Genetic constructs

Abstract

We provide gene constructs and vectors for cancer treatment. [Solution] The present invention provides a gene construct comprising at least one microRNA target site, and a vector comprising such a construct. The gene construct and vector can be used for the diagnosis and treatment of various disorders, including cancer, such as T-cell acute lymphoblastic leukemia (T-ALL).

Description

[Technical Field]

[0001] This invention relates to gene constructs and vectors. The invention also extends to pharmaceutical compositions comprising gene constructs and vectors, as well as methods of using gene constructs and vectors in diagnosis and treatment. In particular, the invention relates, but is not exclusive, to the diagnosis and treatment of T-cell acute lymphoblastic leukemia (T-ALL). [Background technology]

[0002] Over the past few decades, our molecular biological knowledge and understanding of various disorders have deepened significantly. As a result, the use of gene-based approaches in the treatment and diagnosis of conditions such as cancer has increased, enabling more precise treatment of conditions, higher diagnostic specificity, and the identification of subsets of diseases that would otherwise go undetected.

[0003] Thanks to dedicated efforts in cancer research, the mechanisms of cancer development have become more deeply understood. As a result, the approach to cancer treatment has changed, and the search for and development of target-specific drugs has begun (1). However, despite these changes, cancer patients are still predominantly treated with nonspecific cytotoxic drugs. Although many cancer drivers are known, targeting these genes has been extremely difficult. Given that many of these genes have important functions in normal tissues, it is difficult to target them without causing significant toxicity to normal cells. Therefore, the development of alternative therapies is urgently needed.

[0004] T-ALL is an aggressive hematological malignancy often associated with poor prognostic factors, such as central nervous system infiltration. T-ALL accounts for 15% and 25% of acute lymphoblastic leukemia cases in children and adults, respectively (2). Although outcomes for T-ALL patients have improved in recent years, the prognosis for patients with resistance or relapsed disease remains very poor (3, 4). Currently, existing treatments have low response rates and high toxicity, highlighting the need to develop more specific and effective treatment strategies (3).

[0005] MicroRNAs (miRs) are small, non-coding RNAs of about 22 nucleotides that typically regulate gene expression post-transcriptionally by binding to a complementary sequence in the 3'UTR of a target mRNA (6) (5) (see Figure 1). MicroRNAs control not only several biological processes but also human diseases, including cancer (5). Furthermore, functional studies have shown that microRNAs act as tumor suppressors or oncogenes and are involved in tumorigenesis (7).

[0006] Importantly, profiling studies have shown that several microRNAs are expressed in a tissue / cell-specific manner (8, 9). This tissue / cell-specific microRNA expression is being used to negatively regulate transgene expression. Post-transcriptional silencing is induced by adding an artificial sequence recognized by a specific microRNA to the transgene. This concept was first developed by Brown et al. (10). By constructing transgene expression lentiviral vectors incorporating target sequences of hematopoietic cell-specific microRNAs, a system was created that enables transgene expression in non-hematopoietic cells while suppressing expression in the hematopoietic system. Recently, this strategy has been investigated in various disease situations for therapeutic gene delivery (11-13).

[0007] In cancer, microRNA expression profiles have been shown to distinguish between tumors and normal cells and to identify tumors with different developmental origins and differentiation states (8, 9, 14, 15). Therefore, the inventors have begun to utilize T-ALL-specific microRNA expression profiles and have developed a recombinant gene construct that operates as a so-called microRNA "detector system". This gene construct can, firstly, use microRNA profiles to identify cancerous or neoplastic T-ALL cells in a patient and distinguish leukemia cells from the patient's normal healthy cells, and secondly, regulate the expression of therapeutic genes (e.g., apoptosis-inducing genes encoding proteins that cause tumor cell death) or marker genes (e.g., reporter genes encoding protein markers) only in T-ALL cells and not in healthy cells. [Overview of the project]

[0008] Therefore, in a first aspect of the present invention, (i) a first promoter operably ligated to a first nucleic acid sequence encoding a therapeutically active molecule or reporter molecule, wherein the first nucleic acid sequence includes at least one microRNA (miRNA) target site, and (ii) A second promoter operably ligated to a second nucleic acid sequence encoding an inhibitor and / or therapeutically active molecule or reporter molecule of the first promoter, wherein the second nucleic acid sequence comprises at least one miRNA target site, and at least one miRNA target site of the first and second nucleic acid sequences is different, A gene construct containing is provided.

[0009] Advantageously, the constructs of the present invention utilize specific, predetermined microRNA profiles to target and kill unhealthy cells in a patient within a tumor context. When expressed in a patient's cells, the constructs determine whether the cells are unhealthy and, in this case, induce cell death, or whether the cells are healthy and, in this case, do not induce cell death. Therefore, a positive match between the expression of specific microRNAs in unhealthy cells and the constructs results in the delivery of therapeutic genes to target cells and the induction of their death (as shown in Figure 2A). The constructs' endogenous ability to distinguish between unhealthy and healthy cells within a patient's body is a feature of this technology, making it a useful tool for high-precision oncology and a significant advantage over prior art in providing patients with the best efficacy and safety outcomes. Indeed, the constructs of the present invention have the potential to effectively avoid the lack of specificity associated with standard gene therapy technologies.

[0010] Furthermore, the constructs of the present invention enable the delivery of therapeutically active molecules with high specificity and efficiency using only a single vector. In addition, only one construct (i.e., the construct of the present invention) is delivered to the target cells, thereby ensuring co-localization of (i) the therapeutically active molecule or reporter molecule and (ii) the inhibitor of the first promoter and / or the therapeutically active molecule or reporter molecule. This means that significantly lower doses of constructs are required compared to cases where multiple constructs are used to ensure co-delivery to the same cells. Moreover, this single construct does not require external factors or signaling to drive the expression of the therapeutic molecule. Thus, the constructs of the present invention are significantly advantageous over the prior art as they result in a simpler and more effective system.

[0011] Preferably, at least one miRNA target site of the second nucleic acid sequence is a target site of a miRNA different from a miRNA that can target at least one miRNA target site of the first nucleic acid sequence.

[0012] Therefore, it will be understood that the construct of the present invention comprises a first expression cassette comprising a first promoter and a first nucleic acid sequence, and a second expression cassette comprising a second promoter and a second nucleic acid sequence.

[0013] It will be understood that the first nucleic acid sequence may contain two or more species or types of miRNA target sequences. Preferably, the first nucleic acid sequence contains at least one miRNA target site (or at least one species of miRNA target site). Preferably, the first nucleic acid sequence contains at least two miRNA target sites (or at least two species of miRNA target sites). Preferably, the first nucleic acid sequence contains at least three miRNA target sites (or at least three species of miRNA target sites). Preferably, the first nucleic acid sequence contains at least four miRNA target sites (or at least four species of miRNA target sites). Preferably, the first nucleic acid sequence contains at least five miRNA target sites (or at least five species of miRNA target sites). Preferably, the miRNA target sites present in the first nucleic acid sequence are target sites of different miRNA species. The more miRNA target sites present in the first nucleic acid sequence, the more robustly gene expression will be regulated. However, it will be understood that for the construct to function properly, each of the first and second nucleic acid sequences needs to have at least one miRNA target site, provided that the first and second nucleic acid sequences are target sites for different miRNAs.

[0014] It will be understood that there may be two or more copies of each miRNA target site, that is, each miRNA target site species may contain at least one duplicate of the target site. Therefore, preferably, there is at least one copy of each miRNA target site, i.e., each species. Preferably, there are at least two copies of each miRNA target site, i.e., each species. Preferably, there are at least three copies of each miRNA target site, i.e., each species. Preferably, there are at least four copies of each miRNA target site, i.e., each species. Preferably, there are at least five copies of each miRNA target site, i.e., each species.

[0015] Preferably, at least one miRNA target site present in the first nucleic acid sequence is a target site of a miRNA whose expression is absent, reduced, or downregulated in diseased cells compared to healthy cells.

[0016] Identifying miRNAs that are specifically deficient or reduced in cancer cells compared to healthy cells can be achieved by analyzing differentially expressed miRNAs between cancer cells and healthy cells, based on the following criteria.

[0017] 1) The 5th, 10th, 15th, 20th, 25th, 30th, or 35th percentiles of the high-expression group are higher than the 60th, 65th, 70th, 75th, 80th, 85th, 90th, or 95th percentiles of the low-expression group, preferably the 25th percentile of the high-expression group is higher than the 75th percentile of the low-expression group, and 2) miRNA is expressed in at least 60%, 65%, 75%, 80%, 85%, 90%, or 95% of all healthy samples analyzed. Preferably, miRNA is expressed in at least 75% of all healthy samples analyzed.

[0018] Preferably, at least one miRNA target site present in the first nucleic acid sequence is a target site for miRNA that is not expressed, or is expressed at a very low or undetectable level in diseased cells. Preferably, at least one miRNA target site present in the first nucleic acid is a target site for miRNA that is specifically expressed in healthy cells. Preferably, at least one miRNA target site present in the first nucleic acid is a target site for miRNA that is substantially expressed only in healthy cells. Preferably, at least one miRNA target site present in the first nucleic acid is a target site for miRNA that is widely expressed in healthy cells.

[0019] Downregulated or reduced miRNA expression in diseased cells may be at least 5%, 10%, 15%, or 20% lower than that occurring in healthy cells. Preferably, downregulated or reduced miRNA expression in diseased cells may be at least 25%, 30%, 35%, or 40% lower than that occurring in healthy cells. Preferably, downregulated or reduced miRNA expression in diseased cells may be at least 50%, 55%, 60%, or 65% lower than that occurring in healthy cells. Preferably, downregulated or reduced miRNA expression in diseased cells may be at least 70%, 75%, 80%, or 85% lower than that occurring in healthy cells. Preferably, downregulated or reduced miRNA expression in diseased cells may be at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% lower than that occurring in healthy cells.

[0020] Preferably, the diseased cells are cancer cells. More preferably, the diseased cells are T-cell acute lymphoblastic leukemia (T-ALL) cells. For example, miRNA153, miRNA128a, miR-3687, miR-92a-2-5p, miR-20b-3p, miR-6087, miR-106a-3p, miR-7704, miR-5701, miR-766-5p, miR-3609, miR-3615, and / or miR-4746-5p are upregulated (i.e., at higher expression levels than in healthy cells) in T-ALL cells, while miRNA29a, miRNA149 miR-539-5p, miR-487a-3p, miR-655-3p, miR-411-3p, miR-377-5p, miR-337-5p, miR-31-3p, miR-214-5p, miR-1185-5p, miR-483-5p, miR-365a-3p, miR-127-3p, miR-574-3p, and / or miR-125b-5p are downregulated in T-ALL cells (i.e., lower expression levels than in healthy cells).

[0021] Preferably, and therefore, at least one miRNA target site present in the first nucleic acid sequence includes the miRNA29a, miRNA149, miR-539-5p, miR-487a-3p, miR-655-3p, miR-411-3p, miR-377-5p, miR-337-5p, miR-31-3p, miR-214-5p, miR-1185-5p, miR-483-5p, miR-365a-3p, miR-127-3p, miR-574-3p, and / or miR-125b-5p target sites. Preferably, at least one miRNA target site present in the first nucleic acid sequence includes at least one, two, three, four, or five copies of each of the miRNA29a, miRNA149, miR-539-5p, miR-487a-3p, miR-655-3p, miR-411-3p, miR-377-5p, miR-337-5p, miR-31-3p, miR-214-5p, miR-1185-5p, miR-483-5p, miR-365a-3p, miR-127-3p, miR-574-3p, and miR-125b-5p target sites. More preferably, at least one miRNA target site present in the first nucleic acid sequence includes four copies each of the miRNA29a, miRNA149, miR-539-5p, miR-487a-3p, miR-655-3p, miR-411-3p, miR-377-5p, miR-337-5p, miR-31-3p, miR-214-5p, miR-1185-5p, miR-483-5p, miR-365a-3p, miR-127-3p, miR-574-3p, and miR-125b-5p target sites.

[0022] Preferably, at least one miRNA target site present in the first nucleic acid sequence includes a miRNA29a target site, which is provided herein as Sequence ID No. 1, as follows: TAACCGATTTCAGATGGTGCTA [Sequence ID 1] Therefore, preferably, at least one miRNA target site present in the first nucleic acid sequence includes a sequence substantially like that described in Sequence ID No. 1, or a variant or fragment thereof, or a miRNA29a target site consisting of such a sequence.

[0023] Preferably, the at least one miRNA target site present in the first nucleic acid sequence comprises a miR149 target site, which is provided herein as SEQ ID NO: 2 as follows: GGGAGTGAAGACACGGAGCCAGA [SEQ ID NO: 2] Accordingly, preferably the at least one miRNA target site present in the first nucleic acid sequence comprises a miR149 target site comprising, or consisting of, a sequence substantially as set forth in SEQ ID NO: 2, or a variant or fragment thereof.

[0024] Preferably, the at least one miRNA target site present in the first nucleic acid sequence comprises a miR-539-5p target site, which is provided herein as SEQ ID NO: 28 as follows: ACACACCAAGGATAATTTCTCC [SEQ ID NO: 28] Accordingly, preferably the at least one miRNA target site present in the first nucleic acid sequence comprises a miR-539-5p target site comprising, or consisting of, a sequence substantially as set forth in SEQ ID NO: 28, or a variant or fragment thereof.

[0025] Preferably, the at least one miRNA target site present in the first nucleic acid sequence comprises a miR-487a-3p target site, which is provided herein as SEQ ID NO: 29 as follows: AACTGGATGTCCCTGTATGATT [SEQ ID NO: 29] Accordingly, preferably the at least one miRNA target site present in the first nucleic acid sequence comprises a miR-487a-3p target site comprising, or consisting of, a sequence substantially as set forth in SEQ ID NO: 29, or a variant or fragment thereof.

[0026] Preferably, at least one miRNA target site present in the first nucleic acid sequence includes a miR-655-3p target site, which is provided herein as Sequence ID No. 30. AAAGAGGTTAACCATGTATTAT [Sequence ID 30] Therefore, preferably, at least one miRNA target site present in the first nucleic acid sequence includes a sequence substantially like that described in SEQ ID NO: 30, or a variant or fragment thereof, or a miR-655-3p target site consisting of such a sequence.

[0027] Preferably, at least one miRNA target site present in the first nucleic acid sequence includes a miR-411-3p target site, which is provided herein as Sequence ID No. 31. GGTTAGTGGACCGTGTTACATA [Sequence ID 31] Therefore, preferably, at least one miRNA target site present in the first nucleic acid sequence includes a sequence substantially like that described in SEQ ID NO: 31, or a variant or fragment thereof, or a miR-411-3p target site consisting of such a sequence.

[0028] Preferably, at least one miRNA target site present in the first nucleic acid sequence includes a miR-377-5p target site, which is provided herein as Sequence ID No. 32. GAATTCACCAAGGGCAACCTCT [Sequence ID 32] Therefore, preferably, at least one miRNA target site present in the first nucleic acid sequence includes a sequence substantially like that described in SEQ ID NO: 32, or a variant or fragment thereof, or a miR-377-5p target site consisting of such a sequence.

[0029] Preferably, at least one miRNA target site present in the first nucleic acid sequence includes a miR-337-5p target site, which is provided herein as Sequence ID No. 33. AACTCCTGTATGAAGCCGTTC [Sequence ID 33] Therefore, preferably, at least one miRNA target site present in the first nucleic acid sequence includes a sequence substantially like that described in SEQ ID NO: 33, or a variant or fragment thereof, or a miR-337-5p target site consisting of such a sequence.

[0030] Preferably, at least one miRNA target site present in the first nucleic acid sequence includes a miR-31-3p target site, which is provided herein as Sequence ID No. 34. ATGGCAATATGTTGGCATAGCA [Sequence ID 34] Therefore, preferably, at least one miRNA target site present in the first nucleic acid sequence includes a sequence substantially like that described in SEQ ID NO: 34, or a variant or fragment thereof, or a miR-31-3p target site consisting of such a sequence.

[0031] Preferably, at least one miRNA target site present in the first nucleic acid sequence includes a miR-214-5p target site, which is provided herein as Sequence ID No. 35. GCACAGCAAGTGTAGACAGGCA [Sequence ID 35] Therefore, preferably, at least one miRNA target site present in the first nucleic acid sequence includes a sequence substantially like that described in SEQ ID NO: 35, or a variant or fragment thereof, or a miR-214-5p target site consisting of such a sequence.

[0032] Preferably, at least one miRNA target site present in the first nucleic acid sequence includes a miR-1185-5p target site, which is provided herein as Sequence ID No. 36. AACATACAAAGGGTATCCTCT [Sequence ID 36] Therefore, preferably, at least one miRNA target site present in the first nucleic acid sequence includes a sequence substantially like that described in SEQ ID NO: 36, or a variant or fragment thereof, or a miR-1185-5p target site consisting of such a sequence.

[0033] Preferably, at least one miRNA target site present in the first nucleic acid sequence includes a miR-483-5p target site, which is provided herein as Sequence ID No. 37. CTCCCTTCTTTCCTCCCGTCTT [Sequence ID 37] Therefore, preferably, at least one miRNA target site present in the first nucleic acid sequence includes a sequence substantially like that described in SEQ ID NO: 37, or a variant or fragment thereof, or a miR-483-5p target site consisting of such a sequence.

[0034] Preferably, at least one miRNA target site present in the first nucleic acid sequence includes a miR-365a-3p target site, which is provided herein as Sequence ID No. 38. CACATCTGCCCCCAAAAGTCCCT [Sequence ID 38] Therefore, preferably, at least one miRNA target site present in the first nucleic acid sequence includes a sequence substantially like that described in SEQ ID NO: 38, or a variant or fragment thereof, or includes a miR-365a-3p target site consisting of such a sequence.

[0035] Preferably, at least one miRNA target site present in the first nucleic acid sequence includes a miR-127-3p target site, which is provided herein as Sequence ID No. 39. AGCCAAGCTCAGACGGATCCGA [Sequence ID 39] Therefore, preferably, at least one miRNA target site present in the first nucleic acid sequence includes a sequence substantially like that described in SEQ ID NO: 39, or a variant or fragment thereof, or a miR-127-3p target site consisting of such a sequence.

[0036] Preferably, at least one miRNA target site present in the first nucleic acid sequence includes a miR-574-3p target site, which is provided herein as Sequence ID No. 40. TGTGGGTGTGTGCATGAGCGTG [Sequence ID 40] Therefore, preferably, at least one miRNA target site present in the first nucleic acid sequence includes a sequence substantially like that described in SEQ ID NO: 40, or a variant or fragment thereof, or a miR-574-3p target site consisting of such a sequence.

[0037] Preferably, at least one miRNA target site present in the first nucleic acid sequence includes a miR-125b-5p target site, which is provided herein as Sequence ID No. 41. TCACAAGTTAGGGTCTCAGGGA [Sequence ID 41] Therefore, preferably, at least one miRNA target site present in the first nucleic acid sequence includes a sequence substantially like that described in SEQ ID NO: 41, or a variant or fragment thereof, or includes a miR-125b-5p target site consisting of such a sequence.

[0038] Preferably, at least one miRNA target site present in the first nucleic acid sequence includes SEQ ID NOs: 1, 2, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, and / or 41, or a variant or fragment thereof. In some embodiments, the first nucleic acid sequence may include additional miRNA target sites and / or two or more copies of each miRNA target site.

[0039] In one embodiment, miRNA29a is represented by the miR ID number MIMAT0000086. The sequence of miRNA29a may be provided herein as Sequence ID No. 6, as follows. UAGCACCAUCUGAAAUCGGUUA [Sequence ID 6] Therefore, preferably, miRNA29a comprises or consists of a sequence substantially described in SEQ ID NO: 6, or a variant or fragment thereof.

[0040] In one embodiment, miRNA149 is represented by the miR ID number MIMAT0000450. The sequence of miRNA149 may be provided herein as Sequence ID No. 7, as follows. UCUGGCUCCGUGUCUUCACUCCC [Sequence ID 7] Therefore, preferably, miRNA149 includes or consists of a sequence substantially like that described in SEQ ID NO: 7, or a variant or fragment thereof.

[0041] In one embodiment, miR-539-5p is represented by the miR ID number MIMAT0003163. The sequence of miR-539-5p may be provided herein as sequence number 42, as follows. GGAGAAAUUAUCCUUGGUGUGU [Sequence ID 42] Therefore, preferably, miR-539-5p includes or consists of a sequence substantially like that described in Sequence ID No. 42, or a variant or fragment thereof.

[0042] In one embodiment, miR-487a-3p is represented by the miR ID number MIMAT0002178. The sequence of miR-487a-3p may be provided herein as Sequence ID No. 43, as follows. AAUCAUACAGGGACAUCCAGUU [Sequence ID 43] Therefore, preferably, miR-487a-3p comprises or consists of a sequence substantially like that described in Sequence ID No. 43, or a variant or fragment thereof.

[0043] In one embodiment, miR-655-3p is represented by the miR ID number MIMAT0003331. The sequence of miR-655-3p may be provided herein as sequence number 44, as follows. AUAAUACAUGGUUAACCUCUUU [Sequence ID 44] Therefore, preferably, miR-655-3p comprises or consists of a sequence substantially like that described in Sequence ID No. 44, or a variant or fragment thereof.

[0044] In one embodiment, miR-411-3p is represented by the miR ID number MIMAT0004813. The sequence of miR-411-3p may be provided herein as sequence number 45, as follows. UAUGUAACACGGUCCACUAACC [Sequence ID 45] Therefore, preferably, miR-411-3p comprises or consists of a sequence substantially like that described in Sequence ID No. 45, or a variant or fragment thereof.

[0045] In one embodiment, miR-377-5p is represented by the miR ID number MIMAT0004689. The sequence of miR-377-5p may be provided herein as sequence number 46, as follows: AGAGGUUGCCCUUGGUGAAUUC [Sequence ID 46] Therefore, preferably, miR-377-5p comprises or consists of a sequence substantially like that described in Sequence ID No. 46, or a variant or fragment thereof.

[0046] In one embodiment, miR-337-5p is represented by the miR ID number MIMAT0004695. The sequence of miR-337-5p may be provided herein as sequence number 47, as follows. GAACGGCUUCAUACAGGAGUU [Sequence ID 47] Therefore, preferably, miR-337-5p comprises or consists of a sequence substantially like that described in Sequence ID No. 47, or a variant or fragment thereof.

[0047] In one embodiment, miR-31-3p is represented by the miR ID number MIMAT0004504. The sequence of miR-31-3p may be provided herein as sequence number 48, as follows. UGCUAUGCCAACAUAUUGCCAU [Sequence ID 48] Therefore, preferably, miR-31-3p comprises or consists of a sequence substantially described in Sequence ID No. 48, or a variant or fragment thereof.

[0048] In one embodiment, miR-214-5p is represented by the miR ID number MIMAT0004564. The sequence of miR-214-5p may be provided herein as sequence number 49, as follows. UGCCUGUCUACACUUGCUGUGC [Sequence ID 49] Therefore, preferably, miR-214-5p comprises or consists of a sequence substantially like that described in Sequence ID No. 49, or a variant or fragment thereof.

[0049] In one embodiment, miR-1185-5p is represented by the miR ID number MIMAT0005798. The sequence of miR-1185-5p may be provided herein as sequence number 50, as follows. AGAGGAUACCCUUUGUAUGUU [Sequence ID 50] Therefore, preferably, miR-1185-5p comprises or consists of a sequence substantially like that described in Sequence ID No. 50, or a variant or fragment thereof.

[0050] In one embodiment, miR-483-5p is represented by the miR ID number MIMAT0004761. The sequence of miR-483-5p may be provided herein as Sequence ID No. 51, as follows. AAGACGGGAGGAAAGAAGGGAG [Sequence ID 51] Therefore, preferably, miR-483-5p comprises or consists of a sequence substantially like that described in Sequence ID No. 51, or a variant or fragment thereof.

[0051] In one embodiment, miR-365a-3p is represented by the miR ID number MIMAT0009199. The sequence of miR-365a-3p may be provided herein as sequence number 52, as follows. AGGGACUUUUGGGGGCAGAUGUG [Sequence ID 52] Therefore, preferably, miR-365a-3p includes or consists of a sequence substantially described in Sequence ID No. 52, or a variant or fragment thereof.

[0052] In one embodiment, miR-127-3p is represented by the miR ID number MIMAT0000446. The sequence of miR-127-3p may be provided herein as sequence number 53, as follows. UCGGAUCCGUCUGAGCUUGGCU [Sequence ID 53] Therefore, preferably, miR-127-3p comprises or consists of a sequence substantially like that described in Sequence ID No. 53, or a variant or fragment thereof.

[0053] In one embodiment, miR-574-3p is represented by the miR ID number MIMAT0003239. The sequence of miR-574-3p may be provided herein as sequence number 54, as follows: CACGCUCAUGCACACACCCACA [Sequence ID 54] Therefore, preferably, miR-574-3p comprises or consists of a sequence substantially like that described in Sequence ID No. 54, or a variant or fragment thereof.

[0054] In one embodiment, miR-125b-5p is represented by the miR ID number MIMAT0000423. The sequence of miR-125b-5p may be provided herein as sequence number 55, as follows. UCCCUGAGACCCUAACUUGUGA [Sequence ID 55] Therefore, preferably, miR-125b-5p comprises or consists of a sequence substantially like that described in Sequence ID No. 55, or a variant or fragment thereof.

[0055] Preferably, miRNAs including or consisting of sequence numbers 6, 7, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54 and / or 55, or their variants or fragments, target at least one miRNA target site of the first nucleic acid sequence, preferably two different miRNA target sites of the first nucleic acid sequence.

[0056] It will be understood that the first nucleic acid sequence may contain miRNA target sequences from two or more species. Preferably, the second nucleic acid sequence contains at least one miRNA target site (or at least one species of miRNA target site). Preferably, the second nucleic acid sequence contains at least two miRNA target sites (or at least two species of miRNA target sites). Preferably, the second nucleic acid sequence contains at least three miRNA target sites (or at least three species of miRNA target sites). Preferably, the second nucleic acid sequence contains at least four miRNA target sites (or at least four species of miRNA target sites). Preferably, the second nucleic acid sequence contains at least five miRNA target sites (or at least five species of miRNA target sites). Preferably, the miRNA target sites present in the second nucleic acid are target sites of different miRNAs. The more miRNA target sites present in the second nucleic acid sequence, the more firmly gene expression will be regulated. However, for the construct to function properly, it will be understood that each of the first and second nucleic acid sequences must have at least one miRNA target site, provided that the first and second nucleic acid sequences are target sites for different miRNAs.

[0057] It will be understood that there may be two or more copies of each miRNA target site, that is, each species of miRNA target site may contain at least one duplicate of the target site. Therefore, preferably, there is at least one copy of each miRNA target site, i.e., each species. Preferably, there are at least two copies of each miRNA target site, i.e., each species. Preferably, there are at least three copies of each miRNA target site, i.e., each species. Preferably, there are at least four copies of each miRNA target site, i.e., each species. Preferably, there are at least five copies of each miRNA target site, i.e., each species.

[0058] Preferably, at least one miRNA target site in the second nucleic acid sequence is a target site of miRNA expressed in diseased cells. Preferably, at least one miRNA target site in the second nucleic acid is a target site of miRNA specifically expressed in diseased cells. Preferably, at least one miRNA target site in the second nucleic acid is a target site of miRNA that is not expressed in healthy cells, or is expressed at very low or undetectable levels. Preferably, at least one miRNA target site in the second nucleic acid is a target site of miRNA widely expressed in diseased cells.

[0059] Upregulated or increased miRNA expression in diseased cells may be at least 5%, 10%, 15%, or 20% higher than that occurring in healthy cells. Preferably, upregulated or increased miRNA expression in diseased cells may be at least 25%, 30%, 35%, or 40% higher than that occurring in healthy cells. Preferably, upregulated or increased miRNA expression in diseased cells may be at least 50%, 55%, 60%, or 65% higher than that occurring in healthy cells. Preferably, upregulated or increased miRNA expression in diseased cells may be at least 70%, 75%, 80%, or 85% higher than that occurring in healthy cells. Preferably, upregulated or increased miRNA expression in diseased cells may be at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% higher than that occurring in healthy cells.

[0060] Preferably, the diseased cells are cancer cells. More preferably, the diseased cells are T-cell acute lymphoblastic leukemia (T-ALL) cells. Preferably, at least one miRNA target site present in the second nucleic acid includes the miRNA153, miR128a, miR-3687, miR-92a-2-5p, miR-20b-3p, miR-6087, miR-106a-3p, miR-7704, miR-5701, miR-766-5p, miR-3609, miR-3615, and / or miR-4746-5p target sites.

[0061] Preferably, at least one miRNA target site present in the second nucleic acid includes a miRNA153 target site, which is provided herein as Sequence ID No. 3. AGCTGCAACATCACAAAATGA [Sequence ID 3] Therefore, preferably, at least one miRNA target site present in the second nucleic acid sequence includes a sequence substantially like that described in Sequence ID No. 3, or a variant or fragment thereof, or a miRNA153 target site consisting of such a sequence.

[0062] Preferably, at least one miRNA target site present in the second nucleic acid includes a miR128a target site, which is provided herein as Sequence ID No. 4. AAAGAGACCGGTTCACTGTGA [Sequence ID 4] Therefore, preferably, at least one miRNA target site present in the second nucleic acid sequence includes a sequence substantially like that described in Sequence ID No. 4, or a variant or fragment thereof, or a miR128a target site consisting of such a sequence.

[0063] Preferably, at least one miRNA target site present in the second nucleic acid includes a miR-3687 target site, which is provided herein as Sequence ID No. 56. ACGTCGCACGAACGCCTGTCCGGG [Sequence ID 56] Therefore, preferably, at least one miRNA target site present in the second nucleic acid sequence includes a sequence substantially like that described in SEQ ID NO: 56, or a variant or fragment thereof, or a miR-3687 target site consisting of such a sequence.

[0064] Preferably, at least one miRNA target site present in the second nucleic acid includes a miR-92a-2-5p target site, which is provided herein as Sequence ID No. 57. GTAATGCAACAAATCCCCACCC [Sequence ID 57] Therefore, preferably, at least one miRNA target site present in the second nucleic acid sequence includes a sequence substantially like that described in Sequence ID No. 57, or a variant or fragment thereof, or a miR-92a-2-5p target site consisting of such a sequence.

[0065] Preferably, at least one miRNA target site present in the second nucleic acid includes a miR-20b-3p target site, which is provided herein as Sequence ID No. 58. CTGGAAGTGCCCATACTACAGT [Sequence ID 58] Therefore, preferably, at least one miRNA target site present in the second nucleic acid sequence includes a sequence substantially like that described in Sequence ID No. 58, or a variant or fragment thereof, or a miR-20b-3p target site consisting of such a sequence.

[0066] Preferably, at least one miRNA target site present in the second nucleic acid includes a miR-6087 target site, which is provided herein as Sequence ID No. 59. GCTCGCCCCCCCGCCTCA [Sequence ID 59] Therefore, preferably, at least one miRNA target site present in the second nucleic acid sequence includes a sequence substantially like that described in Sequence ID No. 59, or a variant or fragment thereof, or a miR-6087 target site consisting of such a sequence.

[0067] Preferably, at least one miRNA target site present in the second nucleic acid includes a miR-106a-3p target site, which is provided herein as Sequence ID No. 60. GTAAGAAGTGCTTACATTGCAG [Sequence ID 60] Therefore, preferably, at least one miRNA target site present in the second nucleic acid sequence includes a sequence substantially like that described in SEQ ID NO: 60, or a variant or fragment thereof, or includes a miR-106a-3p target site consisting of such a sequence.

[0068] Preferably, at least one miRNA target site present in the second nucleic acid includes a miR-7704 target site, which is provided herein as Sequence ID No. 61. CACGTCGCCGCCGACCCCG [Sequence ID 61] Therefore, preferably, at least one miRNA target site present in the second nucleic acid sequence includes a sequence substantially like that described in SEQ ID NO: 61, or a variant or fragment thereof, or a miR-7704 target site consisting of such a sequence.

[0069] Preferably, at least one miRNA target site present in the second nucleic acid includes a miR-5701 target site, which is provided herein as Sequence ID No. 62. AATCAGAACGTGACAATAA [Sequence ID 62] Therefore, preferably, at least one miRNA target site present in the second nucleic acid sequence includes a sequence substantially like that described in SEQ ID NO: 62, or a variant or fragment thereof, or a miR-5701 target site consisting of such a sequence.

[0070] Preferably, at least one miRNA target site present in the second nucleic acid includes a miR-766-5p target site, which is provided herein as Sequence ID No. 63. AAGACCAGCACCAATTCCTCCT [Sequence ID 63] Therefore, preferably, at least one miRNA target site present in the second nucleic acid sequence includes a sequence substantially like that described in SEQ ID NO: 63, or a variant or fragment thereof, or a miR-766-5p target site consisting of such a sequence.

[0071] Preferably, at least one miRNA target site present in the second nucleic acid includes a miR-3609 target site, which is provided herein as SEQ ID NO: 64. CAGCCAGTATTACTCATCACTTTG [Sequence ID 64] Therefore, preferably, at least one miRNA target site present in the second nucleic acid sequence includes a sequence substantially like that described in SEQ ID NO: 64, or a variant or fragment thereof, or a miR-3609 target site consisting of such a sequence.

[0072] Preferably, at least one miRNA target site present in the second nucleic acid includes a miR-3615 target site, which is provided herein as Sequence ID No. 65. GAGCCGCGAGGAGCCGAGAGA [Sequence ID 65] Therefore, preferably, at least one miRNA target site present in the second nucleic acid sequence includes a sequence substantially like that described in SEQ ID NO: 65, or a variant or fragment thereof, or a miR-3615 target site consisting of such a sequence.

[0073] Preferably, at least one miRNA target site present in the second nucleic acid includes a miR-4746-5p target site, which is provided herein as Sequence ID No. 66. TCTGCAGGTTCTCCTGGGACCGG [Sequence ID 66] Therefore, preferably, at least one miRNA target site present in the second nucleic acid sequence includes a sequence substantially like that described in SEQ ID NO: 66, or a variant or fragment thereof, or includes a miR-4746-5p target site consisting of such a sequence.

[0074] Preferably, at least one miRNA target site present in the second nucleic acid sequence includes SEQ ID NOs: 3, 4, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, and / or 66, or a variant or fragment thereof. In some embodiments, the second nucleic acid sequence may include additional miRNA target sites.

[0075] In one embodiment, miRNA153 is represented by the miR ID number MIMAT0026480. The sequence of miRNA153 may be provided herein as Sequence ID No. 8, as follows. UCAUUUUUGUGAUGUUGCAGCU [Sequence 8] Therefore, preferably, miRNA153 comprises or consists of a sequence substantially like that described in SEQ ID NO: 8, or a variant or fragment thereof.

[0076] In one embodiment, miRNA128a is represented by the miR ID number MIMAT0000424. The sequence of miRNA128a may be provided herein as Sequence ID No. 9, as follows. UCACAGUGAACCGGUCUCUUU [Sequence ID 9] Therefore, preferably, miRNA128a comprises or consists of a sequence substantially like that described in Sequence ID No. 9, or a variant or fragment thereof.

[0077] In one embodiment, miR-3687 is represented by the miR ID number MIMAT0018115. The sequence of miR-3687 may be provided herein as sequence number 67, as follows. CCCGGACAGGCGUUCGUGCGACGU [Sequence ID 67] Therefore, preferably, miR-3687 comprises or consists of a sequence substantially described in Sequence ID No. 67, or a variant or fragment thereof.

[0078] In one embodiment, miR-92a-2-5p is represented by the miR ID number MIMAT0004508. The sequence of miR-92a-2-5p may be provided herein as sequence number 68, as follows. GGGUGGGGAUUUGUUGCAUUAC [Sequence ID 68] Therefore, preferably, miR-92a-2-5p comprises or consists of a sequence substantially like that described in Sequence ID No. 68, or a variant or fragment thereof.

[0079] In one embodiment, miR-20b-3p is represented by the miR ID number MIMAT0004752. The sequence of miR-20b-3p may be provided herein as sequence number 69, as follows. ACUGUAGUAUGGGCACUUCCAG [Sequence ID 69] Therefore, preferably, miR-20b-3p comprises or consists of a sequence substantially like that described in Sequence ID No. 69, or a variant or fragment thereof.

[0080] In one embodiment, miR-6087 is represented by the miR ID number MIMAT0023712. The sequence of miR-6087 may be provided herein as sequence number 70, as follows. UGAGGCGGGGGGGCGAGC [Sequence ID 70] Therefore, preferably, miR-6087 comprises or consists of a sequence substantially described in Sequence ID No. 70, or a variant or fragment thereof.

[0081] In one embodiment, miR-106a-3p is represented by the miR ID number MIMAT0004517. The sequence of miR-106a-3p may be provided herein as sequence number 71, as follows. CUGCAAUGUAAGCACUUCUUAC [Sequence ID 71] Therefore, preferably, miR-106a-3p comprises or consists of a sequence substantially like that described in Sequence ID No. 71, or a variant or fragment thereof.

[0082] In one embodiment, miR-7704 is represented by the miR ID number MIMAT0030019. The sequence of miR-7704 may be provided herein as sequence number 72, as follows: CGGGGUCGGCGGCGACGUG [Sequence ID 72] Therefore, preferably, miR-7704 includes or consists of a sequence substantially described in Sequence ID No. 72, or a variant or fragment thereof.

[0083] In one embodiment, miR-5701 is represented by the miR ID number MIMAT0022494. The sequence of miR-5701 may be provided herein as sequence number 73, as follows. UUAUUGUCACGUUCUGAUU [Sequence ID 73] Therefore, preferably, miR-5701 comprises or consists of a sequence substantially described in Sequence ID No. 73, or a variant or fragment thereof.

[0084] In one embodiment, miR-766-5p is represented by the miR ID number MIMAT0022714. The sequence of miR-766-5p may be provided herein as sequence number 74, as follows: AGGAGGAAUUGGUGCUGGUCUU [Sequence ID 74] Therefore, preferably, miR-766-5p comprises or consists of a sequence substantially like that described in Sequence ID No. 74, or a variant or fragment thereof.

[0085] In one embodiment, miR-3609 is represented by the miR ID number MIMAT0017986. The sequence of miR-3609 may be provided herein as sequence number 75, as follows. CAAAGUGAUGAGUAAUACUGGCUG [Sequence ID 75] Therefore, preferably, miR-3609 includes or consists of a sequence substantially like that described in Sequence ID No. 75, or a variant or fragment thereof.

[0086] In one embodiment, miR-3615 is represented by the miR ID number MIMAT0017994. The sequence of miR-3615 may be provided herein as sequence number 76, as follows. UCUCUCGGCUCCUCGCGGCUC [Sequence ID 76] Therefore, preferably, miR-3615 includes or consists of a sequence substantially described in Sequence ID No. 76, or a variant or fragment thereof.

[0087] In one embodiment, miR-4746-5p is represented by the miR ID number MIMAT0019880. The sequence of miR-4746-5p may be provided herein as sequence number 77, as follows: CCGGUCCCAGGAGAACCUGCAGA [Sequence ID 77] Therefore, preferably, miR-4746-5p comprises or consists of a sequence substantially like that described in Sequence ID No. 77, or a variant or fragment thereof.

[0088] Preferably, miRNAs including or consisting of sequence numbers 8, 9, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76 and / or 77, or their variants or fragments, target at least one miRNA target site of a second nucleic acid sequence, and preferably two different miRNA target sites of the second nucleic acid sequence.

[0089] Therefore, advantageously, the delivery of therapeutic molecules or reporter molecules is possible in patients' T-ALL cells only, and in these cells, microRNAs (e.g., miRNA153, miRNA128a, miR-3687, miR-92a-2-5p, miR-20b-3p, miR-6087, miR-106a-3p, miR-7704, miR-5701, miR-766-5p, miR-3609, miR-3615, and / or miR-4746-5p) are present (or highly expressed). The expression is controlled by both specifically deficient (or significantly underexpressed) miRNAs (e.g., miRNA29a, miRNA149, miR-539-5p, miR-487a-3p, miR-655-3p, miR-411-3p, miR-377-5p, miR-337-5p, miR-31-3p, miR-214-5p, miR-1185-5p, miR-483-5p, miR-365a-3p, miR-127-3p, miR-574-3p, and / or miR-125b-5p). The constructs of the present invention utilize the co-existence and absence of specific microRNAs to positively regulate the expression of a therapeutic molecule or reporter molecule. Preferably, and advantageously, this dual regulation is achieved by the use of a bidirectional expression vector. It will be understood that negative feedback loops mediated by gene expression provide further control.

[0090] The first promoter may be any suitable promoter, including a constitutive promoter, an activatable promoter, an inducible promoter, or a tissue-specific promoter. The first promoter may be a broadly expressed promoter or a cancer cell-specific promoter. Preferably, the first promoter is a broadly expressed promoter.

[0091] The first promoter may be selected from the group consisting of the elongation factor 1α (EF1α) promoter, the EF1α short (EFS) promoter, the phosphoglycerate kinase (PGK) promoter, the cytomegalovirus (CMV) early promoter, the splenic focal formation virus (SFFV) promoter, the CASI promoter, the myeloproliferative sarcoma virus (MPSV) long terminal repeat, the mouse stem cell virus (MSCV) long terminal repeat (LTR), and the combined CAG promoter (consisting of the CMV early enhancer and the chicken β-actin promoter).

[0092] Preferably, the first promoter extends in the 5' to 3' direction. The first nucleic acid coding sequence can encode a reporter molecule or a therapeutic molecule.

[0093] The reporter molecule may be an optical reporter, a nuclear medicine reporter, or an MRI reporter. The optical reporter may be a fluorescent protein or luciferase. The nuclear medicine reporter may be herpes simplex virus type 1 [HSV1] thymidine kinase (TK), human mitochondrial TK2, dopaminergic receptor, dopamine 2 receptor, sodium iodide cotransporter, norepinephrine transporter, somatostatin receptor, or estrogen receptor. The MRI reporter may be a transferrin receptor, β-galactosidase, tyrosinase, ferritin, or lysine-rich protein (LRP).

[0094] The therapeutically active molecules may be therapeutic proteins and / or nucleic acids. The nucleic acid may be DNA, RNA, or a chimeric DNA / RNA molecule. The nucleic acid may also be a gene silencing molecule, such as RNAi molecules including siNA, siRNA, shRNA, miRNA, ribozymes, and antisense molecules. The nucleic acid may also be a guide RNA.

[0095] Those skilled in the art will understand the term guide RNA to refer to the non-coding RNA component of the CRISPR / Cas system that binds to a complementary target DNA sequence. The guide RNA first binds to the Cas enzyme, and the gRNA sequence, via pairing, guides the complex to a specific location on the DNA, where Cas performs endonuclease activity by cleaving the target DNA strand.

[0096] Gene silencing molecules may be antisense molecules (antisense DNA or antisense RNA) or ribozyme molecules. Ribozymes and antisense molecules can be used to inhibit the transcription of essential genes in diseased cells. Antisense molecules are oligonucleotides that bind to nucleic acids such as DNA or RNA in a sequence-specific manner. Antisense RNA inhibits mRNA translation when it binds to mRNA with a complementary sequence. A triple-stranded molecule refers to a single strand of antisense DNA binding to a double-stranded DNA molecule to form a collinear triple-stranded molecule, thereby inhibiting transcription.

[0097] The therapeutically active molecule may be a therapeutic protein, and the first nucleic acid encodes an mRNA molecule that encodes the therapeutic protein. Therapeutic proteins may also be recombinant proteins with therapeutic applications.

[0098] The therapeutic protein may be an endonuclease. For example, the therapeutic protein may be a CRISPR endonuclease such as a CRISPR-related protein (Cas). Preferably, the Cas protein may be Cas9 and / or Cpf1.

[0099] The therapeutic molecule may contain components of both the Cas protein and the CRIPSR / Cas system, including the guide RNA. In this embodiment, the construct may also contain an additional promoter to drive the expression of the Cas protein.

[0100] The therapeutic protein may also be a CAR (chimeric antigen receptor). For example, the therapeutic protein may be a CAR protein that enables the CAR-T cell mechanism for in vivo T cell immunotherapy.

[0101] The therapeutic protein may be an effector protein. Preferably, the therapeutic protein may be capable of inducing an apoptotic cascade within tumor cells. Therefore, preferably, the therapeutic protein may be an apoptosis-driven protein. Preferably, the apoptosis-driven protein may be Bax, Apoptin, E4orf4, and / or Bim.

[0102] Preferably, the apoptosis-driven protein may be Bax, which can be represented by gene number 581, provided herein as SEQ ID NO: 16. MDGSGEQPRGGGPTSSEQIMKTGALLLQGFIQDRAGRMGGEAPELALDPVPQDASTKKLSECLKRIGDELDSNMELQRMIAAVDTDSPREVFFRVAADMFSDGNFNWGRV VALFYFASKLVLKALCTKVPELIRTIMGWTLDFLRERLLGWIQDQGGWGLPLAESLKRLMSLSPGRPPLLLWDAHVADRDHLCGGSAHRLTHHLEEDGLRPPAALDCVFPP [Sequence ID 16] Therefore, preferably, Bax comprises or consists of an amino acid sequence substantially described in SEQ ID NO: 16, or a biologically active variant or fragment thereof.

[0103] In one embodiment, Bax may be encoded by a nucleotide sequence provided herein as Sequence ID No. 17, as follows: gccgctcggc cgcccgcgcg gacccggcga gaggcggcgg cgggagcggc ggtgatggacgggtccggggagcagcccag aggcgggggg cccaccagct ctgagcagat catgaagacatgcttcaggg tttcatccag gatcgagcag ggcgaatggg gggggaggcacccgagctgg ccctggaccc ggtgcctcag gatgcgtcca ccaagaagct gagcgagtgtctcaagcgca tcggggacga actggacagt aacatggagc tgcagaggat gattgccgccgtggacacag actccccccg agaggtcttt ttccgagtgg cagctgacat gtttctgacggcaacttca actggggccg ggttgtcgcc cttttctact ttgccagcaa actggtgctcaaggccctgt gcaccaaggt gccggaactg atcagaacca tcatgggctg gacattggacttctccggg agcggctgtt gggctggatc caagaccagg gtggttgggg gctgcccctggccgagtcac tgaagcgact gatgtccctg tctccaggac ggcctcctct cctactttgggacgcccag tggcagaccg tgaccatctt tgtggcggga gtgctcaccg cctcactcaccatctggaag aagatgggct gaggccccca gctgccttgg actgtgtttt tcctccataaattatggcat tttctggga ggggtggga ttggggacg tgggcatttt tcttacttttgtaattattg gggggtgtgg ggaagagtgg tcttgagggg gtaataaacc tccttcgggacaca [query number 17] Therefore, preferably, the Bax polypeptide or its biologically active variant or fragment may be encoded by a nucleotide sequence, or a variant or fragment thereof, substantially as described in Sequence ID No. 17.

[0104] Preferably, the apoptosis-driven protein may be Apoptin, which can be represented by gene number 1494446, provided herein as SEQ ID NO: 18, as follows. MNALQEDTPPGPSTVFRPPTSSRPLETPHCREIRIGIAGITITLSLCGCANARAPTLRSATADNSESTGFKNVPDLRTDQPKPPSKKRSCDPSEYRVSELKESLITTTPSRPRTARRRIRL [Sequence ID 18] Therefore, preferably, Apoptin comprises or consists of an amino acid sequence substantially described in SEQ ID NO: 18, or a biologically active variant or fragment thereof.

[0105] In one embodiment, Apoptin may be encoded by a nucleotide sequence provided herein as Sequence ID No. 19, as follows: atgaacgctc tccaagaaga tactccaccc ggaccatcaa cggtgttcag gccaccaacaagttcacggc cgttggaaac ccctcactgc agagagatcc ggattggtat cgctggaattacaatcactc tatcgctgtg tggctgcgcg aatgctcgcg ctcccacgct aagatctgcaactgcggaca attcagaaag cactggtttc aagaatgtgc cggacttgag gaccgatcaacccaagcctc cctcgaagaa gcgatcctgc gacccctccg agtacagggt aagcgagctaaaagaaagct tgattaccac tactcccagc cgaccccgaa ccgcaagaag gcgtataagactgtaa [Sequence ID 19] Therefore, preferably, the apoptin polypeptide or its biologically active variant or fragment may be encoded by a nucleotide sequence, or a variant or fragment thereof, substantially as described in SEQ ID NO: 19.

[0106] Preferably, the apoptosis-driven protein may be E4orf4, which can be represented by gene number AC_000007.1 provided herein as Sequence ID No. 20, as follows: MVLPALPAPPVCDSQNECVGWLGVAYSAVVDVIRAAAHEGVYIEPEARGRLDALREWIYYNYYTERAKRRDRRRRSVCHARTWFCFRKYDYVRRSIWHDTTTNTISVVSAHSVQ [Sequence ID 20] Therefore, preferably, E4orf4 comprises or consists of an amino acid sequence substantially described in Sequence ID No. 20, or a biologically active variant or fragment thereof.

[0107] In one embodiment, E4orf4 may be encoded by a nucleotide sequence provided herein as Sequence ID No. 21, as follows: atggttcttccagctcttcccgctcctcccgtgtgtgactcgcagaacgaatgtgtaggttggctgggtgtggcttattctgcggtggtggatgttatcagggcagcggcgcatgaaggagtttacatagaacccgaagccagggggcgcctggatgctttgagagagtgga tatactacaactactacacagagcgagctaagcgacgagaccggagacgcagatctgtttgtcacgcccgcacctggttttgcttcaggaaatatgactacgtccggcgttccatttggcatgacactacgaccaacacgatctcggttgtctcggcgcactccgtacagtag [Sequence ID 21] Therefore, preferably, the E4orf4 polypeptide or its biologically active variant or fragment may be encoded by a nucleotide sequence, or a variant or fragment thereof, substantially as described in Sequence ID No. 21.

[0108] Preferably, the apoptosis-driven protein may be Bim(BCL2L11), which can be represented by gene number 10018, provided herein as Sequence ID No. 25, as follows. MAKQPSDVSSECDREGRQLQPAERPPQLRPGAPTSLQTEPQGNPEGNHGGEGDSCPHGSPQGPLAPPASPGPFATRSPLFIFMRRSSLLSRSSSGYFSFDTDRSPAPMSCDKSTQTPSPPCQAFNHYLSAMASMRQAEPADMRPEIWIAQELRRIGDEFNAYYARRVFLNNYQAAEDHPRMVILRLLRYIVRLVWRMH [Sequence ID 25] Therefore, preferably, Bim comprises or consists of an amino acid sequence substantially described in SEQ ID NO: 25, or a biologically active variant or fragment thereof.

[0109] In one embodiment, Bim may be encoded by a nucleotide sequence provided herein as Sequence ID No. 26, as follows: atggcaaagcaaccttctgatg taagttctga gtgtgaccga gaaggtagac aattgcagcc tgcggagaggcctccccagc tcagacctgg ggcccctacc tccctacaga cagagccaca aggtaatcctgaaggcaatc acggaggtga aggggacagc tgccccccacg gcagccctca gggcccgctggccccacctg ccagccctgg cccttttgct accagatccc cgcttttcat ctttatgagaagatcctccc tgctgtctcg atcctccagt gggtatttct ctttgacac agacaggagcccagcaccca tgagttgtga caaatcaaca caaaccccaa gtcctccttg ccaggccttcaaccactatc tcagtgcaat ggcttccatg aggcaggctg aacctgcaga tatgcgcccagagatatgga tcgcccaaga gttgcggcgt attggagacg agtttaacgc ttactatgcaaggagggtat ttttgaataa ttaccaagca gccgaagacc acccacgaat ggttatcttacgactgttac gttacattgt ccgcctggtg tggagaatgc attga [Sequence ID 26] Therefore, preferably, the Bim polypeptide or its biologically active variant or fragment may be encoded by a nucleotide sequence, or a variant or fragment thereof, substantially as described in SEQ ID NO: 26.

[0110] The second promoter may be any suitable promoter, including a constitutive promoter, an activatable promoter, an inducible promoter, or a tissue-specific promoter. Preferably, the first promoter is a broadly expressive promoter. Preferably, the second promoter is a different promoter from the first promoter.

[0111] The second promoter may be positioned in the same orientation as the first promoter in the construct. The second promoter may extend in the 3' to 5' direction. However, preferably, the second promoter is positioned in the opposite direction to the first promoter in the construct. Preferably, the second promoter extends in the 3' to 5' direction. Preferably, the promoter is bidirectional. The inventors' use of a bidirectional promoter does not require any additional gene elements such as IRES elements, fusion proteins, or self-cleaving 2A peptides, and provides the additional benefit of coordinated expression of two genes, which would result in weak co-expression of the two genes and require additional transgene engineering that could affect protein stability and / or biological function. Furthermore, the use of a bidirectional promoter avoids the problem of unidirectional promoter interference that can occur, for example, when using a lenti vector.

[0112] Therefore, in one embodiment, the first expression cassette is positioned in the same direction as the second expression cassette. However, in a preferred embodiment, the first expression cassette is positioned in the opposite direction to the second expression cassette.

[0113] The second promoter may be selected from the group consisting of the elongation factor 1α (EF1α) promoter, the EF1α short (EFS) promoter, the phosphoglycerate kinase (PGK) promoter, the cytomegalovirus (CMV) early promoter, the splenic focal formation virus (SFFV) promoter, the CASI promoter, the myeloproliferative sarcoma virus (MPSV) long-terminal repeat, the mouse stem cell virus (MSCV) long-terminal repeat (LTR), and the combined CAG promoter (consisting of the CMV early enhancer and the chicken β-actin promoter).

[0114] In one embodiment, the inhibitor encoded by the second nucleic acid sequence is a direct inhibitor of a therapeutically active molecule or reporter molecule. Preferably, however, the inhibitor encoded by the second nucleic acid sequence is an inhibitor of the first promoter. The inhibitor of the first promoter may be part of a tetracycline-controlled operator system or a cumate-controlled operator system, such systems are well known to those skilled in the art.

[0115] The inhibitor of the first promoter may be a Lac operon, the second nucleic acid sequence includes a Lac repressor, and the first promoter includes a Lac operator regulatory region.

[0116] In one embodiment, the first promoter and the first nucleic acid sequence are 5' relative to the second promoter sequence and the second nucleic acid sequence. In another embodiment, the first promoter and the first nucleic acid sequence are 3' relative to the second promoter sequence and the second nucleic acid sequence.

[0117] In one embodiment, the gene construct may include, in this specified order from 5' to 3', a first promoter sequence operably ligated to a first nucleic acid sequence, a first nucleic acid sequence encoding a reporter molecule and / or a therapeutic molecule, a second promoter sequence operably ligated to a second nucleic acid sequence, and a second nucleic acid sequence encoding an inhibitor of the first promoter.

[0118] In a preferred embodiment, however, the gene construct may include, in this specified order from 5' to 3', a second promoter sequence operably ligated to the second nucleic acid, a second nucleic acid sequence encoding an inhibitor of the first promoter, a first promoter sequence operably ligated to the first nucleic acid sequence, and a first nucleic acid sequence encoding a reporter molecule and / or a therapeutic molecule.

[0119] The use of 5' and 3' indicates that the feature is either upstream or downstream, and is not intended to indicate that the feature is necessarily a terminal feature. One embodiment of the gene construct is shown in Figure 8c, which is referred to herein as Sequence ID No. 5. ctctgacacatgcagctcccggagacggtcacagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtgtt ggcgggtgtcggggctggcttaactatgcggcatcagagcagattgtactgagagtgcaccatatgcggtgtgaaataccgcacagatgcgtaa ggagaaaataccgcatcaggcgccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagct ggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgccagggttttcccagtcacgacgttgtaaaacgacggccagtgccaagctg [Sequence ID 5] Preferably, the gene construct comprises a nucleic acid sequence, or a fragment or variant thereof, substantially as described in Sequence ID No. 5.

[0120] Another embodiment of the gene construct is shown in Figure 8H and is referred to herein as Sequence ID No. 78 as follows. [Sequence ID 78] Preferably, the gene construct comprises a nucleic acid sequence, or a fragment or variant thereof, substantially as described in Sequence ID No. 78.

[0121] Another embodiment of the gene construct is shown in Figure 8D, which may include a Bax coding sequence and can be represented by Sequence ID No. 22 as follows: [Sequence ID 22] Preferably, the gene construct comprises a nucleic acid sequence, or a fragment or variant thereof, substantially as described in Sequence ID No. 22.

[0122] Another embodiment of the gene construct containing the Bax coding sequence is shown in Figure 8I and is represented by Sequence ID No. 79 as follows: [Sequence ID 79] Preferably, the gene construct comprises a nucleic acid sequence, or a fragment or variant thereof, substantially as described in Sequence ID No. 79.

[0123] Another embodiment of the gene construct is shown in Figure 8E, which may include an Apoptin coding sequence and can be represented by Sequence ID No. 23 as follows: gatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttccttttttcaatattattgaagcatttatcagggtta ttgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgt ctaagaaaccattattatcatgacattaacctataaaaataggcgtatcacgaggccctttcgtctcgcgcgtttcggtgatgacggtgaaaac ctctgacacatgcagctcccggagacggtcacagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtgtt ggcgggtgtcggggctggcttaactatgcggcatcagagcagattgtactgagagtgcaccatatgcggtgtgaaataccgcacagatgcgtaa ggagaaaataccgcatcaggcgccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagct ggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgccagggttttcccagtcacgacgttgtaaaacgacggccagtgccaagctg [Sequence ID 23] Preferably, the gene construct comprises a nucleic acid sequence, or a fragment or variant thereof, substantially as described in Sequence ID No. 23.

[0124] Another embodiment of the gene construct containing the Apoptin coding sequence is shown in Figure 8J and is represented by Sequence ID No. 80 as follows: [Sequence ID 80] Preferably, the gene construct comprises a nucleic acid sequence, or a fragment or variant thereof, substantially as described in Sequence ID No. 80.

[0125] Another embodiment of the gene construct is shown in Figure 8F, which may include the E4orf4 coding sequence and can be represented by Sequence ID No. 24 as follows: ctcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcgga tacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtctaagaaaccattattatcatgacattaacctataaaaataggcgtatcacgaggccctttcgtctcgcgcgtttcggtgatgacggtgaaaacctctgacacat gcagctcccggagacggtcacagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtgttggcgggtgtcggggctggcttaactatgcggcatcagagcagattgtactgagagtgcaccatatgcggtgtgaaataccgcacagatgcgtaaggagaa aataccgcatcaggcgccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagctggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgccagggtttttcccagtcacgacgttgtaaaacgacggccagtgccaagctg [Sequence ID 24] Preferably, the gene construct comprises a nucleic acid sequence, or a fragment or variant thereof, substantially as described in Sequence ID No. 24.

[0126] Another embodiment of the gene construct containing the E4orf4 coding sequence is shown in Figure 8K and is represented by Sequence ID No. 81 as follows: [Sequence ID 81] Preferably, the gene construct comprises a nucleic acid sequence, or a fragment or variant thereof, substantially as described in Sequence ID No. 81.

[0127] Another embodiment of the gene construct is shown in Figure 8G, which may include a Bim coding sequence and can be represented by Sequence ID No. 27 as follows: ttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtctaagaaaccattattatcatgacattaacctataaaaataggcgtatcacgaggccctttcgtctcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtcacagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtgttggcgggtgtcggggctggcttaactatgcggcatcagagcagattgtactgagagtgcaccatatgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcaggcgccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagctggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgccagggttttcccagtcacgacgttgtaaaacgacggccagtgccaagctg [Sequence ID 27] Preferably, the gene construct comprises a nucleic acid sequence, or a fragment or variant thereof, substantially as described in Sequence ID No. 27.

[0128] Another embodiment of the gene construct containing the Bim coding sequence is shown in Figure 8L and is represented by Sequence ID No. 82 as follows: [Sequence ID 82] Preferably, the gene construct comprises a nucleic acid sequence, or a fragment or variant thereof, substantially as described in Sequence ID No. 82.

[0129] Furthermore, the present invention also extends to recombinant vectors containing gene constructs as vehicles for delivering therapeutic drugs. Therefore, the second embodiment provides a recombinant vector comprising the gene construct described in the first embodiment.

[0130] The vector comprising the gene construct of the first embodiment may be, for example, a plasmid, cosmid or phage and / or viral vector. Such recombinant vectors are very useful in the delivery system of the present invention for transforming cells with nucleotide sequences. Preferably, the vector is a viral vector.

[0131] The viral vector may be selected from adenovirus vectors, adeno-associated virus (AAV) vectors, retrovirus vectors, or lentiviral vectors. The vector may be a recombinant adeno-associated virus (rAAV) vector. The rAAV may be a naturally occurring vector or a vector having a hybrid AAV serotype. The rAAV may be AAV-1, AAV-2, AAV-3A, AAV-3B, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, and AAV-11. AAV-2 is most preferred.

[0132] As used herein, the term “recombinant AAV (rAAV) vector” may mean recombinant AAV-derived nucleic acid containing at least one terminal repeat sequence. Preferably, however, the vector is a recombinant lentiviral vector. The lentiviral vector may be embedded or unembedded. Preferably, the lentiviral vector is unembedded.

[0133] Preferably, the vector of the second embodiment is an episomal vector. Preferably, the vector of the second embodiment is recombinant. Recombinant vectors may contain other functional elements. For example, they may further contain a variety of other functional elements. For example, the vector may be able to autonomously replicate in the nucleus of a host cell, or preferably, the vector may not be able to autonomously replicate in the nucleus of a host cell. In this case, elements that induce or regulate DNA replication may be required in the recombinant vector. Alternatively, the recombinant vector may be designed to be integrated into the genome of a host cell. In this case, DNA sequences that promote targeted integration (e.g., by homologous recombination) are assumed. Suitable promoters include the SV40 promoter, CMV, EF1a, PGK, viral long-end repeats, and inducible promoters, such as the tetracycline-inducible system. The cassette or vector may also contain a terminator such as β-globin, an SV40 polyadenylated sequence, or a synthetic polyadenylated sequence. The recombinant vector may also contain regulatory factors or enhancers to control nucleic acid expression as needed.

[0134] The vector may contain DNA encoding a gene that can be used as a selectable marker in the cloning process, i.e., enabling the selection of transfected or transformed cells, and enabling the selection of cells that carry the vector incorporating heterologous DNA. For example, ampicillin, neomycin, puromycin, or chloramphenicol resistance are conceivable. Alternatively, the selectable marker gene may be present in a different vector for use concurrently with the vector containing the gene construct. The vector may contain DNA involved in regulating the expression of nucleotide sequences, or DNA for targeting expressed polypeptides to specific parts of host cells.

[0135] In a third embodiment, a gene construct according to the first embodiment or a vector according to the second embodiment is provided for use in therapy. In a fourth aspect, a gene construct according to the first aspect or a vector according to the second aspect is provided for use in treating, preventing, or improving cancer.

[0136] A fifth aspect provides a method for treating, preventing or improving cancer in a subject, the method comprising administering, or having administered, a therapeutically effective amount of the gene construct described in the first aspect or the vector described in the second aspect to a patient in need of such treatment.

[0137] Preferably, the cancer is T-ALL. It will be understood that the gene constructs or vectors described in the present invention (collectively referred to herein as “agents”) may be used as monotherapy (e.g., the use of a gene construct or vector alone) for the treatment, preferably the treatment, improvement, or prevention of cancer. Alternatively, the agents described in the present invention may be used as adjuncts to or in combination with known treatments for the treatment, preferably the treatment, improvement, or prevention of cancer.

[0138] The pharmaceuticals described in the present invention may be combined in compositions having a number of different forms, in particular, depending on the method in which the composition is used. For example, the composition may be in the form of a powder, tablet, capsule, liquid, ointment, cream, gel, hydrogel, aerosol, spray, micelle solution, transdermal patch, liposome suspension, or any other suitable form that can be administered to a person or animal requiring treatment. It will be understood that the vehicle of the pharmaceutical described in the present invention should be well acceptable to the subject to which it is administered.

[0139] Pharmaceuticals containing the agent of the present invention can be used in many ways. For example, oral administration may be required, in which case the agent may be contained in a composition that can be taken orally, for example, in the form of tablets, capsules, or liquids. Compositions containing the agent and pharmaceuticals of the present invention may also be administered by inhalation (e.g., nasally). Compositions can also be formulated for topical use. For example, creams or ointments may be applied to the skin.

[0140] The agents and pharmaceuticals described in the present invention may also be incorporated into sustained-release or delayed-release devices. Such devices may be inserted, for example, on or under the skin, and the pharmaceuticals may be released over several weeks or months. The devices may be positioned at least adjacent to the treatment site. Such devices may be particularly advantageous when long-term treatment with the agents used according to the present invention is required, and usually requires frequent administration (e.g., at least daily injections).

[0141] In preferred embodiments, the agents and pharmaceuticals described in the present invention may be administered to a subject by injection into the bloodstream or by direct injection into a site requiring treatment. The injection may be intravenous (bolus or infusion), subcutaneous (bolus or infusion), or intradermal (bolus or infusion).

[0142] The required amount of gene construct or vector (i.e., drug) will be determined by its biological activity and bioavailability, and will then depend on the mode of administration, the physicochemical properties of the drug, and whether it is used as monotherapy or in combination therapy. The frequency of administration will also be influenced by the half-life of the drug within the patient being treated. The optimal dose to be administered will be determined by those skilled in the art and will vary depending on the specific drug used, the strength of the pharmaceutical composition, the mode of administration, and the disease being treated, such as the stage of cancer. Additional factors dependent on the specific patient being treated, including the patient's age, weight, sex, diet, and time of administration, will necessitate dose adjustments.

[0143] Generally, the agents described in the present invention may be used for treatment, particularly for the treatment, improvement, or prevention of cancer, at a daily dose between 0.001 μg / kg body weight and 10 mg / kg body weight, depending on the specific agent. More preferably, the daily dose of the agent is between 0.01 μg / kg body weight and 1 mg / kg body weight, more preferably between 0.1 μg / kg body weight and 100 μg / kg body weight, and most preferably between about 0.1 μg / kg body weight and 10 μg / kg body weight.

[0144] Alternatively, the dose administered to the subject is 0.5 × 10 7 From 5x10 12 The dose may be within the range of transduction units (TU) / kg body weight. More preferably, the dose administered to the subject is 0.5 × 10⁻⁶ 8 From 5x10 11 The dose may be within the range of TU / kg body weight. Most preferably, the dose administered to the subject is 0.5 × 10⁻⁶. 9 From 5x10 10 It is also acceptable to be within the range of TU / Kg body weight.

[0145] This drug may be administered before, during, or after the onset of cancer. The daily dose may be administered as a single dose (e.g., once daily injection). Alternatively, the drug may require two or more doses during the day. For example, the drug may be administered in two daily doses (or more, depending on the disease being treated, e.g., the severity of cancer) ranging from 0.07 μg to 700 mg (i.e., assuming a body weight of 70 kg). Patients receiving treatment may receive the first dose upon waking and the second dose in the evening (in the case of a two-dose regimen), or at intervals of three or four hours thereafter. Alternatively, the drug may need to be administered once a week or once a month. Alternatively, an extended-release device may be used to provide the patient with the optimal dose of the drug described in this invention without the need for repeated administration. Known procedures conventionally used in the pharmaceutical industry (e.g., in vivo experiments, clinical trials, etc.) may be used to form specific formulations and precise treatment regimens (such as the daily dose and frequency of administration) of the drug described in this invention.

[0146] A sixth aspect of the present invention provides a pharmaceutical composition comprising a gene construct described in the first aspect, or a vector described in the second aspect, and optionally a pharmaceutically acceptable vehicle.

[0147] The pharmaceutical composition is preferably an anticancer composition, i.e., a pharmaceutical formulation used for the therapeutic improvement, prevention, or treatment of cancer in a subject, and preferably a pharmaceutical formulation used for the therapeutic improvement, prevention, or treatment of T-ALL in a subject.

[0148] In a seventh aspect, the present invention also provides a process for producing the pharmaceutical composition described in the sixth aspect, the process comprising combining a therapeutically effective amount of the gene construct described in the first aspect or the vector described in the second aspect with a pharmaceutically acceptable vehicle.

[0149] The "subject" may be a vertebrate, mammal, or domesticated poultry. Therefore, the pharmaceuticals described in the present invention may be used to treat any mammal, such as livestock (e.g., horses), pets, or for other veterinary purposes. Most preferably, the subject is human.

[0150] A “therapeutically effective amount” of a gene construct or vector is any amount of the drug that, when administered to a subject, is necessary to treat the disease being treated, such as cancer, or to produce the desired effect.

[0151] For example, the therapeutically effective amount of the gene construct or vector used may be about 0.001 ng to about 1 mg, preferably about 0.01 ng to about 100 ng. The amount of the gene construct or vector is preferably about 0.1 ng to about 10 ng, most preferably about 0.5 ng to about 5 ng.

[0152] As used herein, “pharmaceutically acceptable vehicle” means any known compound or combination of known compounds that is known to those skilled in the art to be useful in formulating pharmaceutical compositions.

[0153] In one embodiment, the pharmaceutically acceptable vehicle may be a solid, and the composition may be in the form of a powder or a tablet. The solid pharmaceutically acceptable vehicle may contain one or more substances that may act as flavorings, lubricants, solubilizers, suspending agents, dyes, fillers, flow promoters, compression aids, inert binders, sweeteners, preservatives, dyes, coatings, or tablet disintegrants. The vehicle may also be an encapsulating material. In the powder form, the vehicle is a finely divided solid that is mixed with the finely divided activator described in the present invention. In the tablet form, the activator may be mixed in an appropriate proportion with a vehicle having the required compressibility and compressed into a desired shape and size. The powder and tablet preferably contain up to 99% activator. Suitable solid vehicles include, for example, calcium phosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, polyvinylpyrrolidine, low-melting-point waxes, and ion-exchange resins. In another embodiment, the pharmaceutical vehicle may be a gel, and the composition may be in the form of a cream or the like.

[0154] However, the pharmaceutical vehicle may be a liquid, and the pharmaceutical composition may be in the form of a solution. Liquid vehicles are used in the preparation of solutions, suspensions, emulsions, syrups, elixirs, and pressurized compositions. The activators described in the present invention may be dissolved or suspended in water, organic solvents, mixtures thereof, or pharmaceutically acceptable liquid vehicles such as pharmaceutically acceptable oils or fats. Liquid vehicles may contain other suitable pharmaceutical additives such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavorings, suspending agents, thickeners, colorants, viscosity modifiers, stabilizers, or osmotic pressure modifiers. Preferred examples of liquid vehicles for oral and parenteral administration are water (partially containing the additives described above, e.g., cellulose derivatives, preferably sodium carboxymethylcellulose solution), alcohols (including monohydric and polyhydric alcohols, e.g., glycols) and their derivatives), and oils (e.g., fractionated coconut oil and peanut oil). For parenteral administration, the vehicle may also be an oily ester such as ethyl oleate and isopropyl myristate. For sterile liquid compositions for parenteral administration, a sterile liquid vehicle is useful. For pressurized compositions, the liquid vehicle may be a halogenated hydrocarbon or other pharmaceutically acceptable spray.

[0155] Liquid pharmaceutical compositions, which are sterile solutions or suspensions, can be administered, for example, by intramuscular, intrathecal, epidural, intraperitoneal, intravenous, and especially subcutaneous injection. The drugs may also be prepared as sterile solid compositions that can be dissolved or suspended at the time of administration using sterile water, saline, or other suitable sterile injection media.

[0156] The drugs and compositions of the present invention may be administered orally in the form of sterile solutions or suspensions containing other solutes or suspensions (e.g., physiological saline or glucose sufficient to make the solution isotonic), bile salts, acacia, gelatin, sorbitan monooleate, polysorbate 80 (oleic acid ester obtained by copolymerizing sorbitol and its anhydrous with ethylene oxide), etc. Furthermore, drugs used according to the present invention may be administered orally in the form of liquid or solid compositions. Suitable compositions for oral administration include solid forms such as pills, capsules, granules, tablets, and powders, and liquid forms such as solutions, syrups, elixirs, and suspensions. Forms useful for parenteral administration include sterile solutions, emulsions, and suspensions.

[0157] As discussed herein, the gene constructs of the present invention may include a reporter gene, which may be specifically expressed in diseased cells. Therefore, advantageously, the gene constructs and vectors of the present invention may also be used as robust diagnostic tools. Advantageously, the constructs of the present invention can be used to detect the miRNA expression profile of a tumor in a patient and identify the tumor subtype, which can then be used to determine appropriate treatment for the patient. Alternatively, the constructs of the present invention may simply be used to detect the presence of cancer.

[0158] Accordingly, the eighth aspect provides a gene construct according to the first aspect or a vector according to the second aspect for use in diagnosis. In a ninth aspect, a gene construct according to the first aspect or a vector according to the second aspect is provided for use in the diagnosis of cancer.

[0159] A tenth embodiment provides a diagnostic or predictive method comprising the step of detecting a reporter molecule of the gene construct described in the first embodiment or a vector described in the second embodiment in a sample obtained from a subject.

[0160] In the eleventh aspect, a method is provided for diagnosing or predicting a target cancer, comprising the step of detecting a reporter molecule of the gene construct described in the first aspect or a vector described in the second aspect in a sample obtained from the target.

[0161] Preferably, the cancer is T-ALL. Prognosis may be relevant to determining the treatment outcome in subjects diagnosed with a disease, preferably cancer. Prognosis may be relevant to predicting the rate and / or duration of disease progression or improvement in subjects, the probability of survival, and / or the effectiveness of various treatment regimens. Therefore, a poor prognosis may indicate disease progression, a low probability of survival, and reduced effectiveness of treatment regimens. A good prognosis may indicate disease remission, a high probability of survival, and high effectiveness of treatment regimens.

[0162] Diagnostic methods may be performed in vivo. However, diagnostic methods are preferably performed in vitro or ex vivo. Preferably, diagnostic methods are performed in vitro. Preferably, diagnostic methods are performed ex vivo.

[0163] Furthermore, the present invention provides a kit for diagnosing patients suffering from cancer. In a twelfth aspect, a kit is provided for diagnosing a subject with cancer or a predisposition thereto, or for providing a prognosis for the subject's condition, the kit comprising the gene construct described in the first aspect or the vector described in the second aspect.

[0164] The sample may be a biological sample or an imaging sample. The image sample may be a PET scan image or an MRI image. Preferably, the sample includes a biological sample. The sample may be any material obtainable from the subject.

[0165] Biological samples may be tissues or biological fluids. Furthermore, samples may be blood, plasma, serum, cerebrospinal fluid, urine, sweat, saliva, tears, mammary gland aspirate, breast milk, prostatic fluid, semen, vaginal fluid, feces, cervical scraping, cytes, amniotic fluid, intraocular fluid, mucus, breath moisture, animal tissue, cell lysates, tumor tissue, hair, skin, cheek scraping, lymph, interstitial fluid, nails, bone marrow, cartilage, prions, bone meal, earwax, lymph, granuloma, cerebrospinal fluid, cancer biopsy, or a combination thereof.

[0166] The sample may be a liquid aspirate. For example, the sample may be bronchoalveolar lavage (BAL), ascites, pleural lavage, or pericardial lavage. The sample may include blood, urine, tissue, etc. In a preferred embodiment, the biological sample includes a blood sample. The blood may be venous or arterial blood. The blood sample may be assayed immediately. Alternatively, the blood sample may be stored at a low temperature, for example in a refrigerator, or frozen, before the method is performed. Alternatively, the blood sample may be stored at room temperature, for example between 18 and 22°C, before the method is performed. The blood sample may include serum. The blood sample may include plasma. Preferably, however, detection is performed with whole blood, and most preferably, the blood sample is peripheral blood.

[0167] Blood samples may contain circulating tumor cells, and the construct can be used to detect the miRNA expression profile of these circulating tumor cells, thus allowing for the identification of the best treatment options for the patient.

[0168] Before performing the diagnostic procedure, the blood may be further processed. For example, anticoagulants such as citrate (e.g., sodium citrate), hirudin, heparin, PPACK, or sodium fluoride may be added. Therefore, the sample collection container may contain an anticoagulant to prevent the blood sample from clotting.

[0169] In one embodiment, the construct of the present invention may be used to detect cancer cells or determine cancer subtypes in cerebrospinal fluid obtained from a patient. Therefore, preferably, the biological sample may include cerebrospinal fluid.

[0170] The present invention extends to any nucleic acid, peptide, or variant, derivative, or analog thereof, and will be understood to substantially include the amino acid sequence or nucleic acid sequence, variant or fragment thereof of any sequence referred to herein. The terms “substantially amino acid / nucleotide / peptide sequence,” “variant,” and “fragment” may be sequences having at least 40% sequence identity with the amino acid / nucleotide / peptide sequence of any one of the sequences referred to herein, for example, sequences identified as SEQ ID NOs. 1 to 82, etc.

[0171] Amino acid / polynucleotide / polypeptide sequences having sequence identity of greater than 65%, more preferably greater than 70%, even more preferably greater than 75%, and still more preferably greater than 80% with any of the referenced sequences are also conceivable. Preferably, the amino acid / polynucleotide / polypeptide sequences have at least 85% identity, more preferably at least 90% identity, even more preferably at least 92% identity, even more preferably at least 95% identity, even more preferably at least 97% identity, even more preferably at least 98% identity, and most preferably at least 99% identity with any of the sequences referred to herein.

[0172] Those skilled in the art will understand how to calculate the percentage of identity between two amino acid / polynucleotide / polypeptide sequences. To calculate the percentage of identity between two amino acid / polynucleotide / polypeptide sequences, it is first necessary to create an alignment of the two sequences and then calculate the sequence identity value. The percentage of identity between two sequences may be different depending on (i) the method used to align the sequences, e.g., structural alignment from ClustalW, BLAST, FASTA, Smith-Waterman (implemented in different programs), or 3D comparison; and (ii) the parameters used by the alignment method, e.g., local vs. global alignment, the pair score matrix used (e.g., BLOSUM62, PAM250, Gonnet, etc.), and the gap penalty, e.g., functional form and constant.

[0173] After alignment, there are many different ways to calculate the percentage of identity between two sequences. For example, the number of identities may be divided by (i) the length of the shortest sequence, (ii) the length of the alignment, (iii) the average length of the sequences, (iv) the number of non-gap positions, or (v) the number of equivalent positions excluding overhangs. Furthermore, it will be understood that the percentage of identity also depends strongly on length. Therefore, the shorter the pair of sequences, the higher the expected sequence identity that would occur by chance may be.

[0174] Therefore, it will be understood that the precise alignment of protein or DNA sequences is a complex process. ClustalW (Thompson et al., 1994, Nucleic Acids Research, 22, pp. 4673-4680; Thompson et al., 1997, Nucleic Acids Research, 24, pp. 4876-4882), a common multiple alignment program, is a preferred method for generating multiple alignments of proteins or DNA according to the present invention. Appropriate parameters for ClustalW are as follows: For DNA alignment: Gap start penalty = 15.0, gap extension penalty = 6.66, and matrix = identity. For protein alignment: Gap start penalty = 10.0, gap extension penalty = 0.2, and matrix = Gonnet. For DNA and protein alignment: ENDGAP = -1, and GAPDIST = 4. Those skilled in the art will recognize that it is necessary to vary these and other parameters to achieve optimal sequence alignment.

[0175] Preferably, the percentage of identity between two amino acid / polynucleotide / polypeptide sequences is calculated from such alignments (N / T). * It may be calculated as 100, where N is the number of positions in which the sequences share identical residues, and T is the total number of compared positions, including gaps and whether or not they include overhangs. Preferably, overhangs are included in the calculation. Therefore, the most preferred method for calculating the identity percentage between two sequences is to (i) prepare the sequence alignment using the ClustalW program with an appropriate set of parameters, for example, as described above; and (ii) use the values ​​of N and T in the following formula: Sequence Identity = (N / T) * This includes inserting into 100.

[0176] Alternative methods for identifying similar sequences will be known to those skilled in the art. For example, substantially similar nucleotide sequences would be encoded by sequences that hybridize to DNA sequences or their complements under strict conditions. By strict conditions, we mean that the nucleotides hybridize to filter-bound DNA or RNA in 3× sodium chloride / sodium citrate (SSC) at about 45°C, and then wash at least once in 0.2× SSC / 0.1% SDS at about 20 to 65°C. Alternatively, substantially similar polypeptides may differ from, for example, the sequences shown in SEQ ID NOs: 1 to 82 by at least one, but less than 5, 10, 20, 50, or 100 amino acids.

[0177] Due to the degeneracy of the genetic code, it is evident that any nucleic acid sequence described herein can be altered or modified without substantially affecting the sequence of the protein it encodes, thereby providing a functional variant. Preferred nucleotide variants have sequences altered by the substitution of different codons encoding the same amino acid in the sequence, thus producing a silent (synonymous) change. Other suitable variants have homologous nucleotide sequences but are altered by the substitution of different codons encoding an amino acid that includes all or part of the sequence and has a side chain with similar biophysical properties to the amino acid it substitutes for, resulting in a conserved change. Examples of small nonpolar hydrophobic amino acids include glycine, alanine, leucine, isoleucine, valine, proline, and methionine. Examples of large nonpolar hydrophobic amino acids include phenylalanine, tryptophan, and tyrosine. Examples of polar neutral amino acids include serine, threonine, cysteine, asparagine, and glutamine. Examples of positively charged (basic) amino acids include lysine, arginine, and histidine. Examples of negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Therefore, it is understood that any amino acid may be substituted with an amino acid having similar biophysical properties, and those skilled in the art will be able to determine the nucleotide sequences encoding these amino acids.

[0178] All features described herein (including the attached claims, abstract and drawings), and / or all steps of any method or process disclosed herein, may be combined in any combination with any of the above embodiments, except in any combination in which at least part of such features and / or steps are mutually exclusive.

[0179] To better understand the present invention and to illustrate how embodiments of the present invention can be carried out, the accompanying figures are now referenced as examples. [Brief explanation of the drawing]

[0180] [Figure 1] This is a schematic diagram of miRNA-mediated silencing of target mRNA. Both mRNA and miRNA are transcribed from gene precursors. Subsequently, miRNAs bind to the complementary region of their target mRNA, leading to degradation. [Figure 2] This is a schematic diagram of one embodiment of the microRNA detector system and related gene construct of the present invention for therapeutic gene delivery in T-ALL cells. [Figure 3] This figure shows the relative microRNA expression in T-ALL cells and non-T-ALL cells, determined by RT-qPCR and calculated using the 2-ΔΔCt method. *p<0.05; ***p<0.0001. [Figure 4] This is a schematic diagram of one embodiment of SBI's pCDH-EF1-MCS-T2A-GFP(PGK-Puro) expression vector. [Figure 5] This is a schematic diagram of a bidirectional lentiviral expression vector ("bdLV") modified to express the Lac operon, illustrating a two-step approach. [Figure 6]This figure shows the results of cloning the Lac operator sequence (LacO) downstream of the EF1 promoter. A) shows the GFP expression of 293T cells transfected with the bdLV vector or the bdLV_LacO vector. B) shows the GFP expression of 293T cells transfected with the bdLV_LacO vector or the bdLV_LacO vector and LacI vector. B.ii) shows the GFP expression of 293T cells co-transfected with the bdLV_LacO vector and LacI vector, and treated with or without IPTG (isopropyl β-D-1-thiogalactopyranoside) for 24 and 48 hours. Unt. = Untransfected. [Figure 7] This figure shows the results of cloning the Lac repressor (LacI) downstream of the PGK promoter. It shows GFP expression (intensity and geometric mean) in 293T cells transfected with either the bdLV_LacO vector or the bdLV_LacI_LacO vector and treated with IPTG. Unt. = Untransfected. [Figure 8] Two embodiments (A and B) of the microRNA detector system and a schematic diagram of the associated gene construct having possible combinations of microRNA target sites (C, D, E, F, G, H, I, J, K, and L) are shown. [Figure 8C] Two embodiments (A and B) of the microRNA detector system and a schematic diagram of the associated gene construct having possible combinations of microRNA target sites (C, D, E, F, G, H, I, J, K, and L) are shown. [Figure 8D] Two embodiments (A and B) of the microRNA detector system and a schematic diagram of the associated gene construct having possible combinations of microRNA target sites (C, D, E, F, G, H, I, J, K, and L) are shown. [Figure 8E] Two embodiments (A and B) of the microRNA detector system and a schematic diagram of the associated gene construct having possible combinations of microRNA target sites (C, D, E, F, G, H, I, J, K, and L) are shown. [Figure 8F]Two embodiments (A and B) of the microRNA detector system and a schematic diagram of the associated gene construct having possible combinations of microRNA target sites (C, D, E, F, G, H, I, J, K, and L) are shown. [Figure 8G] Two embodiments (A and B) of the microRNA detector system and a schematic diagram of the associated gene construct having possible combinations of microRNA target sites (C, D, E, F, G, H, I, J, K, and L) are shown. [Figure 8H] Two embodiments (A and B) of the microRNA detector system and a schematic diagram of the associated gene construct having possible combinations of microRNA target sites (C, D, E, F, G, H, I, J, K, and L) are shown. [Figure 8I] Two embodiments (A and B) of the microRNA detector system and a schematic diagram of the associated gene construct having possible combinations of microRNA target sites (C, D, E, F, G, H, I, J, K, and L) are shown. [Figure 8J] Two embodiments (A and B) of the microRNA detector system and a schematic diagram of the associated gene construct having possible combinations of microRNA target sites (C, D, E, F, G, H, I, J, K, and L) are shown. [Figure 8K] Two embodiments (A and B) of the microRNA detector system and a schematic diagram of the associated gene construct having possible combinations of microRNA target sites (C, D, E, F, G, H, I, J, K, and L) are shown. [Figure 8L] Two embodiments (A and B) of the microRNA detector system and a schematic diagram of the associated gene construct having possible combinations of microRNA target sites (C, D, E, F, G, H, I, J, K, and L) are shown. [Figure 9A]This figure shows the in vitro validation of the efficiency and specificity of microRNA detectors with target sequences for miR-29a and miR-149. T-ALL (A) and non-T-ALL (B) cells were transduced using microRNA detectors BdLV_miR29a_4×T, BdLV_miR149_4×T, and BdLV_miR29a_4×T_miR149_4×T. GFP expression was analyzed by flow cytometry and used as a readout to determine the efficiency of the detectors that suppressed reporter gene expression. [Figure 9B] This figure shows the in vitro validation of the efficiency and specificity of microRNA detectors with target sequences for miR-29a and miR-149. T-ALL (A) and non-T-ALL (B) cells were transduced using microRNA detectors BdLV_miR29a_4×T, BdLV_miR149_4×T, and BdLV_miR29a_4×T_miR149_4×T. GFP expression was analyzed by flow cytometry and used as a readout to determine the efficiency of the detectors that suppressed reporter gene expression. [Figure 10A] This figure shows the in vitro validation of the efficiency and specificity of microRNA detectors with target sequences miR-128a and miR-153. T-ALL cells were transduced with the microRNA detectors BdLV_miR128a_4×T, BdLV_miR153_4×T, and BdLV_miR128a_4×T_miR153_4×T. GFP expression was analyzed by flow cytometry and used as a readout to determine the efficiency of the microRNA detectors in unsuppressing the reporter gene promoter via LacI. [Figure 10B]This figure shows the in vitro validation of the efficiency and specificity of microRNA detectors with target sequences miR-128a and miR-153. T-ALL cells were transduced with the microRNA detectors BdLV_miR128a_4×T, BdLV_miR153_4×T, and BdLV_miR128a_4×T_miR153_4×T. GFP expression was analyzed by flow cytometry and used as a readout to determine the efficiency of the microRNA detectors in unsuppressing the reporter gene promoter via LacI. [Figure 11] This figure shows the in vitro validation of a construct containing dual miRNA target sites. [Figure 12] This figure shows in vitro validation of the efficiency and specificity of microRNA detectors containing two, three, or four miRNA target sites. [Figure 13] This figure shows the in vitro verification of the efficiency and specificity of a microRNA detector in cultures of T-ALL cells transduced with non-integrated lentiviral vectors and non-T-ALL cells. [Modes for carrying out the invention]

[0181] [Examples]

[0182] Example 1: Overview of a novel microRNA detector capable of identifying and selectively inhibiting T-ALL. In cancer, microRNA expression profiles have been shown to distinguish between tumor cells and normal cells, identifying different developmental origins and their differentiation states (8, 9, 14, 15). Therefore, we have developed a microRNA detection system (i.e., the recombinant gene construct of the present invention) that utilizes a T-ALL-specific microRNA expression profile to (1) identify T-ALL cells and then (2) control the expression of therapeutic genes (apoptosis-inducing genes) in leukemia cells. When expressed in host cells, the construct first determines whether the host cell is a leukemia cell based on a predetermined microRNA profile specific only to T-ALL cells. If it is a leukemia cell, the construct induces cell death. Therefore, a positive match between the expression of a specific microRNA in T-ALL cells and the construct results in the delivery of therapeutic genes to those T-ALL cells, inducing their death, while normal, healthy cells remain unaffected (see Figure 2A). The essential ability of this system to distinguish between T-ALL cells and non-T-ALL cells in a patient's body is, therefore, an inherent aspect of the present invention. Therefore, it has a significant advantage over other gene therapy technologies applied to high-precision oncology and is intentionally designed to provide patients with the best efficacy and safety outcomes.

[0183] The delivery of therapeutic genes is controlled by both microRNAs that are present (or highly expressed) only in T-ALL cells and microRNAs that are not specifically present (or are expressed at significantly low levels) in those cells. For example, referring to Figure 2B, assuming that leukemia cells can be distinguished from normal cells by the absence of microRNAs A and B, and the presence of microRNAs C and D, therapeutic genes are delivered only when (1) microRNAs A and B are absent, and (2) microRNAs C and D are expressed in the same cell (see Figure 2B).

[0184] MicroRNAs have traditionally been explored for negatively regulating gene expression. However, this invention utilizes the simultaneous presence and absence of specific microRNAs to positively regulate the expression of therapeutic genes. This dual regulation is achieved through the use of a bidirectional expression vector and a negative feedback loop. Such a system has not been applied before and enhances the specificity of techniques for delivering therapeutic genes only to target cells, maximizing drug efficacy and safety, and patient treatment outcomes (see Figure 2C).

[0185] This innovative technology has the potential to effectively circumvent the lack of specificity in T-ALL treatment. Importantly, it serves as a proof of concept demonstrating that this strategy can be applied to the development of similar personalized therapies in other cancer types where microRNA expression profiles vary. material and method Bioinformatics analysis To define a microRNA expression profile specific to T-ALL cells, the inventors collected microRNA expression data from publicly available human datasets. The inventors initially identified 10 human datasets containing T-ALL samples (1-10). Of these, four were excluded due to the lack of non-tumor samples (1-4), and two were excluded because access to raw data was impossible (5, 6). For each of the analyzed datasets (7-10), the inventors compared non-T-ALL samples (control group) with T-ALL samples (T-ALL group). The samples analyzed in the control group varied by study and included healthy tissue from the lung, colon, bladder, brain, kidney, breast, whole bone marrow (BM) cells, whole thymocytes, hematopoietic CD34+ BM cells, CD4+ T cells, CD8+ T cells, and CD4+CD8+CD3+ T cells. The T-ALL group included primary T-ALL cells and cell lines.

[0186] To be considered upregulated in T-ALL samples, the minimum expression level of a given miRNA in the T-ALL group had to be higher than the maximum expression level of that miRNA in the control group. Similarly, when the minimum expression level of a miRNA in the control group was higher than the maximum expression level in the T-ALL group, that miRNA was considered to be downregulated in T-ALL samples. Using this classification system, the inventors were able to identify 12 miRNAs that were downregulated and upregulated in different datasets.

[0187] Thereafter, the inventors established additional T-ALL-specific microRNA expression profiles via bioinformatics analysis of publicly available microRNA data obtained from human T-ALL cells, normal cells and tissues. These datasets include approximately 140 types of T-ALL cell lines and patient cells, as well as approximately 480 types of normal tissue samples including brain, liver, kidney, lung, heart, breast, bladder, colon, uterus, skin, ovary, pancreas, prostate, stomach, testis, meninges, thyroid, thymus, bone marrow, spleen, lymph node, peripheral blood and the like. Hematopoietic and lymphoid organs covered different lineages and different differentiation stages, including hematopoietic stem cells and progenitor cells.

[0188] Through this analysis, 25 microRNAs were identified, as shown in Table 1 below.

[0189]

Table 1-1

[0190]

Table 1-2

[0191] Based on this list, the inventors listed 16 microRNAs that are most likely to be used for constructing a microRNA detector, as shown in Table 2 below.

[0192]

Table 2

[0193] Verification of microRNA expression profiles Validation of T-ALL-specific microRNA expression profiles was performed using the TaqMan MicroRNA assay (Applied Biosystems) by real-time PCR quantification of each microRNA expression. MicroRNA expression was analyzed in T-ALL cell lines corresponding to different differentiation stages (Jurkat, DND4.1, MOLT4, CEM, SUP-T1, HPB-ALL, TALL-1, P12, and Loucy) as well as in several non-T-ALL cell lines such as 293T, A549, MDA231, CaCO2, U2OS, HCT-116, ACHN, A498, and D458.

[0194] T-ALL cell lines were cultured in RPMI-1640 medium containing L-glutamine supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin (Pen / Strep). Non-T-ALL cell lines were cultured as follows: 293T, A549, MDA231, and CaCO2 were cultured in Dulbecco's Modified Eagle medium supplemented with 10% FBS and 1% Pen / Strep; U2OS and HCT-116 were cultured in McCoy's 5A medium containing 10% FBS and 1% Pen / Strep; ACHN and A498 were cultured in Eagle's Minimum Essential Medium containing 10% FBS and 1% Pen / Strep; and D458 was cultured in Iscove Modified Dulbecco's medium containing 10% FBS and 1% Pen / Strep. All cell lines were stored at 37°C in a 5% CO2 environment.

[0195] Following the manufacturer's protocol, total RNA was extracted from cells using TRIzol reagent. Next, Applied Biosystems' TaqMan MicroRNA was extracted. MicroRNA expression levels were quantified using a combination of Reverse Transcription Kit and TaqMan miRNA Assays. This is a two-step process that begins with reverse transcription of microRNA into cDNA using microRNA RT-specific primers. Following this reaction, microRNA is amplified by real-time PCR using a microRNA-specific TaqMan probe. The PCR reaction was performed using the Vii7 Real Time PCR system. MicroRNA expression was normalized using RNU6b, and relative microRNA expression was determined by 2 -ΔΔCt method. Technical design of the microRNA detector As shown in Figure 4, the pCDH-EF1-MCS-T2A-GFP (PGK-Puro) expression vector (SBI) was used as the backbone to construct the microRNA detector (i.e., the construct of the present invention). This bidirectional promoter expression vector enables GFP expression driven by the constitutive EF1 promoter. In the negative orientation, the PGK promoter drives expression of the puromycin marker. For proof-of-concept purposes, GFP was used as a substitute for the therapeutic gene.

[0196] The present inventors first started with introducing a Lac operon system into the bidirectional lentiviral expression vector (bdLV), which is the vector backbone shown in Figure 4. This process includes the following steps. Step 1. Cloning of the Lac operator sequence (LacO) downstream of the EF1 promoter For this purpose, the inventors amplified an SV40 intron (SV40intron / 3LacO) containing three copies of the Lac Operator sequence from pOPI3CAT (Agilent's LacSwitch II Inducible Mammalian Expression System) and cloned it into bdLV (bdLV_LacO). SV40intron / 3LacO was amplified using primers with EcoRI and NotI restriction sites (Table 3). After amplification, both the PCR product and the BdLV vector were digested with EcoRI and NotI restriction enzymes (Thermo Scientific). The digested SV40intron / 3LacO PCR product and BdLV vector were then ligated using T4 DNA ligase (Fermentas). Correct insertion of the LacO sequence was confirmed by restriction enzyme diagnostic digestion and Sanger sequencing. Step 2. Clone the Lac repressor (LacI) downstream of the PGK promoter.

[0197] This step involves the substitution of puromycin with LacI (in the reverse direction). LacI was PCR-amplified from the pCMVLacI vector (Agilent's LacSwitch II Inducible Mammalian Expression System) and subcloned into a smaller vector (pcDNA3.1+) obtained in the reverse direction. To this end, LacI_NLS was amplified using primers with blunt-end SmaI restriction sites (Table 3). After purification, the LacI_NLS PCR products and pcDNA3.1+ vector were digested with SmaI (NEB). The digested LacI_NLS fragments and pcDNA3.1+ vector were then ligated using T4 DNA ligase (Fermentas). The correct orientation of LacI insertion was confirmed by restriction enzyme digestion and Sanger sequencing. Next, using directional mutagenesis (Agilent's QuickChange Lightning Site-Directed Mutagenesis Kit), we excised the puromycin cDNA from the bdLV_LacO vector and created the restriction enzyme sites necessary to replace it with LacI. Due to the size of bdLV_LacO (approximately 10Kb), the mutagenic region (puromycin and adjacent sequences) of the bdLV_LacO vector was removed and subcloned into a pcDNA3.1+ vector. More specifically, both the bdLV_LacO and pcDNA3.1+ vectors were digested using the restriction enzymes NotI and EcoRI. The puromycin digest and the digested pcDNA3.1+ were ligated using T4 DNA ligase (Fermentas). Using this intermediate small vector (approximately 6.7Kb), one XhoI restriction site was removed to generate an AvrII restriction site. Once mutated, puromycin and the adjacent region were amplified using primers with restriction enzyme sites for HpaI and SpeI (Table 3), and then re-cloned into the bdLV vector by ligation of the PCR product with a vector digested with these enzymes.Next, puromycin was removed from the bdLV_LacO vector using XhoI and AvrII restriction enzymes, and LacI was excised from the pcDNA3.1+ vector_LacI using the same enzymes. The bdLV_LacO vector and LacI were ligated using T4 DNA ligase to create the bdLV_LacI_LacO vector. The correct insertion of LacI into the bdLV_LacO vector was confirmed by restriction enzyme digestion and Sanger sequencing.

[0198] Next, the inventors cloned four target sites for miR-29a, miR-149, or miR-29a and miR-149 (microRNAs specifically absent or downregulated in T-ALL cells) downstream of the reporter gene promoter in the bdLV_LacI_LacO construct. To this end, restriction enzyme sites for XmaI were created downstream of GFP. The four target sites for each microRNA (or both) were synthesized in tandem, digested with XmaI, ligated with T4 DNA ligase, and introduced into the bdLV-LacI_LacO vector.

[0199] In the second step, the inventors cloned four target sites of miR-128a or miR-153 (microRNAs specifically upregulated in T-ALL cells) downstream of the reporter gene promoter repressor in the bdLV_LacI_LacO construct. To this end, the four target sites of each microRNA were synthesized in tandem, digested with the restriction enzyme XhoI, and then ligated with T4 DNA ligase to introduce them into the bdLV-LacI_LacO vector. References for the methods used 1) Landgraf, P. et al. (2007) Cell 29;129(7):1401-142) Rosenfeld, N. et al. (2008) Nat Biotechnol Apr;26(4):462-93) Fulci, V. et al. (2009) Genes, Chromosomes & Cancer 48:1069-10824) de Leeuw, D. et al. (2013) Clin Cancer Res; 19(8); 2187-965) Schotte, D. et al (2009) Leukemia Feb;23(2):313-22. 6) Coskum, E. et al. (2013) Leukemia Research 37:647- 6567) Lu, J. et al (2005) Nature. Jun 9;435 (7043):834-8 8) Mavrakis, KJ et al (2011) Nat Genet. Jun 5;43(7):673-8, 9) Schotte, D. et al. (2011) Haematologica 2011;96(5):703-711 10) Sanghvi, VR eta al. (2014) Sci. Signal. Nov 18;7(352):ra111.

[0200] [Table 3]

[0201] Example 2: Definition and validation of T-ALL-specific miRNA expression profiles To begin constructing the microRNA detector construct of the present invention, the inventors established a preliminary T-ALL-specific microRNA expression profile using publicly available human datasets. Using this approach, the inventors were able to identify 12 miRNAs that are specifically downregulated and upregulated in T-ALL cells.

[0202] As shown in Figure 3, after verifying these 12 microRNAs by real-time PCR, it was confirmed that miR-128a and miR-153 are specifically upregulated in T-ALL cells, and miR-29a and miR-149 are specifically downregulated in T-ALL cells.

[0203] Subsequently, bioinformatics analysis of a larger cohort of publicly available microRNA data obtained from human T-ALL cells, normal cells and tissues identified 25 miRNAs that are specifically downregulated (miR-539-5p, miR-487a-3p, miR-655-3p, miR-411-3p, miR-377-5p, miR-337-5p, miR-31-3p, miR-214-5p, miR-1185-5p, miR-483-5p, miR-365a-3p, miR-127-3p, miR-574-3p and miR-125b-5p) and upregulated (miR-3687, miR-92a-2-5p, miR-20b-3p, miR-6087, miR-106a-3p, miR-7704, miR-5701, miR-766-5p, miR-3609, miR-3615 and miR-4746-5p) in T-ALL cells compared with healthy cells. These were included among the 16 microRNAs most likely to be used for constructing a microRNA detector, wherein in T-ALL cells, 10 microRNAs (miR-539-5p, miR-487a-3p, miR-655-3p, miR-411-3p, miR-377-5p, miR-337-5p, miR-31-3p, miR-214-5p, miR-1185-5p and miR-483-5p) are specifically downregulated, and 6 microRNAs (miR-3687, miR-92a-2-5p, miR-20b-3p, miR-6087, miR-106a-3p and miR-7704) are specifically upregulated.

[0204] Example 3 Design and Construction of MicroRNA Detector / Construct The microRNA detector construct developed by the present inventors consists of a bidirectional lentiviral expression vector, in which a therapeutic gene is expressed in one promoter and a repressor of the therapeutic gene promoter is expressed in the second promoter. The Lac operon system is used as the repressor. The expression of both mRNAs is regulated by T-ALL cell-specific microRNAs. For proof of concept, GFP was used instead of the apoptosis-inducing gene (i.e., GFP expression was used as a substitute for the therapeutic gene).

[0205] As shown in Figure 4, the inventors used SBI's pCDH-EF1-MCS-T2A-GFP(PGK-Puro) expression vector (referred to as BdLV) as the framework of the detector system. This bidirectional promoter expression vector can express GFP with a constitutive EF1 promoter. In the negative direction, the PGK promoter drives the expression of the puromycin marker.

[0206] As shown in Figure 5, the inventors introduced a Lac operon system into the skeletal vector and made it function as an inhibitory system. The process was divided into two steps. i) Cloning the Lac operator sequence (LacO) downstream of the EF1 promoter. As shown in Figure 6, the Lac repressor (LacI) binds to the Lac operator (LacO) regulatory site of the promoter and stops transcription, confirming that insertion of the LacO sequence does not affect GFP expression by the EF1 promoter (Figure 6A).

[0207] The inventors further confirmed the functionality of LacO. They confirmed that the LacI protein can bind to the Lac operator sequence of bdLV_LacO and suppress GFP expression mediated by the EF1 promoter. As shown in Figure 6Bi, transfection of bdLV_LacO-expressing cells with the LacI expression vector resulted in decreased GFP expression. The inventors further confirmed LacO's ability to suppress the EF1 promoter by treating cells co-transfected with LacI and the bdLV_LacO vector with isopropyl β-D-1-thiogalactopyranoside (IPTG) for 24 and 48 hours. IPTG binds to and suppresses LacI. Therefore, the increase in GFP expression observed when cells are treated with IPTG indicates that, in the absence of IPTG, LacI binds to the Lac operator sequence and suppresses GFP transcription mediated by the EF1 promoter (Figure 6Bii). ii) Cloning the Lac repressor (LacI) downstream of the PGK promoter. This step involves the substitution of puromycin with LacI (in reverse direction). LacI was PCR amplified from the pCMVLac vector (Agilent's LacSwitch II Inducible Mammalian Expression System) and subcloned into a smaller vector (pCDN3.1+) for reverse expression. Next, directional mutagenesis was used to excise the puromycin cDNA from the bdLV_LacO vector and create the restriction enzyme sites necessary to substitute it with LacI.

[0208] As shown in Figure 7, the inventors confirmed that the LacI protein expressed by the PGK promoter has the functionality to bind to the Lac operator sequence of bdLV_LacI_LacO and suppress GFP expression mediated by the EF1 promoter. For this reason, cells transfected with the bdLV_LacI_LacO vector were treated with IPTG. If the LacI protein expressed by PGK is functional, treatment with IPTG should increase GFP expression. The inventors confirmed that treatment with IPTG released approximately 50% of the suppression of GFP expression.

[0209] Next, as shown in Figure 8A, the inventors cloned four target sites of miR-29a, miR-149, or miR-29a and miR-149 (microRNAs specifically absent or downregulated in T-ALL cells) downstream of the reporter gene promoter in the bdLV_LacI_LacO construct. In the second step, as shown in Figure 8B, the inventors cloned four target sites of miR-128a or miR-153 (microRNAs specifically upregulated in T-ALL cells) downstream of the repressor of the reporter gene promoter in the bdLV_LacI_LacO construct.

[0210] Example 4: Testing the efficiency and specificity of a microRNA detector in vitro. To evaluate the actual efficiency and specificity of different microRNA detectors for selectively targeting leukemia cells in vitro, we began with cultures of T-ALL ("A" in Figures 9A and 9B) or non-T-ALL ("B" in Figures 9A and 9B) cells transduced with the microRNA detectors BdLV_miR29a_4×T, BdLV_miR149_4×T, and BdLV_miR29a_4×T_miR149_4×T.

[0211] By flow cytometry analysis of GFP expression, the inventors were able to determine the efficiency and specificity of each microRNA detector tested (see Figures 9A and 9B). The results showed that in cells expressing miR29a or miR149, the microRNA detectors BdLV_miR29a_4×T and BdLV_miR149_4×T (respectively) efficiently suppressed reporter gene expression (up to 90% suppression of GFP expression compared to BdLV_LacI_LacO transduced cells) (see Figures 9A and B). In cells not expressing any of these microRNAs, transduction using each microRNA detector did not affect GFP expression ("A" in Figures 9A and 9B). Importantly, in cells expressing both miR29a and miR149, transduction with the BdLV_miR29a_4×T_miR149_4×T detector has an additive effect, inducing a suppressive effect on GFP expression that surpasses that of the BdLV_miR29a_4×T and BdLV_miR149_4×T detectors ("B" in Figures 9A and 9B).

[0212] Referring to Figures 10A and 10B, the inventors then cultured T-ALL cells transduced with the microRNA detectors BdLV_miR128_4×T, BdLV_miR153_4×T, and BdLV_miR128_4×T_miR153_4×T. Flow cytometry analysis of GFP expression revealed that in T-ALL cells expressing miR128 and miR153, the microRNA detectors BdLV_miR128_4×T, BdLV_miR153_4×T, and BdLV_miR128_4×T_miR153_4×T efficiently desuppressed reporter gene expression (GFP expression increased up to 40-fold compared to BdLV_LacI_LacO transduced cells) (see Figures 10A and 10B).

[0213] Referring here to Figure 11, the inventors cultured cells transduced with microRNA detectors regulated by present or upregulated microRNAs and absent or downregulated microRNAs. Transduction with the microRNA detectors BdLV_miR128_4×T_miR29a_4×T, BdLV_miR128_4×T_miR149_4×T, BdLV_miR128_4×T_miR29a_4×T_miR149_4×T, and BdLV_miR153_4×T_miR29a_4×T_miR149_4×T efficiently suppressed reporter genes (up to 90% suppression of GFP expression compared to BdLV_LacI_LacO-transduced cells with microRNA detectors containing both miR29a and 149 target sites).

[0214] In CEM T-ALL cells expressing miR-128, miR-153, and miR-29a, transduction with the microRNA detectors BdLV_miR128_4×T_miR29a_4×T, BdLV_miR128_4×T_miR29a_4×T_miR149_4×T, and BdLV_miR153_4×T_miR29a_4×T_miR149_4×T efficiently suppressed the reporter gene. Conversely, transduction with the construct BdLV_miR128_4×T_miR149_4×T had little effect on reporter gene expression.

[0215] Referring to Figure 12, in 293-T non-T-ALL cells that do not express microRNAs 128 and 153, transduction using the microRNA detectors BdLV_miR-153_4×T-miR-29a_4×T_miR-149_4×T, BdLV_miR-128_4×T-miR-29a_4×T_miR-149_4×T, and BdLVmiR-128_4×T_miR-153_4×T-miR-29a_4×T_miR-149_4×T resulted in efficient suppression of the reporter gene.

[0216] In DND4.1 T-ALL cells expressing microRNAs 128 and 153, transduction using the microRNA detectors BdLV_miR-153_4×T-miR-29a_4×T, BdLV_miR-153_4×T-miR-149_4×T, and BdLV_miR-128_4×T_miR-153_4×T-miR-149_4×T efficiently desuppressed reporter genes. For CEM T-ALL cells expressing miR-128, miR-153, and miR-29a, transduction using the microRNA detectors BdLV_miR-128_4×T-miR-149_4×T, BdLV_miR-153_4×T-miR-149_4×T, and BdLV_miR-128_4×T miR-153_4×T-miR-149_4×T resulted in efficient desuppression of the reporter gene.

[0217] Referring to Figure 13, the inventors then cultured T-ALL cells and non-T-ALL cells transduced with non-integrated lentiviral vectors. As shown in the figure, transduction using all microRNA detectors resulted in efficient suppression of the reporter gene in 293-T non-T-ALL cells that do not express microRNAs 128 and 153. On the other hand, in CEM T-ALL cells, transduction using the microRNA detectors miR-153_4×T-miR-29a_4×T and miR-153_4×T-miR-149_4×T resulted in efficient desuppression of the reporter gene. Discussion and Conclusion As an example of the modulation of effector gene activity in a first nucleic acid sequence encoding a therapeutically active molecule or reporter molecule according to a first aspect of the present invention, the inventors have produced compelling proof-of-concept data on the feasibility and effectiveness of a microRNA detector that modulates GFP expression only in T-ALL cells. The inventors anticipate that such a system could be adapted in the future to other cancer types or other disease types by introducing relevant microRNA target sequences to the target cancer or disease, thereby paving the way for the use of this gene therapy technology in any disease in which cells exhibit diverse miRNA profiles.

[0218] The system's inherent ability to distinguish between T-ALL and non-T-ALL cells within a patient's body is therefore a defining characteristic of this technology and a critically valuable tool in high-precision oncology. It offers significant advantages over competing gene therapy technologies applied to high-precision oncology developed to date and is intentionally designed to provide patients with the best efficacy and safety outcomes. This innovative technology has the potential to effectively circumvent the lack of specificity in T-ALL treatment. Importantly, this strategy serves as a proof-of-concept demonstrating its applicability to the development of similar personalized therapies for other diseases and cancer cell types. Furthermore, this technology can be effectively used to diagnose specific conditions characterized by differing miRNA profiles in diseased cells and tissues compared to healthy cells / tissues.

[0219] Finally, using the system of the present invention, therapeutic molecules can be delivered with high specificity and efficiency using only a single vector, without the need for external factors or signaling. Furthermore, co-localization of molecules encoded within the same cell is achieved at lower doses than is possible using multiple construct approaches. Thus, compared to conventional techniques, these results offer a significant advantage due to the extremely simple system. References 1) Martini, M., Vecchione, L., Siena, S., Tejpar, S., Bardelli, A. (2011) Targeted therapies: how personal should we go. Nature Reviews of Clinical Oncology. Nov 15;9(2):87-97. 2) Pui, C .H., Relling, M.V., Downing, J.R. (2004) Acute lymphoblastic leukemia. New England Journal of Medicine. Apr 8;350(15):1535-48.3) Fullmer, A., O'Brien, S., Kantarjian, H., Jabbour, E. (2009) Novel therapies for relapsed acute lymphoblastic leukemia. Curr Hematol Malig Rep. Jul;4(3):148-56. 4) Smith, M. A. Update on developmental therapeutics for acute lymphoblastic leukemia (2009)C urr Hematol Malig Rep. 2009 Jul;4(3):175-82.5) Bartel, D.P. (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell Jan 23;116(2):281-97. 6) Huntzinger, E., Izaurralde, E. (2011) Gene silencing by microRNAs: contributions of translational repression and mRNA decay. Nat Rev Genet Feb;12(2):99-110.7) Kasinski, AL, Slack, FJ (2011) Epigenetics and genetics. MicroRNAs en route to the clinic: progress in validating and targeting microRNAs for cancer therapy. Nat. Rev. Fr. C cancer Nov 24;11(12):849-64.8) Rosenfeld, N., Aharonov, R., Meiri, E., Rosenwald, S., Spector, Y., Zepeniuk, M., Benjamin, H., Shabes, N., Tabak, S., Levy, A., Lebanony, D., Goren, Y., Silberschein, E., Targan, N., Ben-Ari, A., Gilad, S., Sion-Vardy, N., Tobar, A., Feinmesser, M., Kharenko O, Nativ O, Nass D, Perelman M, Yosepovich A, Shalmon B, Polak-C harcon S, Fridman E, Avniel A, Bentwich I, Bentwich Z, C ohen D, C hajut A, Barshack I. (2008) MicroRNAs accurately identify cancer tissue origin. Nat Biotechnol Apr;26(4):462-9. 9) Landgraf, P., Rusu, M., Sheridan, R., Sewer, A., Iovino, N., Aravin, A., Pfeffer, S., Rice, A., Kamphorst, AO, Landthaler, M., Lin, J., Socci, ND, Hermida, L., Fulci, V., Chiaretti, S., Focaliw, R., Schaliw, J. Fuchs, U., Novosel, A., Muller, RU, Schermer, B., Bissels, U., Inman, J., Phan, Q., Chien, M., Weir, DB, Choksi, R. et al. (2007) A mammalian microRNA expression atlas based on small RNA library sequencing. Cell Jun 29;129(7):1401-14.10) Brown, BD, Venneri, MA, Zingale, A., Sergi Sergi, L., Naldini, L. (2006) Endogenous microRNA regulation suppresses transgene expression in hematopoietic lineages and enables stable gene transfer. Nat Med May;12(5):585-91.11) Gentner, B., Visigalli, I., Hiramatsu, H., Lechman, E., Ungari, S., Giustacchini, A., Schira, G., Amendola, M., Quattrini, A., Martino, S., Orlacchio, A., Diffini, A., Bildini, J., Bildini, A. L. (2010) Identification of hematopoietic stem cell-specific miRNAs enables gene therapy of globoid cell leukodystrophy.Sci Transl Med. Nov 17;2(58):58ra84. 12) Xie, J., Xie, Q., Zhang, H., Ameres, S.L., Hung, J.H., Su, Q., He, R., Mu, X., Seher Ahmed, S., Park, S., Kato, H., Li, C., Mueller, C., Mello, C.C., Weng, Z., Flotte, T.R., Zamore, P.D., Gao, G. (2011) MicroRNA-regulated, systemically delivered rAAV9: a step closer to CNS-restricted transgene expression. Mol Ther. Mar;19(3):526-35. 13) Skalsky, R.L., Cullen, B.R. (2011) Reduced expression of brain-enriched microRNAs in glioblastomas permits targeted regulation of a cell death gene. PLoS One 6(9):e24248. 14) Garzon, R., Fabbri, M., Cimmino, A., Calin, GA, Croce, CM (2006) MicroRNA expression and function in cancer. Trends Mol Med. Dec;12(12):580-7.15) Lu, J., Getz, G., Miska, EA, Alvarez-Saavedra, E., Lamb, J., Peck, D., Sweet- Cordero, A., Ebert, BL, Mak, RH, Ferrando, AA, Downing, JR, Jacks, T. Horvitz, TR, Golub, TR. (2005) MicroRNA expression profiles classify human cancers. Nature. Jun 9;435(7043):834-8.16) Mavrakis, KJ, Van Der Meulen, J., Wolfe, AL, Liu, X., Mets, E., Taghon, T., Khan, AA, Setty, M., Rondou, P., Vandenberghe, P., Delabesse, E., Benoit, N., Socci, Y., Y. Leslie, C .S., Van Vlierberghe, P., Speleman, F., Wendel, HG (2011) A cooperative microRNA tumor suppressor gene network in acute T-cell lymphoblastic leukemia (T-ALL). Nat Genet. 2011 Jun 5;43(7):673-8.17) Schotte, D., C hau, JC ., Sylvester, G., Liu, G., C hen, C ., van der Velden, VH, Broekhuis, MJ, Peters, TC ., Pieters, R., den Boer, ML.(2009) Identification of new microRNA genes and aberrant microRNA profiles in childhood acute lymphoblastic leukemia. Leukemia Feb;23(2):313-22.18) Silva, A., Laranjeira, A.B., Martins, L.R., C ardoso, B.A., Demengeot, J., Yunes, J.A., Seddon, B., Barata, J.T. (2011) IL-7 contributes to the progression of human T-cell acute lymphoblastic leukemias. Cancer Res. Jul 15;71(14):4780-9.

Claims

1. (i) A first promoter operably ligated to a first nucleic acid sequence encoding a therapeutically active molecule or reporter molecule, wherein the first nucleic acid sequence includes at least one microRNA (miRNA) target site, and (ii) A second promoter operably ligated to a second nucleic acid sequence encoding an inhibitor and / or therapeutically active molecule or reporter molecule of a first promoter, wherein the second nucleic acid sequence comprises at least one miRNA target site, and at least one miRNA target site of the first and second nucleic acid sequences is different, A gene construct that includes this.

2. The gene construct according to claim 1, wherein at least one miRNA target site of the second nucleic acid sequence is a target site of a miRNA different from a miRNA that can target at least one miRNA target site of the first nucleic acid sequence.

3. The first nucleic acid sequence includes at least one miRNA target site or species of miRNA target site, at least two miRNA target sites or species of miRNA target site, at least three miRNA target sites or species of miRNA target site, at least four miRNA target sites or species of miRNA target site, or at least five miRNA target sites or species of miRNA target site. Depending on the circumstances, at least one copy of each miRNA target site, at least two copies of each miRNA target site, at least three copies of each miRNA target site, at least four copies of each miRNA target site, or at least five copies of each miRNA target site, or a species of miRNA target site may exist. A gene construct according to either claim 1 or claim 2.

4. The gene construct according to any one of claims 1 to 3, wherein at least one miRNA target site present in the first nucleic acid sequence is a target site of a miRNA not expressed in disease cells.

5. The gene construct according to claim 4, wherein the diseased cells are cancer cells, and in some cases the diseased cells are T-cell acute lymphoblastic leukemia (T-ALL) cells.

6. The second nucleic acid sequence includes at least one miRNA target site or species of miRNA target site, at least two miRNA target sites or species of miRNA target site, at least three miRNA target sites or species of miRNA target site, at least four miRNA target sites or species of miRNA target site, or at least five miRNA target sites or species of miRNA target site. Depending on the circumstances, at least one copy of each miRNA target site, at least two copies of each miRNA target site, at least three copies of each miRNA target site, at least four copies of each miRNA target site, or at least five copies of each miRNA target site, or a species of miRNA target site may exist. A gene construct according to any one of claims 1 to 5.

7. The gene construct according to any one of claims 1 to 6, wherein at least one miRNA target site present in the second nucleic acid sequence is a target site of a miRNA expressed in disease cells.

8. The gene construct according to any one of claims 1 to 7, wherein the reporter molecule is an optical reporter, a nuclear medicine reporter, or an MRI reporter.

9. The gene construct according to any one of claims 1 to 8, wherein the therapeutically active molecule is a therapeutic protein and / or nucleic acid.

10. The gene construct according to claim 9, wherein the nucleic acid is DNA, RNA, or a chimeric DNA / RNA molecule.

11. A gene construct according to any one of claims 1 to 9, wherein the therapeutic protein is selected from the group consisting of endonucleases, chimeric antigen receptors, viral proteins, and apoptosis-driven proteins.

12. The gene construct according to claim 11, wherein the therapeutic protein is an apoptosis-driven protein selected from the group consisting of Bax, Apoptin, E4orf4, and Bim.

13. The gene construct according to claim 12, wherein the therapeutic protein is Bax and comprises or comprises an amino acid sequence substantially described in Sequence ID No. 16, or a biologically active variant or fragment thereof.

14. The gene construct according to claim 12, wherein the therapeutic protein is Apoptin and comprises or consists of an amino acid sequence substantially described in Sequence ID No. 18, or a biologically active variant or fragment thereof.

15. The gene construct according to claim 12, wherein the therapeutic protein is E4orf4 and comprises or consists of a sequence substantially described in Sequence ID No. 20, or a biologically active variant or fragment thereof.

16. The gene construct according to claim 12, wherein the therapeutic protein is Bim and comprises, or consists of, a sequence substantially described in Sequence ID No. 25, or a biologically active variant or fragment thereof.

17. The gene construct according to any one of claims 1 to 16, wherein the second promoter is positioned in the construct in the opposite direction to the first promoter.

18. A gene construct according to any one of claims 1 to 17, wherein the inhibitor encoded by the second nucleic acid sequence is an inhibitor of the first promoter, the inhibitor of the first promoter is a Lac operon, the second nucleic acid sequence comprises a Lac repressor, and the first promoter comprises a Lac operator regulatory factor site.

19. A gene construct according to any one of claims 1 to 18, wherein the gene construct comprises a nucleic acid sequence, or a fragment or variant thereof, substantially described in any one of sequence numbers 5, 22 to 24, 27, or 78 to 82.

20. A recombinant vector comprising a gene construct according to any one of claims 1 to 19.

21. A gene construct according to any one of claims 1 to 19, or a vector according to claim 20, for use in therapeutic purposes.

22. A gene construct according to any one of claims 1 to 19, or a vector according to claim 20, for use in treating, preventing, or improving cancer.

23. A pharmaceutical composition comprising a gene construct according to any one of claims 1 to 19, or a vector according to claim 20, and optionally a pharmaceutically acceptable vehicle.

24. A gene construct according to any one of claims 1 to 19, or a vector according to claim 20, for use in diagnosis.

25. A gene construct according to any one of claims 1 to 19, or a vector according to claim 20, for use in the diagnosis of cancer.

26. A method for diagnosis or prediction, comprising the step of detecting a reporter molecule of a gene construct according to any one of claims 1 to 19, or a vector according to claim 20, in a sample obtained from a subject.

27. A method for diagnosing or predicting a target cancer, comprising the step of detecting a reporter molecule of a gene construct according to any one of claims 1 to 19, or a vector according to claim 20, in a sample obtained from a target.

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