive) or pKI67-GFP LV and investigated after 1 week for the presence of the fluorescent protein. Interestingly, it was found that the vast majority of neurons infected with pKI67-GFP (MAP2 + ) is GFP - , indicating that its transcription was strongly reduced to undetectable levels, whereas constitutive GFP was expressed in all cells of the dish ( Fig.10 c). Surprisingly, a small number of GFP + Cells are also Ki67 + , and are therefore in an active cell cycle, which may correspond to young proliferating astrocytes ( Fig.10 Red arrows in c). Thus, this strategy leads to efficient silencing of SES expression in postmitotic cells in the brain without compromising activation of the SES transgene in cancer cells.

[1099] We then generated other variants of SES using alternative repressor domains, such as the chromoshadow domain from the CBX5 protein (inserted at the 5' position) (SESv2) or the YAF2-RYBP domain from the RYBP protein (inserted at the 3' position) (SESv3), replacing the KRAB and DNMT3A / L catalytic domains ( Fig.11 a). Cancer cells transduced with SESv2 or v3 exhibited rapid loss of proliferation and diffuse cell death, which led to premature termination of the culture within two weeks from initial treatment ( Fig.11bc). Patient-derived classical subtype GBM CSCs better retained primary tumor characteristics and also showed a strong loss of proliferation after SESv3 lentiviral (LV) transduction ( Fig.12 ab).

[1100] These results obtained with SES suggest that reconfiguring activating TFs into epigenetic silencing factors (ESFs) can be extended to establish more epigenetic regulators by engineering other activating cancer-associated TFs. With this goal in mind, we applied the same rational design to TEAD1 and c-MYC, two other transcriptional activators with key oncogenic activities, generating TES and MES factors, respectively. Fig.11 d). For SES, ESFs were generated by removing the transcriptional activation domain and adding the KRAB and DNMT3A / L catalytic domains to the remaining parts of the two TFs ( Fig.11 d). In the case of c-MYC, a repressor domain is inserted at the 5' end to avoid steric hindrance at the 3' end, which interacts directly with MAX to form a heterodimer ( Fig.11 d). Cancer cells transduced with TES or MES exhibited rapid loss of proliferation and diffuse cell death, which led to premature termination of the culture within two weeks from the initial treatment ( Fig.11 ef and Fig.13 ).

[1101] We then evaluated the suppression of tumor development in vivo by TES / MES. When xenografted subcutaneously in NSG mice, both TES- and MES-preinfected CSCs exhibited reduced tumorigenic potential ( Fig.14 TES / MES pre-infected CSCs also showed limited tumor growth in NSG brains (orthotopic transplantation) compared with mock-infected CSCs ( Fig.15 Notably, in situ TES expression was sufficient to strongly reduce the growth of tumor masses formed by transplantation of naive CSCs one week before virus injection ( Fig.16 ).

[1102] Therefore, ESF design can be applied to different oncogenic TFs, generating a family of synthetic factors with strong antitumor activity. ESFs represent a new class of rationally designed factors with pervasive and durable epigenetic functions and can precisely and efficiently reshape entire transcriptional pathways. Targeting ESF expression can inhibit cancer development and represents a new gene-based therapeutic agent for glioblastoma and other cancers.

[1103] discuss

[1104] The activity of developmental TFs is mainly restricted during morphogenesis, playing important roles in stem cell identity, cell lineage commitment, and differentiation. However, these TFs can be reactivated or manipulated by cancer genetic programs to drive tumor development and progression. It is estimated that about 20% of all known oncogenic proteins are represented by TFs, which are essential for the acquisition of malignant cell dedifferentiation, proliferation, and migration. Despite their ubiquitous roles in tumors, interfering with their functions from a translational perspective has proven challenging. In fact, stable and complete gene silencing by various genetic tools or small molecules has been difficult to achieve in cancer cells. In addition, cancer genetic programs have been repeatedly shown to overcome single gene inactivation by reconfiguring transcriptional networks, thereby promoting cancer resistance and recurrence. In this paper, we designed epigenetic repressors (SES) by rationally assembling transcriptional and epigenetic negative regulators of gene transcription to reconfigure the SOX2 TF. This design is modular and versatile and can in principle be applied to other activating oncogenic transcription factors, as we have demonstrated for TEAD1 and c-MYC. Importantly, SES-dependent de novo DNA methylation in SOX2 target genes triggered by the DNMTA / L catalytic domains promotes widespread and stable silencing of the SOX2 downstream network. These widespread transcriptional changes inhibit cell proliferation in cancer cells, which are unable to cope with these changes and eventually die. Here, we show that these domains can reconfigure the transcriptional activity of endogenous TFs while preserving their chromatin occupancy and target selectivity. We also show that by using different epigenetically active protein domains (e.g., the Chromo Shadow domain from CBX5 and the YAF2-RYBP domain from RYBP), differently configured SES can elicit the same functional activity.

[1105] SOX2 expression is essential for controlling self-renewal and malignant phenotypes in GBM cancer cells as well as in many stem cells, both pluripotent and neural types. Importantly, SOX2 plays a major role in promoting tumor development in many other malignancies besides GBM, including medulloblastoma and lung, prostate, and breast cancers.

[1106] Therefore, the use of SES or other ESFs can be extended to the treatment of other cancers. Tumor targeting of ESFs delivered by virus-mediated delivery can be effective in principle for cancers confined to those solid tissues that can be efficiently targeted by in vivo viral transduction. From this point of view, liver cancer, lung cancer, breast cancer and kidney cancer represent reasonable targets for this approach, as known delivery routes and viral strains can achieve broad and high tissue transduction efficiencies. Similarly, the same approach can be proposed to treat metastatic masses in the same organs.

[1107] In this paper, SES was injected directly into the tumor mass to arrest its development. Similar approaches could be used in a clinical setting to treat glioblastomas, which are impractical to surgically remove because they are located in inaccessible locations within the brain or close to vital brain regions. In addition, SES could be delivered as an adjuvant therapy into the brain parenchyma surrounding a resected primary tumor to target remaining cancer cells and inhibit subsequent tumor recurrence.

[1108] In this article, treatment of glioblastoma with ESF is performed by lentiviral transduction injected locally into the affected tissue. However, alternative therapeutic viruses can also be used similarly, in particular adeno-associated virus (AAV) strains, which can spread through brain tissue due to their small size and reduced binding to cell membranes. Maximizing the spread of the virus in the brain parenchyma will increase the targeting efficiency of cancer cells dispersed in the tissue, thereby providing better prevention from tumor recurrence. In addition, non-viral vehicles such as nanoparticles or liposomes can be used to deliver SES mRNA or protein to obtain acute transgene expression, which may still be sufficient to inhibit cancer cells while strongly enhancing the overall safety profile of the procedure.

[1109] We show that SES expression is not deleterious in neuronal cultures and in the murine brain, and we further describe a strategy to restrict its activation to proliferating cells, which are abundantly enriched in cancer and rarely present in the brain parenchyma. The system described here has been shown to efficiently express viral transgenes primarily in cancer cells but not in brain postmitotic cells.

[1110] In conclusion, we assembled an epigenetic repressor that functions as a dominant negative version of the oncogenic SOX2 TF and is able to bind and stably repress the SOX2 transcriptional network. Targeted viral delivery of SES in glioblastoma was sufficient to inhibit tumor development by blocking cell proliferation and inducing cell death. Given its broad applicability to other oncogenic transcription factors and the high efficiency of targeting cancer cells via viral transduction, this approach offers an opportunity to repress glioblastoma and other deadly cancers.

[1111] Materials and methods

[1112] Construct

[1113] KRAB-hSOX2: The full-length human SOX2 gene is fused to the KRAB repressor domain (from the gene encoding zinc finger protein ZNF10, amino acids 1-97) at its N-terminus, while the V5 tag is fused to the C-terminus of SOX2. This transgene is used in a lentiviral construct with Ef1a as the promoter.

[1114] KRAB-hSOX2-D3A&L: The full-length human SOX2 gene is fused to the KRAB repressor domain (from the gene ZNF10 encoding zinc finger protein, amino acids 1-97) at its N-terminus, while the functional domains of DNMT3A (amino acids 388-689) and 3L (amino acids 206-421) are fused to the C-terminus of SOX2, and the V5 tag is fused to the end of the last domain, i.e., the C-terminus of the new chimeric transgene. This transgene is used in a lentiviral construct with Ef1a as the promoter.

[1115] KRAB-hSOX2 1-179 : The initial part of the SOX2 gene, encoding amino acids 1-179 (thus excluding the SOX2 activation domain), is fused at its N-terminus to the KRAB repressor domain (from the gene encoding the zinc finger protein ZNF10, amino acids 1-97), while the V5 tag is fused to the C-terminus of SOX2. This transgene is used in a lentiviral construct with Ef1a as promoter.

[1116] hSOX2 1-179 -D3A&L: the initial part of the SOX2 gene, encoding amino acids 1-179 (thus excluding the SOX2 activation domain), fused to the functional domains of DNMT3A (amino acids 388-689) and 3L (amino acids 206-421), and a V5 tag fused to the C-terminus of SOX2. This transgene was used in a lentiviral construct with Ef1a as promoter.

[1117] SES v1: the initial part of the SOX2 gene, encoding amino acids 1-179 (thus excluding the SOX2 activation domain), is fused at its N-terminus to the KRAB repressor domain (from the gene encoding the zinc finger protein ZNF10, amino acids 1-97), while the functional domains of DNMT3A (amino acids 388-689) and 3L (amino acids 206-421) are fused at the C-terminus of the SOX2 part, and the V5 tag is fused at the end of the last domain, i.e. at the C-terminus of the new chimeric transgene. This transgene is used in a lentiviral construct with Ef1a as promoter.

[1118] SES(R74P / L97P): SES v1 was mutagenized at residues 74 and 97 in the beginning of the SOX2 gene (arginine at position 74 and leucine at position 97 were replaced by proline).

[1119] SES v1.1: The transgene is identical to SES version 1, but the Ef1a promoter is replaced by the proximal promoter of the murine Mki67 gene (-1263 to -1 relative to the Mki67 ATG).

[1120] SES v2: The initial part of the SOX2 gene, encoding amino acids 1-179 (thus excluding the SOX2 activation domain), is fused at its N-terminus to the Chromo Shadow (CS) repressor domain (from the gene CBX5, amino acids 121-179), while the V5 tag is fused to the C-terminus of SOX2.

[1121] SES v3: the initial part of the SOX2 gene, encoding amino acids 1-179 (thus excluding the SOX2 activation domain), is fused at its C-terminus to the YAF2-RYBP (YR) repressor domain (from the gene RYBP, amino acids 145-189) and the V5 tag is fused to the C-terminus of YR.

[1122] TES: the initial part of the TEAD1 gene, encoding amino acids 1-166 (thus excluding the TEAD1 activation domain), fused at its N-terminus to the KRAB repressor domain (from the gene encoding the zinc finger protein ZNF10, amino acids 1-97), while the functional domains of DNMT3A (amino acids 388-689) and 3L (amino acids 206-421) were fused at the C-terminus of the TEAD1 part, and the V5 tag was fused at the end of the last domain, i.e., at the C-terminus of the new chimeric transgene. This transgene was used in a lentiviral construct with Ef1a as promoter.

[1123] MES: The C-terminal part of the MYC gene, encoding amino acids 144-454 (thus excluding the MYC activation domain), fused in tandem to the N-terminus of the MYC part with the KRAB repressor domain (from the gene encoding the zinc finger protein ZNF10, amino acids 1-97) and DNMT3A (amino acids 388-689) and 3L (amino acids 206-421), and a V5 tag fused to the C-terminus. This transgene was used in a lentiviral construct with Ef1a as promoter.

[1124] Lentiviral production

[1125] Replication-incompetent, VSVg-coated lentiviral particles were packaged in 293T cells. Cells were transfected with 30 μg of vector and packaging construct according to a conventional CaCl2 transfection protocol. After 30 hours, the culture medium was collected, filtered through 0.44 μm cellulose acetate and centrifuged at 20,000 rpm for 2 hours at 20°C to concentrate the virus.

[1126] Cell culture

[1127] U-87, U-251 and SNB-19 (human glioblastoma cell lines), HeLa (human cervical cancer cell line), DU145 (human prostate cancer cell line) and HepG2 (human liver cancer cell line) were cultured under plastic adherence conditions in DMEM medium (Dulbecco's Modified Eagle's Medium-high glucose, Sigma-Aldrich) containing 10% fetal bovine serum (FBS, Sigma-Aldrich), 1% Pen / Strept (Sigma-Aldrich), 2mM glutamine (Sigma-Aldrich), 1% non-essential amino acids (MEMNEAA, ThermoFisher Scientific), 1% sodium pyruvate solution (Sigma-Aldrich), and passaged twice a week using trypsin-EDTA solution (Sigma-Aldrich).

[1128] BxPC3 (human pancreatic cancer cell line) were cultured in RPMI-1640 (Sigma-Aldrich) containing 10% FBS, 1% Pen / Strept, 2 mM glutamine under plastic adherent conditions. All cell lines were passaged twice a week using trypsin-EDTA solution (Sigma-Aldrich).

[1129] Cancer stem cells (CSCs) from classical (L0627) and mesenchymal (1312) glioblastoma tumors were maintained as spheroids in suspension culture in DMEM / F12 (Sigma-Aldrich) supplemented with a hormone cocktail (DMEM / F12, 0.6% glucose (Sigma-Aldrich)) (30% in phosphate buffered saline (PBS) (Euroclone)), insulin (Sigma-Aldrich) 250 μg / ml, putrescine powder (Sigma-Aldrich) 97 μg / ml, apotransferrin powder (Sigma-Aldrich), sodium selenite 0.3 μM, progesterone 0.2 μM, 1% Pen / Strept, 2 mM glutamine, 0.66% glucose (30% in phosphate buffered saline (PBS) (Euroclone)) and heparin (4 mg / ml, Sigma-Aldrich); bFGF (20 ng / ml, ThermoFisher Scientific) and EGF (20 ng / ml, ThermoFisher Scientific) were freshly added to the culture medium. Spheroid cultures were passaged weekly by mechanical dissociation of spheroids into single cell suspensions.

[1130] All cultures were maintained at 37°C under atmospheric oxygen in a humidified atmosphere of 5% CO2.

[1131] Growth curve analysis

[1132] On day 0, 5x10^5 cancer cell lines were seeded in 6-well plates under adherent conditions; on day 1, the cultures were infected with lentiviral vectors, and on day 3, the cells were detached, live cells were stained with trypan blue solution (0.4%, ThermoFisherScientific), and counted using Countess TM II automated cell counter (ThermoFisherScientific); after this passage, 3x10^5 cells were seeded again. Repeat 3 time points every 3-4 days; repeat the experiment 3 times at each time point. Take bright field representative photos at each time point.

[1133] On day 0, 25x10^4 CSCs were seeded into 24-well plates as single cell suspensions; on day 1, the cultures were infected with lentiviral vectors (expressing SES or GFP). On days 4, 7, 10, and 13, CSC spheres were dissociated into single cell suspensions, and live cells were stained with trypan blue solution and counted as described previously. The number of live cells and the percentage of dead cells at each time point are reported on the graph; the experiment was repeated 3 times for each time point. Bright field representative photos were taken at each time point.

[1134] Western blot analysis

[1135] Cells were homogenized in RIPA buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, SDS (0.1% for cells, 1% for 3D cultures), 1% Triton X-100, Roche Complete EDTA-free Protease Inhibitor Cocktail, Roche PhosSTOP EASYpack) and incubated with primary antibodies overnight at 4°C in a blocking solution consisting of 5% BSA (Sigma-Aldrich) or 5% nonfat dry milk in PBS-TWEEN 0.1% (Sigma-Aldrich) according to the antibody datasheet for Western blot analysis. The primary antibodies used were as follows: anti-V5 (mouse, 1:1000, ThermoFisher Scientific, R96025), anti-SOX2 (clone #245610, mouse, 1:500, R&D system, MAB2018), anti-histone H3 (rabbit, 1:2000, Abcam, ab1791). The band density relative to the control was calculated using Fiji software (NIH, USA) and normalized on housekeeper (H3).

[1136] Clonogenic assay

[1137] On day 0, 25x10^4 CSCs were inoculated into 24 multiwell plates in a single cell suspension; on day 1, the culture was infected or not infected with a lentiviral vector (expressing SES or GFP). On day 6, the spheres were dissociated into a single cell suspension, and the live cells were counted as described previously, and 25x10^4 CSCs were inoculated again to allow the cells to grow and form spheres until day 10. Bright field images were taken on days 6 and 10, and the resulting number of spheres for each condition (uninfected, GFP infected, or SES infected) was calculated; the sphere diameter was measured, and the percentage of spheres with a diameter <100 mm was reported on the graph for each condition and time point. The experiment was repeated 3 times at each time point.

[1138] RNA isolation and real-time RT-qPCR

[1139] RNA was extracted using the TRI Reagent Isolation System (Sigma-Aldrich) according to the manufacturer's instructions. For quantitative RT-PCR (qRT-PCR), 1 microgram of RNA was reverse transcribed using the ImProm-II Reverse Transcription System (Promega). Thereafter, qRT-PCR was performed in triplicate using the CFX96 Real-Time PCR Detection System (Bio-Rad, USA) and Titan HotTaq EvaGreen qPCR Mix (BIOATLAS) using custom-designed oligonucleotides. The obtained cDNA was diluted 1:10 and amplified in a 16 μl reaction mixture containing 2 μl of diluted cDNA, 1× Titan Hot Taq EvaGreen qPCR Mix (Bioatlas, Estonia) and 0.4 mM of each primer. Relative expression was analyzed using the ΔΔCt method, 18S rRNA as a housekeeping gene, and CFX Manager software (Bio-Rad, USA).

[1140] RNA sequencing

[1141] RNA libraries were generated from 1 mg of total RNA (derived from U87, SNB19 and mouse hippocampus) and their quality was assessed using a TapeStation instrument (Agilent). To avoid overrepresentation of 30 ends, only high-quality RNA with an RNA integrity number (RIN) R 8 was used. RNA was processed according to the TruSeq standard mRNA library preparation kit protocol. The library was sequenced on an Illumina HiSeq 3000 with a 76 bp chain read using Illumina TruSeq technology. Image processing and base calling were performed using Illumina real-time analysis software. Fastq files were aligned to hg19 or mm10 human or mouse reference genomes using splice junction maps per TopHat. Differential gene expression and functional enrichment analysis were performed using DESeq2 and GSEA, respectively.

[1142] ChIP sequencing

[1143] Chromatin was isolated from SNB19. Cells were plated under adherent conditions at a density of 6x10^6 per plate using Matrigel-coated 15 mm plates. When the plates reached 90% confluence, cells were fixed by adding formaldehyde directly to the cell culture medium to a final concentration of 1% and then incubated at RT for 10 minutes. Glycine was added to a final concentration of 125 mM to quench the reaction and incubated at RT for 5 minutes. The culture medium was then removed and the cells were washed 3 times with cold sterile PBS + protease inhibitors, then gently scraped and collected and centrifuged at 4°C, 1200 rpm for 50. For ChIP experiments, the collected cell pellets were lysed in lysis buffer (50 mM Tris-HCl pH 8, 0.1% SDS, 10 mM EDTA pH 8, 1 mM phenylmethylsulfonyl fluoride (PMSF, Sigma # P7626), protease inhibitor cocktail (Roche # 04693159001)) and the chromatin was sonicated using a Branson D250 sonicator (30 seconds, 20% amplitude, 4 cycles) to achieve an average fragment size of 0.1-0.5 kb. After quantification, 100 mg of sonicated chromatin was used in each immunoprecipitation and incubated with 4 mg of V5 antibody (mouse, 1:5, ThermoFisherScientific, R96025) at 4°C overnight.

[1144] ChIP-seq libraries were generated using 5ng of each immunoprecipitated and purified DNA. End repair of DNA fragments was achieved by continuous incubation for 15 minutes with 0.15U / ml T4 PNK (NEB#M0201L), 0.04U / ml T4 POL (NEB#M0203L) and 0.1mM dNTP (NEB#N0446S) at 12°C and 25°C. A base addition was performed by incubation for 30 minutes with 0.25U / ml Klenow fragment (NEB#M0212L) and 167mM dATP (NEB N0440S) at 30°C. Linker connection was achieved by using a quick ligation kit (NEB#M2200L) and incubation at 25°C for 15 minutes. DNA fragments were finally amplified for 14 cycles using a PfuUltraII Fusion HSDNAPol kit (Agilent#600674). Agencourt AMPure XP SPRI beads (Beckman #A63882) were used for the DNA purification step after each enzymatic reaction. Before sequencing using Illumina HiSeq 2000, the obtained library was quality controlled on an Agilent bioanalyzer (Agilent Technologies #G2943CA). The sequencing read quality was assessed using fastQC (https: / / www.bioinformatics.babraham.ac.uk / projects / fastqc / ) and the total read was aligned to the human genome (hg19) using Bowtie2 2.2.3 version (http: / / bowtie-bio.sourceforge.net / bowtie2 / index.shtml). Only uniquely located reads were used in subsequent analysis, with an average locatability of >96% of the initial total read. Normalized BigWig tracks of ChIP-seq experiments were generated using bedtools 2.24.0 (https: / / bedtools.readthedocs.io / en / latest / ) and the bedGraphTo-BigWig program (https: / / www.encodeproject.org / software / bedgraphtobigwig / ) and visualized in the UCSC GenomeBrowser (http: / / genome.ucsc.edu / ).To find regions enriched by ChIP-seq relative to background, we used SICER V1.1 (https: / / home.gwu.edu / $wpeng / Software.html) (window size = 200; gap size = 200; for all ChIP-seq data, FDR < 0.01 parameters were used. Density plots (±10 kb) were generated using ngsplot 2.47 (https: / / github.com / shenlab-sinai / ngsplot) command ngs.plot.r and finally re-plotted using GraphPad Prism.

[1145] MeDIP sequencing

[1146] 1 μg of purified genomic DNA (gDNA) was used with the qiAMP DNAmini kit (Qiagen, catalog number 51304). In short, for methylated DNA immunoprecipitation and purification, the MagMeDIPseq kit (Diagenode, cod.C02010040) was used. First, gDNA was sonicated to obtain fragments between 150-300bp in size, which were then denatured into ssDNA and immunoprecipitated using the α-methylcytosine antibody provided by the kit. The next day, the immunoprecipitated DNA and input were purified and eluted. Library preparation was performed using Illumina's NEBNext Ultra II kit (cod.E7645) according to the manufacturer's instructions. Each library was double indexed using Illumina's NEBNext Multiplex Oligos (cod.E6440), and each library was sequenced with Illumina HiSeq 2000 at a depth of 30 million pairs of ends. First, Trimmomatic (http: / / www.usadellab.org / cms / ?page=trimmomatic) was used for linker trimming. The trimmed reads were then aligned to the reference Hg19 genome using Bowtie2 (http: / / bowtie-bio.sourceforge.net / bowtie2 / index.shtml). To obtain coverage tracks, bamCoverage (https: / / deeptools.readthedocs.io / en / develop / content / tools / bamCoverage.html) was used, and the BAM files were converted to BigWigs using CPM normalization and effective genome size parameters, with a bin size of 10. Peaks were called using Macs2 (https: / / github.com / macs3-project / MACS). Normal peak calling mode was used in paired-end mode with q value set to 0.05. Differential peaks were called using Macs2 and the BAM files of the treated and control conditions were compared using the obtained BedGraph. The differential peaks in each condition were then intersected with the normal peaks to filter the differential peaks that were actually present in at least one of the two conditions. Density plots (±10 kb) were generated using ngsplot2.47 (https: / / github.com / shenlab-sinai / ngsplot) command ngs.plot.r and finally replotted using GraphPad Prism.

[1147] Xenografts

[1148] GBM cell lines or cancer stem cells L0627 were seeded in 6-well dishes and infected with 10 μl LV-EIF1α-SES / TES / MES or 5 μl LV-EIF1α-GFP per well for 48 h as described in the infection section.

[1149] Heterotopic xenografts. Infected cells were counted and 3x10^6 cells were resuspended in 100 μl of matrigel (matrigel reduced by growth factors, Corning). By using a 1 ml syringe pre-cooled at 20°C, GFP infected cells were subcutaneously injected into the left flank of NOD-SCID mice (NOD.cg-Prkdc scid Il2rg tm1Wjl / SzJ), while SES / TES / MES infected cells were subcutaneously injected into the right flank of the same animal. Mice were killed 1-3 months after injection (according to growth rate), and subcutaneous tumors were extracted and fixed in 4% PFA for at least 24 hours. Tumor samples were sized and stored overnight in PBS containing 30% sucrose, then embedded in OCT for cryopreservation. Histological sections were cut into 50 μm sections on a cryostat (CM1850 UV, Leica). Subsequently, sections were processed for immunofluorescence or mounted on gelatin-coated slides and Nissl-stained.

[1150] Intracranial xenografts. Infected cells were counted and 3x10^5 cells were resuspended in 3μl 1X PBS and then injected unilaterally into the striatum of NOD-SCID mice (AP+0.5; ML±1.8; DV-3.3 from the skull). Mice were sacrificed after general observation or 40 days after injection of U87 or 3-5 weeks after injection of CSC; after anesthesia, mice were perfused cardiacly with PBS containing 4% PFA, and the brain was removed from the skull and stored in the same solution for overnight fixation. After fixation, the brain was stored in PBS containing 30% sucrose overnight and then embedded in OCT for cryopreservation. The samples were cut coronally into 50μm sections on a cryostat (CM1850 UV, Leica). Subsequently, the sections were processed for immunofluorescence or mounted on gelatin-coated slides and Nissl stained.

[1151] In vivo treatment (using U87). U87 orthotopic xenografts were induced as described above, but 75,000 initial cells were used. Four days later, the mice were randomly divided into two groups and injected with LV carrying GF or SES at the same topological coordinates. One group of animals was killed 26 days after LV injection, and the other group of animals was kept alive for survival rate and killed after observing the general condition or 90 days after the first surgery; after anesthesia, the mice were perfused by heart with PBS containing 4% PFA, and then the brain was removed from the skull and stored in the same solution for overnight fixation. After fixation, the brain was stored in PBS containing 30% sucrose overnight and then embedded in OCT for cryopreservation. The samples were cut coronally into 50 μm slices on a cryostat (CM1850 UV, Leica). Subsequently, the slices were processed for immunofluorescence or mounted on gelatin-coated slides and Nissl stained.

[1152] In vivo treatment (using CSC). As described above, classic initial CSC orthotopic xenografts (3x10^5 cells) were induced. After 7 days, mice were randomly divided into two groups, injected with LV carrying GFP or TES at the same topological coordinates, and killed 3 weeks after LV injection; after anesthesia, mice were perfused by heart with PBS containing 4% PFA, and then the brain was removed from the skull and stored in the same solution for O / N fixation. After fixation, the brain was kept O / N in PBS containing 30% sucrose and then embedded in OCT for cryopreservation. The samples were coronally cut into 50 μm slices on a cryostat (CM1850 UV, Leica). Subsequently, the slices were processed for immunofluorescence or mounted on gelatin-coated slides and Nissl stained.

[1153] SES delivery in WT animals

[1154] LV carrying GFP or SES was injected into the hippocampus of Wt C57BL / 6 animals (2 injections per hippocampus, AP-2.8, ML+ / -3, DV-3.5;-2.5., 0.8 μl each time). One month after surgery, the animals were tested on behavioral tasks and sacrificed for molecular and histological analysis.

[1155] Immunostaining

[1156] Cells were seeded on glass coverslips (previously coated with Matrigel for CSCs to allow cell adhesion) and fixed in 4% paraformaldehyde (PFA, Sigma) in phosphate-buffered saline (PBS, Euroclone) for 20 min on ice. They were then washed twice with PBS and permeabilized for 30' in a blocking solution containing 0.2% Triton X-100 (SigmaAldrich) and 5% donkey serum (Euroclone) and incubated overnight at 4°C with primary antibodies diluted in blocking solution. The primary antibodies used were as follows: anti-V5 (mouse, 1:500, ThermoFisher Scientific, R96025), anti-GFP (chicken, 1:1000, Thermo Fisher Scientific, A10262), anti-MAP2 (chicken, 1:1000, Abcam, ab92434), anti-phospho-histone H3 (Ser10, rabbit, 1:200, Sigma-Aldrich, 06-570), anti-cleaved caspase-3 (Asp175, rabbit, 1:200, Cell Signaling Technology, 9661), anti-Ki-67 (clone SP6, rabbit, 1:500, ImmunologicalSciences, MAB-90948), anti-human nucleus (mouse, 1:500, Millipore, MAB1281). The next day, cells were washed 3 times with PBS for 5 minutes each time and incubated in blocking solution with Hoechst 33342 (ThermoFischer Scientific) and secondary antibody (ThermoFisher Scientific) at room temperature for 1 hour. Brain sections were blocked in 10% donkey serum and 0.2% Triton X-100 at room temperature for 1 hour. Incubated overnight at 4°C with primary antibody. Secondary antibody was applied to sections for 2 hours at RT in blocking solution containing Hoechst 33342. Finally, sections were washed and mounted in fluorescent mounting medium (DakoCytomation). Images were collected using an epifluorescence microscope Nikon DS-Qi2 and analyzed using Fiji software.

[1157] Nissl stain

[1158] Brain sections were rinsed in distilled water for 1 min and then stained in a 0.1% cresyl violet solution boiled at 50°C for 7 min. Afterwards, they were first rinsed in distilled water for 3 min and then washed in a gradient dilution of 70% to 100% ethanol for 1 min. Finally, they were cleared in xylene for 2 h and mounted with mounting solution (Eukitt, Sigma Aldrich).

[1159] MRI acquisition

[1160] MRI was performed on a 7T scanner dedicated to small animals (30 / 70 BioSpec; Bruker, Ettlingen, Germany). The animal protocol included high-resolution T2 sequences. Tumor volume analysis was performed using MIPAV software (https: / / mipav.cit.nih.gov).

[1161] GBM-cortical organoids

[1162] To generate brain organoids, WT iPSCs at 70-80% confluence were detached by incubation in Accutase solution for 10 min at 37°C to obtain a single cell suspension. Cells were centrifuged, counted, and a total of 9000 cells were plated in DMEM / F12, 20% KnockOut TM Serum replacement (KSR, Thermo Fisher Scientific), 2mM glutamine, 1% Pen / Strept, 1% non-essential amino acids, 50nM β-mercaptoethanol (ThermoFisher Scientific) and 4ng / ml bFGF in each well of an ultra-low attachment 96-well plate (Corning). After seeding, the plate was briefly centrifuged to allow single EB formation in each well; ROCK inhibitor Y27632 (50uM) was included in the first 24 hours. EBs were maintained in 96-well plates for 6 days and then transferred to ultra-low attachment 24-well plates (Corning) in neural induction medium containing DMEM / F12, 1X N-2 supplement, 1% non-essential amino acids, 2mM glutamine and 1μg / ml heparin (Sigma-Aldrich) by vigorously pipetting up and down in the wells (with the cut end of a P200 pipette tip). On day 10, EBs were embedded in matrigel (matrigel growth factor reduced, Corning) with CSC spheres (previously infected with RFP alone or SES+RFP) to fuse in the same matrigel droplet and then gelled at 37°C for 30-60 minutes. The embedded EB-CSCs were then cultured in neural maturation medium containing 50% DMEM / F12, 50% Neurobasal A, 0.5X N-2 supplement, 0.5X B-27 supplement (without vitamin A), 2mM glutamine, 2.5ng / ml human insulin, 1% non-essential amino acids and 25nM β-mercaptoethanol. The droplets were cultured in static conditions in a 6cm suspension dish for 4 days and then transferred to an orbital shaker (Orbit) that rotated continuously at 60rpm. TMLS Low Speed ​​Orbital Shaker); in this, the 0.5XB-27 supplement containing vitamin A in the neural maturation medium was replaced.

[1163] Behavioral testing

[1164] Animals were housed at a constant temperature of 23°C with a 12-hour light / dark cycle (lights off at 19:00) and had free access to food and water. We analyzed WT C57BL / 6 mice, both male and female, at the adult stage (range 2 to 4 months old) 4 weeks before infection with GFP (mock) or SES in their hippocampus (all tests). This section was recorded by the video tracking software Ethovision XT (Noldus).

[1165] Spontaneous alternation test. To test exploratory behavior and cognitive functions related to spatial learning and memory, mice were placed in a 4-arm maze and a 10-min video was recorded to evaluate: the total number of entries into all arms, the percentage of entries into different arms, and the number of consecutive entries into different arms. The latter allows the identification of behavioral patterns (see also Fig. 9 e): spontaneous alternating presentation (SAP), a scoring index in which visits to 4 different arms without duplication are scored as 1, while at least one duplication in a series of 4 entries is scored as 0; alternating arm returns, a scoring index in which at least one duplication in a series of 3 entries is scored as 1; same arm returns (SAR), a scoring index in which two consecutive entries in the same arm are scored as 1.

[1166] Radial maze test. The eight-arm radial maze consists of eight identical arms extending radially from an octagonal platform. It is elevated 80 cm above the ground and is surrounded by external cues. A cup with food is placed at the end of each arm. The protocol is divided into different phases: Day 1 - 10 min acclimatization on the apparatus (no food at the end of the arm). Day 2 - fasting until the animals reach 80%-85% of their initial body weight; mice must maintain this weight during the experiment. Day 3 - training: the food is divided in half and placed at the end of each arm. The mouse is released in the center of the arena and it must eat two of the eight pellets placed at the end of the arm. Days 4-13 ( Fig.10 Experimental Days 1-10 in f) - Test: Pellets were placed only at the ends of the eight arms. The mouse was released into the center of the arena and (i) the time required to consume the eight pellets and (ii) the percentage of incorrect choices (mice chose the empty arm) to the total entries were calculated. The maze was cleaned with water and 70% ethanol before the next mouse was placed on the apparatus.

[1167] Morris Water Maze Test. Mice are placed in a circular pool with a platform that allows them to escape from the water (maximum length per trial 120”). The release point can be located in different quadrants of the pool (see Fig. 9 g), the platform position was the same for the first 3 days of the protocol and reversed for the last 2 days of the protocol. The time to complete each trial and the time spent in the platform zone and the opposite quadrant were quantified.

[1168] Neurons from iPSCs

[1169] WT iPSCs were maintained in feeder-free conditions in mTeSR1 (Stem Cell Technologies) supplemented with Pen / Strept and seeded on six-well plates coated with human embryonic stem cells (HESC) qualified matrigel (Corning); cells were fed daily and passaged in cell clumps weekly using Accutase solution (Sigma-Aldrich). On differentiation day -2, 90% confluent iPSC cultures were infected overnight with the lentiviral vector TetO-Ngn2-T2A-Puro in mTeSR1 medium supplemented with doxycycline (2 μg / ml, Sigma-Aldrich). The next day, the medium was replaced with fresh mTeSR1 medium supplemented with antibiotic selection (puromycin 1 μg / ml, Sigma-Aldrich) and doxycycline; doxycycline was maintained in all experiments. At day 0, the medium was replaced with differentiation medium "mTeSR1+LSBX". Differentiation medium was changed daily according to the following scheme: Day 0, 1: mTeSR1 + LSBX; Day 2, 3: mTeSR1 + LSBX + PSD; Day 4, 5: 2 / 3mTeSR1 + 1 / 3N-2 medium + LSX + PSD; Day 6, 7: 1 / 3mTeSR1 + 2 / 3N-2 medium + PSD. On day 8, cells were detached by incubation with Accutase solution at 37°C for 20 min to obtain a single cell suspension. Cells were centrifuged, counted, and centrifuged at 55,000 cells / cm 2 The cells were seeded at a density of 100 μM on poly-L-lysine / laminin / fibronectin coated plates or coverslips in neuronal maturation medium supplemented with ROCK inhibitor Y27632 (10 μM, Selleckchem) for the first 24 hours. The medium was changed the next day to remove the ROCK inhibitor, and then half of the medium was replaced with fresh neuronal maturation medium twice a week.

[1170] LSBX: LDN193189 (Stemgent, 250nm), SB431542 (Sigma-Aldrich, 10μM), XAV939 (Sigma-Aldrich, 5μM). PSD: PD0325901 (Sigma-Aldrich, 8μM), SU5402 (Sigma-Aldrich, 10μM), DAPT (Sigma-Aldrich, 10μM). N-2 medium: DMEM / F12 with B-27 supplement (0.5X, ThermoFisher Scientific) and N-2 supplement (0.5X, ThermoFisher Scientific). Neuronal maturation medium: Neurobasal A (ThermoFisher Scientific), supplemented with 1X B-27 supplement, 2 mM glutamine, 1% Pen / Strept, BDNF (Peprotech, 20 ng / ml), ascorbic acid (Sigma-Aldrich, 100 nM), laminin (1 μg / μl), DAPT (10 μM), dbcAMP (Selleckchem, 250 μM).

[1171] Primary mouse neuron culture

[1172] Primary cultures of mouse embryonic cortical neurons were prepared from E17.5 C57BL / 6 wild-type mice. Briefly, after dissection, the cortex was enzymatically digested with 0.025% trypsin (GIBCO) in Hank's balanced salt solution (HBSS) (Euroclone) at 37°C for 20 minutes. Subsequently, the HBSS containing trypsin was removed, and the hippocampus was washed with plating medium (Neurobasal A medium supplemented with 1X B-27 supplement, 3.3mM glucose, 2mM glutamine and 1% penicillin / streptomycin) and mechanically dissociated with a P1000 pipette to obtain a uniform cell suspension. The cells were then plated on glass coverslips coated with poly-L-lysine (PLL) (0.1mg / ml).

[1173] Example B

[1174] Materials and methods

[1175] Construct

[1176] TES v2: The first part of the TEAD1 gene, encoding amino acids (aa) 1-166 (thus excluding the TEAD1 activator domain), is fused at its N-terminus to the Chromo Shadow (CS) repressor domain (from the gene CBX5, encoding amino acids 121-179), while the V5 tag is fused at the end of the last domain, i.e., at the C-terminus of the new chimeric transgene. This transgene is used in a lentiviral construct with Ef1a as promoter.

[1177] TES v2 (SEQ ID NO: 136):

[1178] Ef1a::CS-hTEAD 1-166 –V5[TESv2]

[1179]

[1180]

[1181] Ef1a promoter

[1182] CS

[1183] hTEAD 1-166

[1184] V5

[1185] TES v3: The beginning part of the TEAD1 gene, encoding amino acids 1-166 (thus excluding the TEAD1 activation domain), is fused at its C-terminus to the YAF2-RYBP (YR) repressor domain (from the gene RYBP, amino acids 145-189), and the V5 tag is fused to the C-terminus of YR. This transgene is used in a lentiviral construct with Ef1a as promoter.

[1186] TES v3 (SEQ ID NO: 137):

[1187] Ef1a::hTEAD 1-166 -YR–V5[TESv3]

[1188]

[1189]

[1190] Ef1a promoter

[1191] hTEAD 1-166

[1192] Y

[1193] V5

[1194] TES v4:TES v2 is fused at its C-terminus to the YAF2-RYBP (YR) repressor domain (from the gene RYBP, amino acids 145-189) and a V5 tag is fused to the C-terminus of YR. This transgene is used in a lentiviral construct with Ef1a as promoter.

[1195] TES v4 (SEQ ID NO: 138):

[1196] Ef1a::CS-hTEAD 1-166 –YR–V5[TESv4]

[1197]

[1198]

[1199]

[1200] Ef1a promoter

[1201] CS

[1202] hTEAD 1-166

[1203] yr

[1204] V5

[1205] MES v2: The C-terminal part of the MYC gene, encoding amino acids 144-454 (thus excluding the MYC activation domain), is fused at its N-terminus to the Chromo Shadow (CS) repressor domain (from the gene CBX5, amino acids 121-179) and the V5 tag is fused at the C-terminus of the new transgene. This transgene is used in a lentiviral construct with Ef1a as promoter.

[1206] MES v2 (SEQ ID NO: 139):

[1207] Ef1a::CS-hMYC 144-454 –V5[MESv2]

[1208]

[1209]

[1210]

[1211] Ef1a promoter

[1212] CS

[1213] HkDJ 144-454

[1214] V5

[1215] MES v3: The C-terminal part of the MYC gene, encoding amino acids 144-454 (thus excluding the MYC activation domain), is fused at its N-terminus to the YAF2-RYBP (YR) repressor domain (from the gene RYBP, amino acids 145-189), while the V5 tag is fused at the C-terminus of the new transgene. This transgene is used in a lentiviral construct with Ef1a as promoter.

[1216] MES v3 (SEQ ID NO: 140):

[1217] Ef1a::YR-hMYC 144-454 –V5[MESv3]

[1218]

[1219]

[1220]

[1221] Ef1a promoter

[1222] Y

[1223] HkDJ 144-454

[1224] V5

[1225] MES v4: The C-terminal part of the MYC gene, encoding amino acids 144-454 (thus excluding the MYC activation domain), fused at its N-terminus to the Chromo Shadow (CS) repressor domain (from the gene CBX5, amino acids 121-179) and the YAF2-RYBP (YR) repressor domain (from the gene RYBP, amino acids 145-189) in tandem, and a V5 tag fused at the C-terminus of the new transgene. This transgene was used in a lentiviral construct with Ef1a as promoter.

[1226] MES v4 (SEQ ID NO: 141):

[1227] Ef1a::CS-YR-hMYC 144-454 –V5[MESv4]

[1228]

[1229]

[1230]

[1231] Ef1a promoter

[1232] CS

[1233] yr

[1234] HkDJ 144-454

[1235] V5

[1236] ESF-Tmir: Tmir box, composed of miR124 ( atattgccttatttc[SEQ ID NO:1]) 、MiR338-3p( caacaaaatcactgatgctgga[SEQ ID NO:2]) and miR31 The target sequences are composed of four tandem sequences, generated synthetically and cloned into the 3'UTR of ESF.

[1237] The triple miRNA target sequence (Tmir) is shown in SEQ ID NO:4:

[1238]

[1239]

[1240] miR124 target sequences

[1241] miR338-3p target sequences

[1242]

[1243] The Tmir cassettes containing the ESFs are listed in the following order.

[1244] SES-Tmir (SEQ ID NO: 142):

[1245] Ef1a::KRAB-hSOX2 1-179 -DNMT3a3L-V5-Tmir[SES-Tmir]

[1246]

[1247]

[1248]

[1249]

[1250] Ef1a promoter

[1251] KRAB

[1252] hSOX2 1-179

[1253] DNMT3a3L

[1254] V5

[1255] miR124 target sequences

[1256] miR338-3p target sequence

[1257]

[1258] SES(SEQ ID NO:155):

[1259] Ef1a::KRAB-hSOX2 1-179 -DNMT3a3L-V5[SES]

[1260]

[1261]

[1262]

[1263]

[1264] Ef1a promoter

[1265] KRAB

[1266] hSOX2 1-179

[1267] DNMT3a3L

[1268] V5

[1269] SESv2-Tmir(SEQ ID NO:143):

[1270] Ef1a::CS-hSOX2 1-179 –V5-Tmir[SESv2-Tmir]

[1271]

[1272]

[1273]

[1274] Ef1a promoter

[1275] CS

[1276] hSOX2 1-179

[1277] V5

[1278] miR124 target sequences

[1279] miR338-3p target sequences

[1280]

[1281] SESv2 (SEQ ID NO: 156):

[1282] Ef1a::CS-hSOX2 1-179 –V5[SESv2]

[1283]

[1284]

[1285]

[1286] Ef1a promoter

[1287] CS

[1288] hSOX2 1-179

[1289] V5

[1290] SESv3-Tmir (SEQ ID NO: 144):

[1291] Ef1a::SOX2 1-179 -YR-V5-Tmir[SESv3-Tmir]

[1292]

[1293]

[1294]

[1295] Ef1a promoter

[1296] hSOX2 1-179

[1297] Y

[1298] V5

[1299] miR124 target sequences

[1300] miR338-3p target sequence

[1301]

[1302] SESv3 (SEQ ID NO:157):

[1303] Ef1a::SOX2 1-179 -Y-R-V5 [SESv3]

[1304]

[1305]

[1306]

[1307] Ef1a promoter

[1308] hSOX2 1-179

[1309] Y

[1310] V5

[1311] TES-Tmir (SEQ ID NO:145):

[1312] Ef1a::KRAB-hTEAD 1-166 -DNMT3a3L-V5-Tmir [TES-Tmir]

[1313]

[1314]

[1315]

[1316]

[1317] Ef1a promoter

[1318] KRAB

[1319] hTEAD 1-166

[1320] DNMT3a3L

[1321] V5

[1322] miR124 target sequences

[1323] miR338-3p target sequence

[1324]

[1325] TES (SEQ ID NO:153):

[1326] Ef1a::KRAB-hTEAD 1-166 -DNMT3a3L-V5[TES]

[1327]

[1328]

[1329]

[1330]

[1331] Ef1a promoter

[1332] KRAB

[1333] hTEAD 1-166

[1334] DNMT3a3L

[1335] V5

[1336] TESv2-Tmir (SEQ ID NO:146):

[1337] Ef1a::CS-hTEAD 1-166 –V5-Tmir[TESv2-Tmir]

[1338]

[1339]

[1340]

[1341] Ef1a promoter

[1342] CS

[1343] hTEAD 1-166

[1344] V5

[1345] miR124 target sequences

[1346] miR338-3p target sequence

[1347]

[1348] TESv3 - Tmir (SEQ ID NO:147):

[1349] Ef1a::hTEAD 1-166 -YR–V5 - Tmir[TESv3 - Tmir]

[1350]

[1351]

[1352]

[1353] Ef1a promoter

[1354] hTEAD 1-166

[1355] Y - R

[1356] V5

[1357] miR124 target sequences

[1358] miR338-3p target sequence

[1359]

[1360] TESv4 - Tmir (SEQ ID NO:148):

[1361] Ef1a::CS - hTEAD 1-166 –Y - R - V5 - Tmir[TESv4 - Tmir]

[1362]

[1363]

[1364]

[1365] Ef1a promoter

[1366] CS

[1367] hTEAD 1-166

[1368] y-r

[1369] V5

[1370] miR124 target sequences

[1371] miR338-3p target sequence

[1372]

[1373] MES-Tmir(SEQ ID NO:149):

[1374] Ef1a::KRAB-DNMT3a3L-hMYC 144-454 -V5-Tmir[MES-Tmir]

[1375]

[1376]

[1377]

[1378]

[1379]

[1380] Ef1a promoter

[1381] KRAB

[1382] DNMT3a3L

[1383] hMYC 144-454

[1384] V5

[1385] miR124 target sequences

[1386] miR338-3p target sequence

[1387]

[1388] MES(SEQ ID NO:154):

[1389] Ef1a::KRAB-DNMT3a3L-hMYC 144-454 -V5[MES]

[1390]

[1391]

[1392]

[1393]

[1394] Ef1a promoter

[1395] KRAB

[1396] DNMT3a3L

[1397] hMYC 144-454

[1398] V5

[1399] MESv2-Tmir (SEQ ID NO:150):

[1400] Ef1a::CS-hMYC 144-454 –V5-Tmir[MESv2-Tmir]

[1401]

[1402]

[1403]

[1404] Ef1a promoter

[1405] CS

[1406] hMYC 144-454

[1407] V5

[1408] miR124 target sequences

[1409] miR338-3p target sequence

[1410]

[1411] MESv3-Tmir (SEQ ID NO:151):

[1412] Ef1a::YR-hMYC 144-454 –V5-Tmir[MESv3-Tmir]

[1413]

[1414]

[1415]

[1416] Ef1a promoter

[1417] Y

[1418] HkDJ 144-454

[1419] V5

[1420] miR124 target sequences

[1421] miR338-3p target sequence

[1422]

[1423] MESv4-Tmir (SEQ ID NO: 152):

[1424] Ef1a::CS-YR-hMYC 144-454 –V5-Tmir[MESv4-Tmir]

[1425]

[1426]

[1427]

[1428]

[1429] Ef1a promoter

[1430] CS

[1431] yr

[1432] HkDJ 144-454

[1433] V5

[1434] miR124 target sequences

[1435] miR338-3p target sequence

[1436]

[1437] Cell culture

[1438] U-251 and CT26 cells were cultured in DMEM medium (Dulbecco's Modified Eagle's Medium-high glucose, Sigma-Aldrich) containing 10% fetal bovine serum (FBS, Sigma-Aldrich), 1% Pen / Strept (Sigma-Aldrich), 2 mM glutamine (Sigma-Aldrich), 1% non-essential amino acids (MEM NEAA, ThermoFisher Scientific), and 1% sodium pyruvate solution (Sigma-Aldrich) under plastic adherence conditions and passaged twice a week using trypsin-EDTA solution (Sigma-Aldrich).

[1439] BxPC-3 and CFPAC-1 were cultured in RPMI-1640 (Sigma-Aldrich) containing 10% FBS, 1% Pen / Strept, 2 mM glutamine under plastic adherent conditions. All cell lines were passaged twice a week using trypsin-EDTA solution (Sigma-Aldrich).

[1440] Cancer stem cell (CSC) glioblastoma tumors were maintained under plastic adherent conditions in DMEM / F12 (Sigma-Aldrich) supplemented with a hormone cocktail (DMEM / F12, 0.6% glucose (Sigma-Aldrich)) (30% in phosphate buffered saline (PBS) (Euroclone)), insulin (Sigma-Aldrich) 250 μg / ml, putrescine powder (Sigma-Aldrich) 97 μg / ml, apotransferrin powder (Sigma-Aldrich), sodium selenite 0.3 μM, progesterone 0.2 μM, 1% Pen / Strept, 2 mM glutamine, 0.66% glucose (30% in phosphate buffered saline (PBS) (Euroclone)) and heparin (4 mg / ml, Sigma-Aldrich); bFGF (20 ng / ml, ThermoFisherScientific) and EGF (20 ng / ml, ThermoFisher Scientific) were freshly added to the culture medium.

[1441] All cultures were maintained under atmospheric oxygen conditions at 37°C in a humidified atmosphere of 5% CO2.

[1442] Growth curve analysis

[1443] On day 0, 5x10 5Cells were seeded in 6-well plates under adherent conditions; on day 1, the cultures were infected with lentiviral vectors, and on day 3, the cells were detached, live cells were stained with trypan blue solution (0.4%, ThermoFisher Scientific), and counted using Countess TM II automatic cell counter (ThermoFisher Scientific); after this passage, 1.5x10^5 cells were seeded again. Repeat 3-4 time points every 3-4 days; repeat the experiment 3 times for each time point.

[1444] Primary mouse neuron culture

[1445] Primary cultures of mouse embryonic cortical neurons were prepared from E17.5 C57BL / 6 wild-type mice. Briefly, after dissection, the cortex was enzymatically digested with 0.025% trypsin (GIBCO) in Hank's balanced salt solution (HBSS) (Euroclone) at 37°C for 20 minutes. Subsequently, the HBSS containing trypsin was removed, and the hippocampus was washed with plating medium (Neurobasal A medium supplemented with 1X B-27 supplement, 3.3mM glucose, 2mM glutamine and 1% penicillin / streptomycin) and mechanically dissociated with a P1000 pipette to obtain a uniform cell suspension. The cells were then plated on glass coverslips coated with poly-L-lysine (PLL) (0.1mg / ml).

[1446] Immunostaining

[1447] Cells were seeded on glass coverslips and fixed on ice for 20 minutes in a solution of 4% paraformaldehyde (PFA, Sigma) in phosphate-buffered saline (PBS, Euroclone). They were then washed twice with PBS and permeabilized for 30' in a blocking solution containing 0.2% Triton X-100 (SigmaAldrich) and 5% donkey serum (Euroclone), and incubated overnight at 4°C with primary antibodies diluted in blocking solution. The primary antibodies used were as follows: anti-V5 (mouse, 1:500, ThermoFisher Scientific, R96025), anti-GFP (chicken, 1:1000, Thermo Fisher Scientific, A10262), anti-MAP2 (chicken, 1:1000, Abcam, ab92434). The next day, cells were washed three times with PBS for 5 minutes each time and incubated in blocking solution with Hoechst 33342 (ThermoFischer Scientific) and secondary antibodies (ThermoFisherScientific) at room temperature for 1 hour. Finally, slides were washed and mounted in fluorescent mounting medium (DakoCytomation). Images were collected using an epifluorescence microscope Nikon DS-Qi2 and analyzed using Fiji software.

[1448] AAV production and infection

[1449] Replication-incapable recombinant virus particles were produced in 293T cells by co-transfection of the following three different plasmids by polyethyleneimine (PEI) (Polyscience): a packaging plasmid containing a transgenic plasmid, rep and cap genes, and pHelper (Agilent) for three adenovirus helper genes. Cells and supernatants were harvested at 120hr. Cells were lysed at 37°C for 1hr in a hypertonic buffer (40mMTris, 500mM NaCl, 2mM MgCl2, pH=8) containing 100U / ml salt active nuclease (SAN, Arcticzymes), and the virus particles present in the supernatant were concentrated by precipitation with 8% PEG8000 (polyethylene glycol 8000, Sigma-Aldrich), and then added to the supernatant for additional incubation of 30min at 37°C. In order to clarify the lysate, cell debris was separated by centrifugation (4000g, 30min). The virus phase was separated by an iodixanol step gradient (15%, 25%, 40%, 60% Optiprep, Sigma-Aldrich) in the 40% fraction and concentrated in PBS (phosphate buffered saline) using a 100K cutoff concentrator (Amicon Ultra15, MERCK-Millipore). Virus titer was determined using AAVpro Titration Kit Ver2 (TaKaRa).

[1450] 2,5x10^4 CSC L0627 were infected with adenoviral vector expressing GFP (5 μl / well) and seeded on glass coverslips previously coated with Matrigel in 24-well plates. After 4 days, the cells were fixed and used for immunostaining studies.

[1451] Example B1

[1452] The inventors attempted to construct a set of ESF variants by using TEAD1 and c-MYC, two transcriptional activators with oncogenic activity. ESFs were generated by rational design, in which the transcriptional activation domains of the relevant transcription factors (TFs) were removed and alternative repression domains (i.e., the chromo shadow (CS) domain from the CBX5 protein and the YAF2-RYBP (YR) domain from the RYBP protein) were added to them. TEAD epigenetic silencers (TESs) were generated by fusing CS to the N-terminus of human TEAD amino acids 1-166 (TES v2); fusing YR to the C-terminus of human TEAD amino acids 1-166 (TES v2); fusing CS to the N-terminus of human TEAD amino acids 1-166 and fusing YR to the C-terminus of human TEAD amino acids 1-166 (TES v2) ( Fig.17 a). MYC epigenetic silencers (MES) were generated by fusing CS or YR or both to the N-terminus of amino acids 144-454 of human MYC to avoid steric hindrance at the C-terminus of the protein, which directly interacts with MAX to form heterodimers (MESv2-4) ( Fig.17 b).

[1453] Example B2

[1454] ESF should not have any obvious and harmful effects on healthy brain cells, which do not express the oncogene of interest and are not proliferative cells. In such healthy cells, the reduction of some key cell cycle genes induced by ESF may be harmful to the cells. However, ESF-dependent chromatin changes may be able to change the performance of neurons in vivo over a longer period of time, for example, if it is used for human treatment. Therefore, the inventors designed a strategy to limit ESF expression in cancer cells after viral inoculation of the brain. The strategy is based on a microRNA (miRNA) detargeting system that enables transgene silencing by including binding / target sites (TS) of endogenously expressed miRNAs in specific cell types in which expression is not desired. To this end, a cassette was generated in which the 3'-UTR downstream of the transgene (e.g., ESF cDNA) contained a cluster of 4x TS of each of miRNA-124, miRNA-338-3p and miRNA-31 (which are specifically expressed in neurons, astrocytes and oligodendrocytes, respectively). Therefore, exogenous transgene expression, for example Fig.18 SES expression, as shown, was silenced in all aforementioned cell types through miRNA-dependent post-transcriptional silencing and protein translation blockade ( Fig.18a). Notably, miRNA-124, miRNA-338-3p, and miRNA-31 were not expressed in GBM, as demonstrated by GFP-Tmir and SES-Tmir lentiviruses (LVs), which displayed unaffected transgene expression in cancer cells ( Fig.18 b). The data herein further demonstrate that the presence of Tmir does not compromise SES activity, as the construct induced significant cell loss in transduced cancer cells in vitro ( Fig.18 b, lower right). The specificity of the miRNA de-targeting system is Fig.18 c, where the data show that, as expected and due to miRNA detargeting, no cells expressed SES when applied to primary cortical cultures from mice ( Fig.18 c).

[1455] Example B3

[1456] It is expected that ESF will be used with a delivery system that allows for easy and widespread distribution of the vector over brain regions that may contain residual tumor cells after surgery. This will reduce the likelihood of tumor recurrence, which is a significant problem in GBM. To this end, the inventors tested the use of adeno-associated virus (AAV) as a shuttle vector for the constructs of the invention. Recombinant serotypes 2 and 5 performed better than serotype 9 for transgene delivery within patient-derived cancer stem cells as assessed by GFP expression ( Fig.19 ).

[1457] Example B4

[1458] Since SOX2, TEAD1 and MYC are widely expressed in many cancer types, it is envisioned that it may be possible to expand the use of the ESFs disclosed herein to other cancer types, particularly where these types can be effectively targeted by viral transduction in vivo. For example, the inventors show herein that SES are able to reduce the growth of a murine cell line (CT26) derived from liver metastases of colon adenocarcinoma ( Fig. 20 a). In addition, TES was also effective in reducing the growth of human pancreatic ductal adenocarcinoma (PDAC) cell lines, including BxPC-3 and CFPAC-1 ( Fig. 20 b and 20c).

[1459] discuss

[1460] The activity of developmental transcription factors (TFs) is mainly restricted during morphogenesis, playing important roles in stem cell identity, cell lineage commitment, and differentiation. However, these TFs can be reactivated or manipulated by cancer genetic programs to drive tumor development and progression. It is estimated that approximately 20% of all known oncoproteins are represented by TFs, which are essential for the acquisition of malignant cell dedifferentiation, proliferation, and migration. Despite their ubiquitous roles in tumors, interfering with their functions from a translational perspective has proven challenging. In fact, stable and complete gene silencing by various genetic tools or small molecules has been difficult to achieve in cancer cells. In addition, cancer genetic programs have been repeatedly shown to overcome the inactivation of single genes by reconfiguring transcriptional networks, thereby promoting cancer resistance and recurrence. In this paper, we designed additional versions of ESFs to target ESF expression or the expression of any relevant transgenes from brain-resident cells by rationally assembling transcriptional and epigenetic negative regulators of gene transcription and miRNA-based cassettes to reconfigure SOX2, TEAD1, and MYC TFs.

[1461] Herein, the inventors demonstrate that ESF configurations utilizing different epigenetically active protein domains (e.g., the ChromoShadow domain from CBX5 and the YAF2-RYBP domain from RYBP) can elicit the same functional activity as those ESFs carrying KRAB and DNMT3A / L domains. Furthermore, by using miRNA target sequences within the 3'UTR, it was demonstrated to target exogenous ESF expression from healthy brain cells (e.g., neurons, astrocytes, and oligodendrocytes), thereby providing safety and specificity. By utilizing miRNA target sequences that are highly expressed in brain cells but not expressed in tumor cells, the inventors provide polynucleotides and transgenic expression cassettes with high specificity, for example, for use as anti-GBM treatments.

[1462] Herein, the inventors also provide alternative therapeutic viruses to lentiviruses that can be used similarly. Particularly useful are adeno-associated virus (AAV) strains that can diffuse through brain tissue due to their small size and exhibit reduced binding to cell membranes. Maximizing the spread of the virus in the brain parenchyma will increase the targeting efficiency of cancer cells dispersed in the tissue, which provides better protection from tumor recurrence. Herein, the inventors demonstrate that AAV2 diffuses in the brain parenchyma when injected directly into the organ and is able to infect GBM CSCs in vitro with high efficiency.

[1463] The inventors further provide evidence that, due to the important role played by selected TFs in promoting tumor development, ESFs can serve as effective anticancer agents / treatments in a variety of cancers that share common vulnerabilities. In particular, herein, the inventors demonstrate that SES is able to reduce the proliferation of cells derived from liver metastases of colon adenocarcinoma in mice, and that TES is effective in blocking the growth of human PDAC cell lines.

[1464] In summary, the disclosure herein provides a group of epigenetic repressors that function as dominant negative versions of oncogenic TFs SOX2, TEAD1, and MYC and are able to bind and stably repress their respective transcriptional networks. Targeted viral delivery of these ESFs in glioblastoma cells is sufficient to inhibit tumor development by blocking cell proliferation and inducing cell death. The assembly of miRNA target sequences provides specificity and safety, and these target sequences are able to target, i.e., inhibit the expression of ESFs from specific brain cells, while ESFs are expressed in tumor cells. In view of the broad applicability to other oncogenic TFs and the high efficiency of targeting cancer cells by viral transduction using viral vectors including AAV, this approach provides an opportunity to repress glioblastoma and other deadly and difficult-to-treat cancers.

[1465] Example C1

[1466] result

[1467] We further tested SESv3 in SNB19 glioma cells, in which lentivirus infection reached approximately 100% ( Fig.21 a). We demonstrate that SESv3 has similar efficacy to the parental SES factor in terms of cell number and cell death ( Fig.21 b and c).

[1468] On the same cell lines, we challenged the efficacy of TES compared with: (i) a version of TES carrying the inactivating mutation R85K (TESmut), which destabilizes TEAD DNA binding ability (Cao et al. (2008) Genes Dev 22:3320-34), (ii) verteporfin, a chemical inhibitor of YAP / TAZ signaling (Liu-Chittenden et al. (2012) Genes Dev 26:1300-5), and (iii) GFP and (iv) uninfected cells as controls ( Fig. 22 We show that TES has activity similar to that of chemical inhibitors, whereas a version with limited DNA binding activity has only a minor effect on cell growth ( Fig. 22 a, b), which demonstrates TES specificity.

[1469] We next set out to assess the overall TES transcriptional output compared to mock (GFP) infected cells. TES-treated SNB19 cells exhibited dramatic transcriptional changes ( Fig.23 a), where known YAP / TAZ targets were generally downregulated ( Fig.23 b), which shows a high presence of TEAD binding motifs ( Fig.23 c). Interestingly, among the downregulated genes, we found signatures of DNA repair, cell division and migration, whereas among the upregulated Gene Ontologies, we demonstrated several categories related to neural differentiation ( Fig.23 d). Gene set enrichment analysis (GSEA) confirmed a substantial downregulation of genes associated with cell migration and motility ( Fig.24 a), which has been associated with YAP / TAZ function (Zhang et al. (2018) J Mol Neurosci 64:262-272). Therefore, we directly measured the inhibition of cell migration ability after SNB19 cell treatment using an in vitro wound healing assay. We found that wounds of uninfected cells healed efficiently 48 hours after incision, while cells treated with TES or verteporfin healed more slowly ( Fig.24 b). We also found that, in this context, inhibition of the DNA binding activity of TES is essential for limiting the ability of cells to migrate ( Fig.24 b).

[1470] To expand our knowledge of SES activity on cancer types other than GBM, we tested SES efficacy in cell lines established from liver metastases arising from both colorectal cancer (CRC) and pancreatic adenocarcinoma (PDAC) in genetically engineered mouse models that are commonly used for preclinical applications on these cancer types. We generated stable lines carrying conditional constructs to express SES in a CRE-dependent manner (Flex-SES, Fig.25 a). After CRE transduction, both cancer cell lines showed extreme sensitivity to SES already 6 days after their induction ( Fig.25 b and c).

[1471] Materials and methods

[1472] Cell growth analysis

[1473] On day 0, a total of 1.5 × 10 5SNB19 were seeded in six-well plates under adherent conditions; on day 1, the cultures were infected with LV vectors (MOI = 0.7), and on day 3, the cells were detached and live cells were stained with trypan blue solution (0.4%, ThermoFisher Scientific) and counted using a Countess II automated cell counter (ThermoFisher Scientific); after this passage, 1.5 x 10 5 cells. Three time points were repeated every 3 to 4 days; the experiment was repeated at least three times for each time point. The YAP / TAZ inhibitor Vertoporfin was used at a concentration of 2 uM instead of LV infection.

[1474] RNA-seq and analysis

[1475] RNA libraries were generated starting from 1 mg of total RNA and their quality was assessed using a TapeStation instrument (Agilent). To avoid overrepresentation of 30 ends, only high-quality RNA with an RNA integrity number >8 was used. RNA was processed according to the TruSeq Stranded mRNA Library Prep Kit protocol. Libraries were sequenced on an Illumina HiSeq3000 with 76 bp stranded reads using Illumina TruSeq technology. Image processing and base calling were performed using Illumina Real-Time Analysis Software. FASTQ files were aligned to the hg38 human reference genome by using a splice junction map per TopHat. Differential gene expression and functional enrichment analyses were performed using DESeq2 and gene set enrichment analysis, respectively. De novo enriched motifs were found in the promoters of downregulated genes using Homer software with the following settings: +1,000–100 from TSS.

[1476] Wound healing assay

[1477] Wound healing assay was performed according to Zhang et al. (2018) J Mol Neurosci 64:262-272. At the specified time, five randomly selected fields of view at the lesion boundary were collected under an inverted microscope (Olympus, IX71). The average number of cells in the control group was defined as 100%, and the average number of cells in other groups was normalized by the control group.

[1478] Flex-SES

[1479] The SES transgene (for SES v1) is inserted in reverse orientation between two tandem LoxP sites (LoxP and Lox2272) in a LV vector with Ef1a as promoter. The CRE recombinase is necessary to flip the transgene to the correct orientation for productive expression.

[1480] Implementation

[1481] Various preferred features and embodiments of the invention will now be described with reference to the following numbered paragraphs.

[1482] 1. A polynucleotide comprising at least one miR-124 target sequence, and / or at least one miR-338-3p target sequence, and / or at least one miR-31 target sequence, wherein the miRNA target sequence is operably linked to a transgene.

[1483] 2. The polynucleotide of paragraph 1, wherein the number of copies of each miRNA target sequence is independently selected from the group consisting of: one, two, three, and four.

[1484] 3. The polynucleotide of paragraph 1 or paragraph 2, wherein the polynucleotide comprises four miR-124 target sequences, four miR-338-3p target sequences, and four miR-31 target sequences, wherein the target sequences are operably linked to a transgene.

[1485] 4. The polynucleotide of any one of paragraphs 1 to 3, wherein:

[1486] (a) the miR-124 target sequence comprises or consists of: a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 1;

[1487] (b) the miR-338-3p target sequence comprises or consists of a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 2; and / or

[1488] (c) The miR-31 target sequence comprises or consists of a nucleotide sequence having at least 90% sequence identity to SEQ ID NO:3.

[1489] 5. The polynucleotide of any one of paragraphs 1 to 4, wherein the miRNA target sequence is located after the transgene in the 5' to 3' direction.

[1490] 6. The polynucleotide of any one of paragraphs 1 to 5, wherein the miRNA target sequence or the cluster of copies of the miRNA target sequence is arranged in the following order from 5' to 3': one or more miR-124 target sequences, one or more miR-338-3p target sequences and one or more miR-31 target sequences.

[1491] 7. The polynucleotide of any one of claims 1 to 6, wherein the miRNA target sequence is separated by a spacer sequence.

[1492] 8. The polynucleotide of any one of paragraphs 1 to 7, wherein the polynucleotide comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO:4.

[1493] 9. The polynucleotide of any one of paragraphs 1 to 8, wherein the transgene encodes an epigenetic silencing factor (ESF) comprising a transcription factor DNA binding domain operably linked to at least one epigenetic effector domain, wherein the transcription factor is an oncogenic transcription factor or a cancer-associated transcription factor.

[1494] 10. The polynucleotide of any of paragraph 9, wherein the transcription factor is selected from the group consisting of SOX2, MYC, MYCN, TEAD1, TEAD2, TEAD3, TEAD4, FOXA1, FOXA2, ELK1, ELK3, ELK4, SRF, FOXM1, FOXC1, FOXC2, TWIST1, SALL4, ELF1, HIF1A, SOX9, SOX12, SOX18, ETS1, PAX3, PAX8, GLI1, GLI2, GLI3, ETV1, ETV2, ETV3, RUNX1, RUNX2, RUNX3, MAFB, TFAP2C and E2F1.

[1495] 11. The polynucleotide of paragraph 9 or paragraph 10, wherein the transcription factor is SOX2, TEAD1 or MYC.

[1496] 12. The polynucleotide of paragraphs 9 to 11, wherein the epigenetic effector domain is selected from the group consisting of a KRAB domain, a DNMT3A domain, a DNMT3L domain, a ZIM3-KRAB (Z-KRAB) domain, a Chromo Shadow (CS) domain, a YAF2-RYBP (YR) domain, a Jagged repressor (En-R) domain, a MeCP2 domain, a GLI3RD domain, and a MAD1RD domain.

[1497] 13. The polynucleotide of any one of paragraphs 9 to 12, wherein the ESF comprises: (a) a CS domain; (b) a YR domain; (c) a CS domain and a YR domain; (d) a KRAB domain; and / or (e) a DNMT3A domain and a DNMT3L domain.

[1498] 14. The polynucleotide of any one of paragraphs 9 to 13, wherein the ESF comprises:

[1499] (a) KRAB domain, SOX2 DNA binding domain, DNMT3A domain, and DNMT3L domain;

[1500] (b) Chromo Shadow (CS) domain and SOX2 DNA binding domain;

[1501] (c) SOX2 DNA binding domain and YAF2-RYBP (YR) domain;

[1502] (d) KRAB domain, TEAD1 DNA binding domain, DNMT3A domain, and DNMT3L domain;

[1503] (e) KRAB domain, DNMT3A domain, DNMT3L domain and MYC DNA binding domain;

[1504] (f) Chromo Shadow (CS) domain and TEAD1 DNA binding domain;

[1505] (g) TEAD1 DNA binding domain and YAF2-RYBP (YR) domain;

[1506] (h) Chromo Shadow (CS) domain, TEAD1 DNA binding domain and YAF2-RYBP (YR) domain;

[1507] (i) Chromo Shadow (CS) domain and MYC DNA binding domain;

[1508] (j) YAF2-RYBP (YR) domain and MYC DNA binding domain; or

[1509] (k) Chromo Shadow (CS) domain, YAF2-RYBP (YR) domain and MYC DNA binding domain.

[1510] 15. The polynucleotide of any one of paragraphs 9 to 14, wherein the polynucleotide comprises a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 114-119 and 126-131.

[1511] 16. The polynucleotide of any one of paragraphs 1 to 15, wherein the polynucleotide further comprises a promoter operably linked to a transgene, optionally wherein the promoter is a tissue-specific promoter or a constitutive promoter, optionally a cancer cell-specific promoter or a proliferating cell-specific promoter.

[1512] 17. The polynucleotide of paragraph 16, wherein the promoter is Ef1a promoter or Mki67 promoter.

[1513] 18. A vector comprising the polynucleotide of any one of paragraphs 1 to 17, optionally wherein the vector is a viral vector, optionally wherein the vector is a lentiviral vector or an adeno-associated viral (AAV) vector.

[1514] 19. A protein encoded by the polynucleotide of any one of paragraphs 1 to 17 or the vector of paragraph 18.

[1515] 20. An epigenetic silencing factor (ESF) comprising a transcription factor DNA binding domain operably linked to at least one epigenetic effector domain, wherein the transcription factor is an oncogenic transcription factor or a cancer-associated transcription factor, wherein the ESF comprises:

[1516] (a) Chromo Shadow (CS) domain and TEAD1 DNA binding domain;

[1517] (b) TEAD1 DNA binding domain and YAF2-RYBP (YR) domain;

[1518] (c) Chromo Shadow (CS) domain, TEAD1 DNA binding domain and YAF2-RYBP (YR) domain;

[1519] (d) Chromo Shadow (CS) domain and MYC DNA binding domain;

[1520] (e) YAF2-RYBP (YR) domain and MYC DNA binding domain; or

[1521] (f) Chromo Shadow (CS) domain, YAF2-RYBP (YR) domain and MYC DNA binding domain.

[1522] 21. A nanoparticle comprising the polynucleotide of any one of paragraphs 1 to 17, the vector of paragraph 18, the protein of paragraph 19, or the ESF of paragraph 20.

[1523] 22. A cell comprising the polynucleotide of any one of paragraphs 1 to 17, the vector of paragraph 18, the protein of paragraph 19, the ESF of paragraph 20, or the nanoparticle of paragraph 21.

[1524] 23. A composition comprising the polynucleotide of any one of paragraphs 1 to 17, the vector of paragraph 18, the protein of paragraph 19, the ESF of paragraph 20, the nanoparticle of paragraph 21, or the cell of paragraph 22.

[1525] 24. The polynucleotide of any one of paragraphs 1 to 17, the vector of paragraph 18, the protein of paragraph 19, the ESF of paragraph 20, the nanoparticle of paragraph 21, the cell of paragraph 22, or the composition of paragraph 23 for use in therapy.

[1526] 25. The polynucleotide of any one of paragraphs 1 to 17, the vector of paragraph 18, the protein of paragraph 19, the ESF of paragraph 20, the nanoparticle of paragraph 21, the cell of paragraph 22, or the composition of paragraph 23, for use in the treatment of cancer.

[1527] 26. Use of the polynucleotide of any one of paragraphs 1 to 17, the vector of paragraph 18, the protein of paragraph 19, the ESF of paragraph 20, the nanoparticle of paragraph 21, the cell of paragraph 22, or the composition of paragraph 23 for reducing the transcription and / or expression of at least one target gene in a cell.

[1528] 27. A method for reducing the transcription and / or expression of at least one target gene in a cell, the method comprising introducing into the cell the polynucleotide of any one of items 1 to 17, the vector of item 18, the protein of item 19, the ESF of item 20, the nanoparticle of item 21, or the composition of item 23.

[1529] 28. An epigenetic silencing factor (ESF) or a polynucleotide encoding the same for use in the treatment of cancer, wherein the ESF comprises a transcription factor DNA binding domain operably linked to at least one epigenetic effector domain, wherein the transcription factor is an oncogenic transcription factor or a cancer-associated transcription factor, wherein the cancer is selected from the group consisting of glioma, glioblastoma, medulloblastoma, astrocytoma, neuroblastoma, ependymoma, meningioma, retinoblastoma, rhabdomyosarcoma, lung cancer, prostate cancer, breast cancer, liver cancer, pancreatic cancer (e.g., human pancreatic ductal adenocarcinoma), bladder cancer, oropharyngeal cancer, kidney cancer, colon cancer (e.g., colon adenocarcinoma), or metastasis of any of the above.

[1530] Additional preferred features and embodiments of the invention will now be described with reference to the following numbered paragraphs.

[1531] B1. A polynucleotide comprising at least one miR-124 target sequence, and / or at least one miR-338-3p target sequence, and / or at least one miR-31 target sequence, wherein the miRNA target sequence is operably linked to a transgene.

[1532] B2. The polynucleotide of paragraph B1, wherein the polynucleotide comprises at least one miR-124 target sequence, at least one miR-338-3p target sequence, and at least one miR-31 target sequence, wherein the miRNA target sequence is operably linked to a transgene.

[1533] B3. The polynucleotide of paragraph B1 or paragraph B2, wherein:

[1534] (a) the miR-124 target sequence comprises or consists of: a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 1;

[1535] (b) the miR-338-3p target sequence comprises or consists of a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 2; and / or

[1536] (c) The miR-31 target sequence comprises or consists of a nucleotide sequence having at least 90% sequence identity to SEQ ID NO:3.

[1537] B4. The polynucleotide of any of paragraphs B1 to B3, wherein the polynucleotide comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO:4.

[1538] B5. The polynucleotide of any of paragraphs B1 to B4, wherein the transgene encodes an epigenetic silencing factor (ESF) comprising a transcription factor DNA binding domain operably linked to at least one epigenetic effector domain, wherein the transcription factor is an oncogenic transcription factor or a cancer-associated transcription factor.

[1539] B6. The polynucleotide of any of paragraph B5, wherein the transcription factor is selected from the group consisting of SOX2, MYC, MYCN, TEAD1, TEAD2, TEAD3, TEAD4, FOXA1, FOXA2, ELK1, ELK3, ELK4, SRF, FOXM1, FOXC1, FOXC2, TWIST1, SALL4, ELF1, HIF1A, SOX9, SOX12, SOX18, ETS1, PAX3, PAX8, GLI1, GLI2, GLI3, ETV1, ETV2, ETV3, RUNX1, RUNX2, RUNX3, MAFB, TFAP2C and E2F1.

[1540] B7. The polynucleotide of paragraph B1 or paragraph B6, wherein the epigenetic effector domain is selected from the group consisting of a KRAB domain, a DNMT3A domain, a DNMT3L domain, a ZIM3-KRAB (Z-KRAB) domain, a Chromo Shadow (CS) domain, a YAF2-RYBP (YR) domain, a Jagged repressor (En-R) domain, a MeCP2 domain, a GLI3RD domain, and a MAD1RD domain.

[1541] B8. The polynucleotide of any one of paragraphs B1 to B7, wherein the polynucleotide further comprises a promoter operably linked to a transgene, optionally wherein the promoter is a tissue-specific promoter or a constitutive promoter, optionally a cancer cell-specific promoter or a proliferating cell-specific promoter.

[1542] B9. The polynucleotide of paragraph B8, wherein the promoter is Ef1a promoter or Mki67 promoter.

[1543] B10. A vector comprising the polynucleotide of any one of paragraphs B1 to B9, optionally wherein the vector is a viral vector, optionally wherein the vector is a lentiviral vector or an adeno-associated viral (AAV) vector.

[1544] B11. An epigenetic silencing factor (ESF) comprising a transcription factor DNA binding domain operably linked to at least one epigenetic effector domain, wherein the transcription factor is an oncogenic transcription factor or a cancer-associated transcription factor, wherein the ESF comprises:

[1545] (a) Chromo Shadow (CS) domain and TEAD1 DNA binding domain;

[1546] (b) TEAD1 DNA binding domain and YAF2-RYBP (YR) domain;

[1547] (c) Chromo Shadow (CS) domain, TEAD1 DNA binding domain and YAF2-RYBP (YR) domain;

[1548] (d) Chromo Shadow (CS) domain and MYC DNA binding domain;

[1549] (e) YAF2-RYBP (YR) domain and MYC DNA binding domain; or

[1550] (f) Chromo Shadow (CS) domain, YAF2-RYBP (YR) domain and MYC DNA binding domain.

[1551] B12. A nanoparticle comprising the polynucleotide of any of paragraphs B1 to B9, the vector of paragraph B10, or the ESF of paragraph B11.

[1552] B13. A cell comprising the polynucleotide of any of paragraphs B1 to B9, the vector of paragraph B10, the ESF of paragraph B11, or the nanoparticle of paragraph B12.

[1553] B14. The polynucleotide of any of paragraphs B1 to B9, the vector of paragraph B10, the ESF of paragraph B11, the nanoparticle of paragraph B12, or the cell of paragraph B13, for use in therapy.

[1554] B15. An epigenetic silencing factor (ESF) or a polynucleotide encoding the same, for use in the treatment of cancer, wherein the ESF comprises a transcription factor DNA binding domain operably linked to at least one epigenetic effector domain, wherein the transcription factor is an oncogenic transcription factor or a cancer-associated transcription factor, wherein the cancer is selected from the group consisting of: glioma, glioblastoma, medulloblastoma, astrocytoma, neuroblastoma, ependymoma, meningioma, retinoblastoma, rhabdomyosarcoma, lung cancer, prostate cancer, breast cancer, liver cancer, pancreatic cancer (e.g., human pancreatic ductal adenocarcinoma), bladder cancer, oropharyngeal cancer, kidney cancer, colon cancer (e.g., colon adenocarcinoma), or metastasis of any of the above.

Claims

1. An epigenetic silencing factor (ESF) or a polynucleotide encoding the same for use in cancer treatment, wherein the ESF comprises a transcription factor DNA binding domain operably linked to at least one epigenetic effector domain, wherein the transcription factor is an oncogenic transcription factor or a cancer-associated transcription factor, wherein the cancer is selected from the group consisting of glioma, glioblastoma, medulloblastoma, astrocytoma, neuroblastoma, ependymoma, meningioma, retinoblastoma, rhabdomyosarcoma, lung cancer, prostate cancer, breast cancer, liver cancer, pancreatic cancer (e.g., human pancreatic ductal adenocarcinoma), bladder cancer, oropharyngeal cancer, renal cancer, colon cancer (e.g., colon adenocarcinoma), colorectal cancer (CRC), or metastasis of any of the above.

2. The ESF or polynucleotide for use according to claim 1, wherein the transcription factor is selected from the group consisting of SOX2, MYC, MYCN, TEAD1, TEAD2, TEAD3, TEAD4, FOXA1, FOXA2, ELK1, ELK3, ELK4, SRF, FOXM1, FOXC1, FOXC2, TWIST1, SALL4, ELF1, HIF1A, SOX9, SOX12, SOX18, ETS1, PAX3, PAX8, GLI1, GLI2, GLI3, ETV1, ETV2, ETV3, RUNX1, RUNX2, RUNX3, MAFB, TFAP2C and E2F1.

3. The ESF or polynucleotide for use according to claim 1 or 2, wherein the epigenetic effector domain is selected from the group consisting of a KRAB domain, a DNMT3A domain, a DNMT3L domain, a ZIM3-KRAB (Z-KRAB) domain, a Chromo Shadow (CS) domain, a YAF2-RYBP (YR) domain, a Jagged repressor (En-R) domain, a MeCP2 domain, a GLI3RD domain and a MAD1RD domain.

4. The ESF or polynucleotide for use according to any one of the preceding claims, wherein the ESF comprises: (a) Chromo Shadow (CS) domain and TEAD1 DNA binding domain; (b) TEAD1 DNA binding domain and YAF2-RYBP (YR) domain; (c) Chromo Shadow (CS) domain, TEAD1 DNA binding domain and YAF2-RYBP (YR) domain; (d) Chromo Shadow (CS) domain and MYC DNA binding domain; (e) YAF2-RYBP (YR) domain and MYC DNA binding domain; or (f) Chromo Shadow (CS) domain, YAF2-RYBP (YR) domain and MYC DNA binding domain.

5. The ESF or polynucleotide for use according to any one of claims 1 to 3, wherein the ESF comprises: (a) a KRAB domain, a SOX2 DNA binding domain, a DNMT3A domain and a DNMT3L domain; (b) a CS domain and a SOX2 DNA binding domain; (c) a SOX2 DNA binding domain and a YR domain; (d) a KRAB domain, a TEAD1 DNA binding domain, a DNMT3A domain and a DNMT3L domain; or (e) a KRAB domain, a DNMT3A domain, a DNMT3L domain and a MYC DNA binding domain.

6. The polynucleotide for use according to any one of the preceding claims, wherein the polynucleotide comprises at least one miR-124 target sequence, and / or at least one miR-338-3p target sequence, and / or at least one miR-31 target sequence, wherein the miRNA target sequence is operably linked to a transgene encoding the ESF.

7. The polynucleotide for use according to any one of the preceding claims, wherein the polynucleotide comprises at least one miR-124 target sequence, at least one miR-338-3p target sequence and at least one miR-31 target sequence, wherein the miRNA target sequence is operably linked to a transgene encoding the ESF.

8. The polynucleotide for use according to claim 6 or 7, wherein: (a) the miR-124 target sequence comprises or consists of: a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 1; (b) the miR-338-3p target sequence comprises or consists of a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 2; and / or (c) the miR-31 target sequence comprises or consists of a nucleotide sequence having at least 90% sequence identity with SEQ ID NO:

3.

9. The polynucleotide for use according to any one of the preceding claims, wherein the polynucleotide comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO:

4.

10. The polynucleotide for use according to any one of the preceding claims, wherein the polynucleotide further comprises a promoter operably linked to a transgene encoding ESF, optionally wherein the promoter is a tissue-specific promoter or a constitutive promoter, optionally a cancer cell-specific promoter or a proliferating cell-specific promoter.

11. The polynucleotide for use according to claim 10, wherein the promoter is Ef1a promoter or Mki67 promoter.

12. A polynucleotide for use according to any one of the preceding claims, wherein the polynucleotide is contained in a vector, a nanoparticle, a cell or a composition, optionally wherein the vector is a viral vector, optionally wherein the vector is a lentiviral vector or an adeno-associated viral (AAV) vector.

13. An epigenetic silencing factor (ESF) comprising a transcription factor DNA binding domain operably linked to at least one epigenetic effector domain, wherein the transcription factor is an oncogenic transcription factor or a cancer-associated transcription factor, wherein the ESF comprises: (a) Chromo Shadow (CS) domain and TEAD1 DNA binding domain; (b) TEAD1 DNA binding domain and YAF2-RYBP (YR) domain; (c) Chromo Shadow (CS) domain, TEAD1 DNA binding domain and YAF2-RYBP (YR) domain; (d) Chromo Shadow (CS) domain and MYC DNA binding domain; (e) YAF2-RYBP (YR) domain and MYC DNA binding domain; or (f) Chromo Shadow (CS) domain, YAF2-RYBP (YR) domain and MYC DNA binding domain.

14. Nanoparticles comprising the ESF of claim 13.

15. A cell comprising the ESF of claim 13 or the nanoparticle of claim 14.

16. The ESF of claim 13, the nanoparticle of claim 14, or the cell of claim 15, for use in therapy.

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