Functional nucleic acid molecules and methods

Abstract

A construct configured to express a payload that is harmful (e.g., cytotoxic and immunomodulatory) intracellularly, which can be used to treat or prevent a primary cancer / tumor and to stimulate, induce, generate, or promote an effective immune response against recurrent / metastatic cancer / tumor that may arise from this primary cancer / tumor, is disclosed.

Description

Technical Field

[0001] The present disclosure relates to constructs for use in the treatment or prevention of recurrent / secondary cancer / tumors. The constructs may include a deleterious payload for killing target (cancer) cells. Such constructs may include a synthetic super-enhancer activated by one or more transcription factors, including a synthetic super-enhancer activated by one or more SOX family transcription factors.

Background Art

[0002] There has been great interest in the identification of cell-type specific enhancers and promoters that can be incorporated into gene therapy, especially in gene therapies where it is necessary to highly cell-selectively express therapeutic protein cargo to ensure maximum dosage and reduced off-target effects. However, current strategies are of limited effectiveness and generally require compromises in size, selectivity, or strength.

[0003] Two distinct but complementary strategies can be used to identify cell-type specific enhancers. First, strong and selective native enhancers for a given cell type can be revealed from a systematic and functional genetic dissection of the regulatory network for the cell type or lineage of interest. Second, fully synthetic enhancers can be constructed based on artificial synthetic DNA-binding motifs for known key transcription factors (TFs). Typically, databases of such motifs, approximately 5–10 base pairs in length, are defined based on biochemical assays for the strongest binding; for example, JASPAR (Jolma et al. 2013). Such transcription factor binding motifs can be synthesized and highly thoroughly screened using a library pool (Jolma et al. 2013). However, each approach has drawbacks. Individual native enhancers are typically not strong enough to be useful in phenotypic assays and are also limited by size constraints or inappropriate selectivity. Artificial promoters constructed on concatamers of transcription factor binding motifs can induce expression but lack the appropriate “grammar,” i.e., spacing, order, and appropriate affinities, present in native enhancer sequences and required for the recruitment of collaborating TFs and cofactors (Farley et al. 2015). Thus, they lack selectivity and stability.

[0004] Promoter arrays, enhancer arrays, and methods for identifying such arrays are described in the art, but these suffer from the drawbacks discussed above. WO 2014 / 066848 and US 2014 / 0296218 describe natural super-enhancers that are claimed to be useful for regulating the expression of cell-type specific genes and methods for identifying such super-enhancers. WO 2017 / 155973 describes synthetic enhancers for use in recombinant parvoviruses, particularly enhancers that have been reduced in size to enable delivery of large gene payloads. WO 2012 / 101191 describes the design of promoters for the selective expression of genes by using predictions of transcription factor binding site motifs to generate minimized promoters based on such motifs.

[0005] US Patent No. 5,952,221 describes an adeno-associated virus vector comprising first and second nucleic acid sequences.

[0006] US Patent No. 6,218,180 describes gene therapy for the treatment of solid tumors using recombinant adeno-associated virus vectors.

[0007] Govaerts et al. describe the introduction of genes encoding interleukin 2 and interleukin 12 into fibroblasts mediated by retroviruses (Cancer Gene Therapy, Vol. 6, pp. 447-455 (1999)).

[0008] Zhang et al. describe methods for enhancing the antitumor effects of chemotherapy by existing antitumor immunity (Cancer Immunology, Immunotherapy, Vol. 62, pp. 1061-1071 (2013)).

[0009] Tugues et al. presented new insights into IL-12-mediated tumor suppression (Cell Death & Differentiation, Vol. 22, pp. 237-246 (2015)).

[0010] Therefore, in this technical field, there remains a need to provide options for the treatment of recurrent / secondary cancers and to provide enhancers having selectivity and activity suitable for use in treatment methods, such as gene therapy.

Prior Art Documents

Patent Documents

[0011]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Patent Document 5

Patent Document 6

Patent Document 7

Non-Patent Documents

[0012]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non - Patent Document 5

Non - Patent Document 6

Non - Patent Document 7

Non - Patent Document 8

Non - Patent Document 9

Non - Patent Document 10

Non - Patent Document 11

Non - Patent Document 12

Non - Patent Document 13

Non - Patent Document 14

Non - Patent Document 15

Non - Patent Document 16

Non - Patent Document 17

Non - Patent Document 18

Non - Patent Document 19

Non - Patent Document 20

Non - Patent Document 21

Non - Patent Document 22

Non-Patent Document 23

Non-Patent Document 24

Non-Patent Document 25

Non-Patent Document 26

Non-Patent Document 27

Non-Patent Document 28

Non-Patent Document 29

Non-Patent Document 30

Non-Patent Document 31

Non-Patent Document 32

Non-Patent Document 33

Non-Patent Document 34

Non-Patent Document 35

Summary of the Invention

[0013] This disclosure is based on the finding that recurrent / secondary cancer / tumor can be treated or prevented using constructs designed and configured to express a harmful (e.g., cytotoxic and immunomodulatory) payload intracellularly.

[0014] In one teaching, any construct of the present disclosure can be used not only for the treatment, elimination or prevention of primary cancer / tumor, but also for stimulating, inducing, generating or promoting an immune response effective against recurrent / secondary cancer / tumor that may arise from this primary cancer / tumor.

[0015] The present disclosure is presented in the context of constructs for use, but the present disclosure (i) A method of treating recurrent / secondary tumor / cancer using the disclosed construct or stimulating, inducing, generating or promoting an immune response effective against recurrent / secondary cancer / tumor, and (ii) The use of the disclosed construct in the manufacture of a medicament for treating recurrent / secondary tumor / cancer or for stimulating, inducing, generating or promoting an immune response effective against recurrent / secondary cancer / tumor should be noted to further extend to.

[0016] In the context of the present disclosure, the terms cancer (primary and / or recurrent / secondary) / tumor (primary and / or recurrent / secondary), i.e., a disease characterized by abnormally proliferating (cancerous) cells, cancer and such a deposit or mass of cells, tumor, are interchangeable. Thus, the terms "recurrent / secondary cancer" or "recurrent / secondary tumor" include cancer (or tumor) associated with primary cancer (or tumor). Recurrent / secondary cancer can be the result of a re-growth of primary cancer or a recurrence from cancer stem cells. In this regard, recurrent / secondary cancer may have the same cancer type as primary cancer and may include the same cancer cells / the same cancer cell type (e.g., cells having the same type, morphology, and / or the same tumor marker). Primary cancer and recurrent / secondary cancer may be located in the same or different tissues. Recurrent / secondary cancer can occur some time after the occurrence of primary cancer or some time after treating, preventing or curing primary cancer.

[0017] An immune response "effective against recurrent / secondary tumors (or cancers)" includes an antibody or cellular response that promotes clearance or removal from recurrent / secondary cancer cells and / or the tissue of recurrent / secondary tumors. Without being bound by theory, it is suggested that in a subject who has already been treated for a primary cancer / tumor at the time of onset of a recurrent / secondary cancer / tumor, an immune response can be induced (via immune memory) using the constructs of the present disclosure. The immune response can include a measurable change in at least one cell or one cell type or one endocrine or exocrine pathway of the immune system (including, but not limited to, a cellular response, a humoral response, a cytokine response, a chemokine response). Thus, the constructs of the present disclosure are capable of treating a primary cancer and rely on the induction or stimulation of an immune response (induced / stimulated by an initial treatment with the construct) without readministering the construct to the subject (or any other optional treatment option, e.g., chemotherapy) to provide an option for treating, curing, and / or clearing any recurrent / secondary tumor / cancer that may arise from the primary tumor / cancer.

[0018] An immune response (as described above) effective against recurrent / secondary tumors / cancers arising from a particular primary tumor / cancer can be referred to as an "anti-recurrent / secondary cancer immune response" or an "anti-recurrent / secondary tumor immune response".

[0019] Accordingly, the present invention provides constructs of the present disclosure for use in establishing an anti-recurrent / secondary cancer immune response or an anti-recurrent / secondary tumor immune response in a subject (suffering from or having a predisposition / susceptibility to a primary cancer / tumor).

[0020] In addition, the present disclosure provides a method for establishing an anti-recurrent / metastatic cancer immune response or an anti-recurrent / metastatic tumor immune response in a subject, wherein the recurrent / metastatic cancer / tumor potentially arises from a primary cancer, and the method includes treating the primary cancer / tumor with the construct of the present disclosure, and the treatment of the primary cancer / tumor with the construct of the present disclosure induces, establishes or stimulates an immune response effective against any recurrent / metastatic cancer / tumor that may arise from the primary cancer (already treated). In one teaching, the method of the present disclosure provides a method for (i) treating or preventing a recurrent / metastatic cancer / tumor and / or (ii) generating, stimulating or inducing an anti-recurrent / metastatic cancer / tumor immune response, the method including administering to a patient suffering from a primary cancer the construct of the present disclosure (optionally packaged in a viral vector), whereby treating or preventing the primary cancer prevents or treats any recurrent / metastatic cancer / tumor that may arise from the already treated primary cancer and / or generates, stimulates or induces an anti-recurrent / metastatic cancer / tumor immune response effective for the elimination, treatment or prevention of any recurrent / metastatic cancer / tumor that may arise from the already treated primary cancer.

[0021] The present disclosure further provides the use of the construct of the present disclosure for the manufacture of a medicament for establishing, generating or stimulating an anti-recurrent / metastatic cancer immune response or an anti-recurrent / metastatic tumor immune response.

[0022] Subjects to which the constructs of the present disclosure are administered can include human or animal subjects suffering from primary cancer or having a predisposition or susceptibility thereto. In such subjects, any of the disclosed constructs can be used to treat, eliminate or prevent primary cancer. As described above, if recurrent / secondary cancer occurs due to an immune response being induced or stimulated by initial treatment (using the construct), it has been found that a defensive anti-tumor / cancer immune response is induced, and this immune response cures or eliminates the recurrent / secondary cancer / tumor. In this specification, this phenomenon is described as immune memory, and (although not wishing to be bound by theory) by using a construct that treats or prevents primary cancer, this immune memory (of cancer) is induced or stimulated, and if recurrent / secondary cancer or a tumor occurs, it is suggested that this immune memory is activated to facilitate clearance and / or treatment of the recurrent / secondary cancer / tumor.

[0023] In the remainder of the present disclosure, the characteristics of the constructs are described, but it should be understood that any such constructs are for the various uses, compositions, methods, and uses in medicaments described herein. Thus, when referring in the specification to some products "for use" (e.g., constructs or functional nucleic acids), as presented in the present disclosure, it should be construed as relating not only to the relevant products for use in the treatment or prevention of recurrent / secondary cancer (by the generation, induction or stimulation of an anti-recurrent / secondary cancer / tumor immune response), but also to the relevant products for use in the manufacture of the methods or medicaments of the present disclosure.

[0024] The constructs of the present disclosure can contain a harmful payload. In the context of the present disclosure, the term "harmful payload" means an element (e.g., a gene or a transgene) that can be expressed within a target cell and that has a negative effect on this cell. The negative effect can be, for example, a negative action on the health or viability of the cell and / or on the cell's ability to divide, proliferate, and / or differentiate. The harmful action can be direct (e.g., a gene or transgene that encodes a substance that harms the cell, such as a pro-apoptotic gene or a suicide gene) or indirect (e.g., a gene or transgene that encodes a substance that mobilizes an exogenous factor that causes an action that harms the cell).

[0025] In view of the above, the constructs for the various uses, methods, and medicaments described herein can be any of the following: (i) A suicide gene (e.g., a gene that expresses or encodes a protein that causes cell death), (ii) A suicide gene that encodes a protein capable of converting an inactive prodrug into a cytotoxic drug, (iii) An element that stimulates an immune response that has a negative effect on the target cell, (iv) A protein that stimulates an immune response that causes activation of cytotoxic immune cells, (v) A chemokine, (vi) A cytokine (including a pro-inflammatory cytokine), (vii) An antibody or an antigen-binding fragment thereof, (viii) An immunomodulatory protein, (ix) IL-12, (x) IL-10, (xi) IL-2, (xii) IFN-α, (xiii) GM-CSF, (xiv) A cytotoxic substance, (xv) Herpes simplex virus thymidine kinase (HSV-TK), and (xvi) Cytosine deaminase (CD) can contain one or more (i.e., 1, 2, 3, 4 or more) of these.

[0026] In one teaching, the harmful payload may include IL-12.

[0027] Without wishing to be bound by theory, IL-12 has anti-cancer activity and not only induces IFN-γ production from resting and / or activated CD4+ T cells, CD8+ T cells, and natural killer (NK) cells, but also enhances the proliferation of activated T cells and NK cells, increases the lytic activity of NK / lymphokine-activated killer cells, and can promote specific cytotoxic T lymphocyte (CTL) responses. On the other hand, IL-12 is toxic, and this particular payload can be used as a targeted therapy that controls expression so that it occurs only within specific target cells, such as specific cancer cells or primary cancer cells.

[0028] Accordingly, the present disclosure provides a construct comprising an element capable of expressing IL-12 intracellularly for use in stimulating, inducing, generating, or promoting an effective immune response against recurrent / secondary cancer / tumor. The cell can be a cancer cell. The cell may be a primary cancer cell, and this primary cancer cell is associated with recurrent / secondary cancer for which the immune response is effective.

[0029] In a further teaching, the harmful payload may include or further include a suicide gene.

[0030] The harmful payload may include or further include herpes simplex virus thymidine kinase (HSV-TK).

[0031] The harmful payload may include any two of the harmful payloads listed / described above.

[0032] In one example, constructs for the various uses described herein may include elements capable of expressing cytokines (e.g., IL12) and suicide genes (e.g., HSV-TK) within target cells. Again, the cells can be cancer cells. The cells can be primary cancer cells, and these primary cancer cells are associated with recurrent / metastatic cancer where the immune response is effective.

[0033] In view of the above, the present disclosure provides constructs containing elements capable of expressing IL-12 and HSV-TK intracellularly for use in stimulating, inducing, generating, or promoting an effective immune response against recurrent / metastatic cancer / tumor. The cells can be cancer cells. The cells can be primary cancer cells, and these primary cancer cells are associated with recurrent / metastatic cancer where the immune response is effective.

[0034] When the construct contains a suicide gene, the construct can be used in combination with a prodrug. For example, when the suicide gene contains HSV-TK, the construct can be administered in combination with ganciclovir (and / or its analogs, e.g., acyclovir and valacyclovir). When the suicide gene contains cytosine deaminase, the construct can be administered in combination with 5-fluorocytosine (5FC).

[0035] Constructs for any of the uses, methods, or medicaments of the present disclosure may further include super-enhancer elements.

[0036] The super-enhancer element may be activated by one or more transcription factors and may contain two or more enhancer sequences derived from different genomic loci, and each of these enhancer sequences contains a binding site for a transcription factor.

[0037] In an alternative teaching, a synthetic super-enhancer for use in the constructs of the present disclosure may be activated by one or more transcription factors, the synthetic super-enhancer comprising two or more enhancer sequences derived from different genomic loci, each enhancer sequence comprising a binding site for a transcription factor and 20 to 400 nucleotides upstream and / or downstream of the binding site for the transcription factor that are present at that genomic locus.

[0038] The term "synthetic" may encompass super-enhancer constructs, and typically two or more of these enhancer sequences located at different genomic loci are isolated and combined in a new construct. Thus, a synthetic super-enhancer may not exist in nature and may be described as an artifact or a "manufacture." Accordingly, the present disclosure describes the use of a construct activated by one or more transcription factors, the construct comprising two or more enhancer sequences derived from different genomic loci in addition to any harmful payload, each enhancer sequence comprising a binding site for a transcription factor. For convenience, the term "synthetic super-enhancer" is used herein to encompass any of the super-enhancers / constructs described herein.

[0039] The present disclosure further provides for the use of a functional nucleic acid encoding any of the constructs described herein. Accordingly, the term "construct" as used herein refers to a product comprising a plurality of elements configured for expression within a cell (e.g., a cancer cell, including a primary cancer cell, etc.). Thus, the present disclosure provides a functional nucleic acid molecule comprising a transgene that itself comprises a harmful payload described herein. The transgene may be operably linked to a promoter designed to ensure expression of the transgene within a target cell. Thus, in one teaching, the transgene may be operably linked to a synthetic super-enhancer of the type described herein.

[0040] In one teaching, the present disclosure provides a functional nucleic acid molecule comprising a super-enhancer construct described herein operably linked to a transgene encoding a harmful payload for use in inducing an anti-tumor immune response. Also, the payload encoded by the transgene can include cytokines (e.g., IL12) and suicide genes (e.g., HSK-TK). The functional nucleic acid can be used in combination with a prodrug that acts as a substrate for the product of the suicide gene.

[0041] According to a further aspect, there is provided the use of a functional nucleic acid molecule comprising a synthetic super-enhancer activated by one or more transcription factors expressed in a target abnormal cell (e.g., a cancer cell or a primary cancer cell) and a harmful payload, wherein the payload is expressed upon activation of the synthetic super-enhancer by the transcription factor. In a further teaching, there is provided the use of a functional nucleic acid molecule comprising a super-enhancer construct activated by one or more transcription factors expressed in a target abnormal cell (e.g., a cancer cell or a primary cancer cell) and a harmful payload, wherein the payload is expressed upon activation of the super-enhancer construct by the transcription factor.

[0042] According to a further aspect, the construct for use described herein may comprise a synthetic super-enhancer activated by one or more SOX family transcription factors, the synthetic super-enhancer comprising two or more enhancer sequences and at least one SOX motif and / or SOX dimer motif. In one teaching, there is provided a super-enhancer construct that is a super-enhancer construct activated by one or more SOX family transcription factors and comprises two or more enhancer sequences and at least one SOX motif and / or SOX dimer motif.

[0043] According to a further aspect, a construct for any of the uses presented in the present disclosure may include four enhancer sequences derived from different genomic loci, each of the enhancer sequences including a synthetic super-enhancer activated by SOX2 and including at least one SOX dimer motif. In one teaching, a construct of the present disclosure may include four enhancer sequences derived from different genomic loci, each of the enhancer sequences including a super-enhancer construct activated by SOX2 and including at least one SOX dimer motif.

[0044] According to a further aspect, a construct of the present disclosure may be provided in the form of a vector.

[0045] According to a further aspect, the present disclosure provides for the use of a composition, such as a pharmaceutically acceptable composition (optionally including a pharmaceutically acceptable excipient), including any of the constructs described herein.

[0046] In one teaching, a method for treating recurrent / secondary glioblastoma that may arise from primary glioblastoma, including administering a functional nucleic acid molecule and an inactive prodrug to a patient suffering from or having a predisposition / susceptibility to primary glioblastoma, thereby treating the primary glioblastoma and inducing or stimulating an immune response effective against any recurrent / secondary glioblastoma, wherein the functional nucleic acid includes a synthetic super-enhancer activated by a SOX transcription factor and a suicide gene encoding a protein capable of converting the inactive prodrug into a cytotoxic drug, the present disclosure presents a method wherein the suicide gene is expressed in glioblastoma cells upon activation of the synthetic super-enhancer by the SOX transcription factor.

[0047] One teaching is a method for treating recurrent / secondary glioblastoma, comprising administering a functional nucleic acid molecule and an inactive prodrug to a patient suffering from or having a predisposition / susceptibility to primary glioblastoma, thereby treating the primary glioblastoma and inducing or stimulating an immune response effective against any recurrent / secondary glioblastoma that may arise from the primary glioblastoma. This functional nucleic acid comprises a super-enhancer construct activated by a SOX transcription factor and a suicide gene encoding a protein capable of converting the inactive prodrug into a cytotoxic drug. Provided is a method wherein this suicide gene is expressed in glioblastoma cells upon activation of the super-enhancer construct by a SOX transcription factor.

Brief Description of the Drawings

[0048]

Figure 1A

Figure 1B

Figure 1C

Figure 1D

Figure 1E

Figure 1F

Figure 1G

Figure 1H

Figure 1I

Figure 1J

Figure 1K

Figure 1L

Figure 2A

Figure 2B

Figure 2C

Figure 2D

Figure 2E

Figure 2F

Figure 2G

Figure 2H

Figure 2I

Figure 2J

Figure 2K

Figure 2L

Figure 3A

Figure 3B

Figure 3C

Figure 3D

Figure 3E

Figure 4A

Figure 4B

Figure 4C

Figure 4D

Figure 4E

Figure 4F

Figure 4G

Figure 4H

Figure 4I

Figure 4J

Figure 4K

Figure 5A

Figure 5B

Figure 5C

Figure 5D

Figure 5E

Figure 5F

Figure 5G

Figure 5H

Figure 6A

Figure 6B

Figure 6C

Figure 6D

Figure 6E

Figure 7A

Figure 7B

Figure 7C

Figure 8A

Figure 8B

Figure 8C

Figure 8D

Figure 8E

Figure 8F

Figure 8G

Figure 8H

Figure 8I

Figure 8J

Figure 8K

Figure 8L

Figure 8M

Figure 9A

Figure 9B

Figure 9C

Figure 9D

Figure 9E

Figure 9F

Figure 9G

Figure 10A

Figure 10B

Figure 10C

Figure 10D

Figure 11A

Figure 11B

Figure 11C

Figure 11D

Figure 12A

Figure 12B

Figure 13

Figure 14

Figure 15

Figure 16

Figure 17

Mode for Carrying Out the Invention

[0049] In the present disclosure, (i) for use in the treatment, prevention, elimination, and / or cure of recurrent / secondary cancer, (ii) for use in the treatment, prevention, elimination, and / or cure of recurrent / secondary cancer in a subject under primary cancer treatment, (iii) for use in generating, stimulating or inducing an anti-recurrent / secondary cancer immune response, (iv) for use in generating, stimulating or inducing an anti-recurrent / secondary cancer immune response in a subject undergoing primary cancer treatment, (v) for use in a method of treating, preventing, eradicating, and / or curing recurrent / secondary cancer, (vi) for use in a method of treating, preventing, eradicating, and / or curing recurrent / secondary cancer in a subject undergoing primary cancer treatment, (vii) for use in a method of generating, stimulating or inducing an anti-recurrent / secondary cancer immune response, (viii) for use in a method of generating, stimulating or inducing an anti-recurrent / secondary cancer immune response in a subject undergoing primary cancer treatment, (ix) for use in the manufacture of a medicament for treating, preventing, eradicating, and / or curing recurrent / secondary cancer, (x) for use in the manufacture of a medicament for treating, preventing, eradicating, and / or curing recurrent / secondary cancer in a subject undergoing primary cancer treatment, (xi) for use in the manufacture of a medicament for generating, stimulating or inducing an anti-recurrent / secondary cancer immune response, (xii) for use in the manufacture of a medicament for generating, stimulating or inducing an anti-recurrent / secondary cancer immune response in a subject undergoing primary cancer treatment to provide a construct.

[0050] The present disclosure provides for the use of novel (synthetic) super enhancers (SSEs) and the delivery of transgenes, such as therapeutic and harmful payloads. Building on advances in the knowledge of cell-type specific gene regulation, the inventors considered that it might be possible to generate SSEs by artificially (or synthetically) associating enhancers that retain central functional elements and transcription factor motifs within full-length enhancers. This may involve utilizing natural and functional flanking sequences that surround direct transcription factor motifs within such enhancers. This strategy aims to enable the retention of the binding relationship of native, localized transcription factors (TFs) to key cell-type specific TFs (at appropriate spacing with low affinity), while promoting high concentrations of TF binding to cause strong transcription. That is, it captures the appropriate syntax (co-binding protein spacing, affinity, and orientation) of TF motifs within cell-type specific enhancers, while pushing transcription activity forward by forming an array of multiple parts in combination. As described herein, the construction of SSEs using assemblies of functionally annotated enhancers has been found to result in potent and more highly cell-type specific regulatory elements, most notably with many applications in gene therapy.

[0051] Synthetic super enhancer In the present disclosure, there is provided the use of a construct comprising a super-enhancer activated by one or more transcription factors, wherein the super-enhancer comprises two or more enhancer sequences derived from different genomic loci, and each enhancer sequence comprises a binding site for a transcription factor. In another teaching, there is provided by the present disclosure the use of a construct comprising a synthetic super-enhancer activated by one or more transcription factors, wherein the synthetic super-enhancer comprises two or more enhancer sequences derived from different genomic loci, and each enhancer sequence comprises a binding site for a transcription factor and 20 to 400 nucleotides upstream and / or downstream of the binding site for the transcription factor present at that genomic locus. In a further teaching, there is provided by the present disclosure the use of a super-enhancer construct activated by one or more transcription factors, wherein the super-enhancer construct comprises two or more enhancer sequences derived from different genomic loci, and each enhancer sequence comprises a binding site for a transcription factor.

[0052] As used herein, "enhancer" refers to a DNA sequence (e.g., an "enhancer sequence") to which a protein (e.g., a transcription factor) binds to enhance the transcription of a gene. The term "enhancer" can encompass, for example, a specific short region of DNA (usually 50 - 1500 bp) to which a transcription factor binds. Nevertheless, an enhancer sequence can further include a portion of the sequence flanking or surrounding the direct transcription factor motif. Enhancers typically act as cis-regulatory elements, but can also act in trans and activate two or more target genes. They can be located distally from the transcription start site (TSS) of the gene they regulate, for the purpose of promoting DNA folding and three-dimensional structure or multimolecular compartments (also called transcription hubs or condensates, which can be formed by phase separation). In contrast, a promoter is present proximal to the transcription start site, upstream of the TSS on the same DNA sequence. A promoter binds RNA polymerase II to initiate transcription.

[0053] H3K27Ac is a histone modification commonly found in enhancers and can be used to predict enhancer active regions. The determination of whether a sequence enhances the transcription of a gene (and thus can be called an "enhancer") can be made using methods known in the art, most typically using a plasmid-based reporter assay.

[0054] Super enhancers are also involved in the activation or regulation of transcription. Originally, the term "super enhancer" refers to a sequence that contains a cluster of multiple enhancers and usually spans a region of 5 - 15 kb. Such "super enhancers" are highly occupied by transcriptional activation cofactors, such as the mediator complex, compared to average enhancers. They exhibit greater activity than average enhancers and are frequently involved in driving the expression of cell-type specific genes involved in maintaining cell identity or disease state (see, for example, US Patent Application Publication No. 2014 / 0296218 and US Patent Application Publication No. 2014 / 0287932, which are hereby incorporated by reference in their entirety). As used herein, when referring to "super enhancer", "synthetic super enhancer" (or "SSE") or "super enhancer construct", it is understood to refer to a super enhancer that is not found in nature. That is, it is an artificially constructed super enhancer designed for the purposes described herein and may exhibit characteristics associated with natural super enhancers. In particular, an SSE is an artificial construct that contains two or more enhancer sequences (which may be derived from different genomic loci). Also, to avoid misunderstanding, the term "SSE" is used to encompass any of the super enhancers, synthetic super enhancers, and / or super enhancer constructs described herein. An SSE can have a high concentration of transcription factor motifs to stimulate the activation of transcription. This allows for the SSE sequence to be shorter than a natural super enhancer because the high concentration of transcription factors binding to the SSE promotes transcriptional activity.

[0055] In one embodiment, the present disclosure presents the use of one or more transgenes by coupling them to an SSE that is activated by a transcription factor or a specific combination of transcription factors that are expressed within a target cell (e.g., a cancer cell or a primary cancer cell). Thus, the endogenous expression of the transcription factor within the target cell is used to highly selectively initiate the expression of the exogenous transgene. Preferably, the transcription factor has a cell type- or lineage-specific expression pattern and thus is minimally expressed in cells other than the target cell. That is, it is not ubiquitously expressed. This enables cell type- or cell state-selective activation of the SSE and expression of the transgene, and thus minimizes off-target toxicity. In one embodiment, the transcription factor is specifically expressed within the target cell, i.e., as compared to other cell types. Also, the use of the SSE preferably results in high expression of the transgene that is close to or equivalent to the expression of the transgene when using a strong promoter, e.g., the full-length cytomegalovirus (CMV) promoter. The SSEs are highly selective, potent, and can be small in size (preferably less than 500 base pairs). These provide a “switch” in gene expression of the associated open reading frame / transgene from minimal to very strong expression depending on the cell type identity or state.

[0056] The function of enhancers and super-enhancers may include the presence of a promoter, e.g., a minimal promoter (e.g., minimal CMV), which includes a key region that promotes the recruitment of the transcriptional machinery and typically includes the TSS. In an alternative embodiment, the SSEs of the present disclosure may not include a promoter and / or a minimal CMV promoter.

[0057] As used herein, the term "transcription factor" refers to a protein that binds to regulatory elements of a target gene to regulate, for example, an increase or decrease in the expression of the target gene. Transcription factors (TFs) are generally grouped based on the type of DNA-binding domain present in the TF. In eukaryotes, the most common DNA-binding domains are the zinc finger domain, the homeodomain, the basic leucine zipper domain, and the basic helix-loop-helix domain. TF paralogs having the same DNA-binding domain generally recognize similar DNA sequences. The synthetic super-enhancers described herein can bind one or more transcription factors. That is, one or more transcription factors bind to the SSE, thereby causing activation. Activation occurs by recruitment of RNA polymerase II, which activates the expression of the associated gene (i.e., the transgene) and enhances transcription beyond the expression level by the promoter alone. Binding of a transcription factor to the SSE can induce the associated transcriptional and chromatin regulatory mechanisms.

[0058] In one embodiment, the transcription factor is a developmental or stem cell-related transcription factor. Transcription factors of this type are associated with high levels of activation in stem cells or cell development.

[0059] In one embodiment, the transcription factor is a SOX family transcription factor. Transcription factors of the SOX (SRY-related HMG box) gene family include 20 members in the human and mouse genomes, all of which share a high-mobility group (HMG) box domain. SOX family members with high homology are classified into groups: (SoxA, SoxB1, SoxB2, SoxC, SoxD, SoxE, SoxF, SoxG, and SoxH). In one embodiment, the transcription factor is a SoxB1 (i.e., SOX1, SOX2, SOX3) or SoxE (i.e., SOX8, SOX9, SOX10) transcription factor.

[0060] SOX2 has been identified as an important regulator of cancer stem cells (Bulstrode et al. 2017, Gangemi et al. 2009, Guerra-Rebollo et al. 2019, Lujan et al. 2012, D. K. Singh et al. 2017, S. K. Singh et al. 2004, and Boumahdi et al. 2014). ChIP-seq datasets for glioblastoma stem cells (GSCs) and their differentiated progeny have been reported by Suva et al. 2014, who classified histone H3 enhancer (H3K27ac) marks and SOX2 binding specific to GSCs. In particular, the inventors have identified a set of candidate SOX2 regulatory enhancers that are operable within GSCs across diverse patient lineages. Thus, in one embodiment, the transcription factor is SOX2.

[0061] The transcription factor binds by virtue of the presence of the DNA binding domain of the transcription factor (i.e., the transcription factor binding site) that binds to a specific motif within the target enhancer. Typically, the transcription factor binding site sequence is 5 to 15 bp in length.

[0062] In one embodiment, the synthetic super enhancer (SSE) contains at least one SOX dimer motif. Such motifs may be important for the function of the SSE in the target cells. For example, many of the enhancer sequences described herein have been found to contain SOX dimer sites required for the maintenance of activity in glioblastoma stem cells. The enhancer element may contain a SOX binding site (which may be a palindromic sequence), and this SOX transcription factor may homodimerize or heterodimerize. Typically, the spacing between each individual monomer binding motif is approximately 8 to 12 bp.

[0063] In one embodiment, the SOX dimer motif is SEQ ID NO:1: ACAAAGRGSVBYTKK In the sequence, R represents A or G, S represents C or G, V represents A, C or G, B represents C, G or T, Y represents C or T, and K represents G or T.

[0064] In a further embodiment, the SOX dimer motif is SEQ ID NO: 2: RRRRASARAGRRRBBHDDBWH In the sequence, R represents A or G, S represents C or G, B represents C, G or T, H represents A, C or T, D represents A, G or T, and W represents A or T.

[0065] In one embodiment, the synthetic super enhancer comprises at least one SOX motif. In a further embodiment, the SOX motif is a SOX2 motif.

[0066] In one embodiment, the SOX2 motif is SEQ ID NO: 3: WSARAGRSMYMHTBB In the sequence, W represents A or T, S represents C or G, R represents A or G, M represents A or C, Y represents C or T, H represents A, C or T, and B represents C, G or T.

[0067] In one embodiment, the synthetic super enhancer comprises a sequence selected from a SOX motif and / or a SOX dimer motif.

[0068] As described herein, a super enhancer refers to a cluster of enhancer sequences. Thus, in one embodiment, the synthetic super enhancer comprises two or more enhancer sequences. In a further embodiment, the synthetic super enhancer comprises 2 to 8 enhancer sequences. In yet a further embodiment, the synthetic super enhancer comprises 4 enhancer sequences.

[0069] Enhancer elements can be identified using techniques known in the art. For example, in the methods described herein, ChIP-seq datasets for specific cell types identifying enhancers bound by the transcription factor of interest, or chromatin proximity assays (e.g., ATAC-seq or MNase profiling) were used. Public resources can be utilized to aid in the genome-wide identification of active enhancers in various organisms and cell types, such as, for example, the ENCODE or The Cancer Genome Atlas (TCGA) datasets.

[0070] Preferably, the enhancer sequences present in the SSE do not all have the same sequence (i.e., the SSE is not a tandem array of identical enhancer elements). Thus, in one embodiment, the SSE comprises at least two different enhancer sequences. In one embodiment, each of the enhancer sequences has a different sequence. In one embodiment, each of the enhancer sequences is activated by the same transcription factor.

[0071] The data presented herein surprisingly show that the association of four-part arrays of enhancers (≈640 bp) from different genomic loci caused a orders-of-magnitude increase in these activities without compromising selectivity. Thus, each of the enhancer sequences used in the SSE can be derived from a different gene. In a further embodiment, each of the enhancer sequences is derived from a different genomic locus. All such enhancer sequences are still preferably activated by a common set of transcription factors or cell type-related transcription factors. In one embodiment, each of the enhancer sequences is activated by a transcription factor and is derived from a different genomic locus. In one embodiment, the synthetic super-enhancer comprises two or more, e.g., four, enhancer sequences derived from different genomic loci.

[0072] In one embodiment, each enhancer sequence comprises a binding site for a transcription factor and 20 to 400 nucleotides upstream and / or downstream of the binding site for the transcription factor that are present at its genomic locus. Thereby, the enhancer sequence includes the relationship surrounding the transcription factor binding site (i.e., immediately upstream and / or immediately downstream), which enables the retention of enhancer function. The upstream and / or downstream sequences typically present in natural enhancer sequences that maintain spacing, order, orientation, and / or affinity may be useful for the recruitment of cooperating transcription factors and cofactors. In addition, the sequence relationship surrounding the transcription factor binding motif has the potential to promote binding and act cooperatively to enhance transcription efficiency. Those skilled in the art can identify / obtain the sequences of 20 to 400 nucleotides upstream and / or downstream of the binding site of interest from public genomic databases. Those skilled in the art can determine the activity of transcription factors by functional analysis, such as the reporter assay described herein or other methods widely employed in the art. In one embodiment, each enhancer sequence comprises a binding site for a transcription factor and 20 to 400 nucleotides upstream and / or downstream of the binding site for the transcription factor that are present at its genomic locus, and these upstream and / or downstream sequences are sufficient to ensure the retention of the function of the enhancer. In a further embodiment, the enhancer sequence comprises 20 to 400 nucleotides upstream and / or downstream of the binding site for the transcription factor, for example, 20 to 300 nucleotides upstream and / or downstream of the binding site for the transcription factor, or preferably 20 to 200 nucleotides upstream and / or downstream of the binding site for the transcription factor. In a further embodiment, the enhancer sequence comprises 50 to 400 nucleotides upstream and / or downstream of the binding site for the transcription factor, for example, 50 to 300 nucleotides upstream and / or downstream of the binding site for the transcription factor, or preferably 50 to 200 nucleotides upstream and / or downstream of the binding site for the transcription factor.In a further embodiment, the enhancer sequence comprises from 80 to 400 nucleotides upstream and / or downstream of the binding site for the transcription factor, such as from 80 to 300 nucleotides upstream and / or downstream of the binding site for the transcription factor, or preferably from 80 to 200 nucleotides upstream and / or downstream of the binding site for the transcription factor.

[0073] In one embodiment, each of the enhancer sequences within the synthetic super enhancer is less than 500 nucleotides in length, such as less than 450, 400, 350, 300, 250 or 200 nucleotides in length. In a further embodiment, each of the enhancer sequences within the SSE is less than 300 nucleotides in length. In one embodiment, each of the enhancer sequences within the synthetic super enhancer is from 20 to 500 nucleotides in length, such as from 50 to 250 nucleotides in length or from 100 to 200 nucleotides in length.

[0074] It is understood that such embodiments can be combined. Thus, in a further embodiment, the enhancer sequence comprises from 20 to 400 nucleotides upstream and / or downstream of the binding site for the transcription factor, and this enhancer sequence is less than 500 nucleotides in length. In yet a further embodiment, the enhancer sequence comprises from 20 to 200 nucleotides upstream and / or downstream of the binding site for the transcription factor, and this enhancer sequence is less than 300 nucleotides in length.

[0075] In a further embodiment, each of the enhancer sequences within the SSE is approximately 160 nucleotides in length. In particular, 160 base pairs (bp) were selected because it provides a size sufficient for nucleosome binding and is considered an important property in the evolution of grammar in functional enhancers. Moreover, 160 bp is a convenient size for the synthesis of oligonucleotide library pools that incorporate 20 bp ends for subsequent plasmid library array construction.

[0076] In one embodiment, the synthetic super enhancer is less than 2000 nucleotides in length. In a further embodiment, the synthetic super enhancer is less than 1500 nucleotides in length, such as less than 1400, 1300, 1200, 1100, 1000, 950, 900, 850, 800, 750 or 700 nucleotides in length. In one embodiment, the synthetic super enhancer is less than 1200 nucleotides in length. In a further embodiment, the synthetic super enhancer is less than 700 nucleotides in length. Preferably, the synthetic super enhancer is less than 650 nucleotides in length, such as less than 640 nucleotides in length.

[0077] The motifs described herein may be present in one or more of the enhancers of the SSE (see, for example, FIG. 8E). Thus, in one embodiment, one or more of the enhancers comprise a sequence selected from the SOX motif and / or the SOX dimer motif.

[0078] In one embodiment, each of the enhancer sequences within the synthetic super enhancer comprises a SOX motif, such as a SOX2 motif. In one embodiment, each of the enhancer sequences within the synthetic super enhancer comprises a SOX dimer motif.

[0079] In one embodiment, the synthetic super-enhancer is activated by SOX2, the synthetic super-enhancer includes four enhancer sequences derived from different genomic loci, and each of the enhancer sequences includes at least one SOX motif and / or at least one SOX dimer motif. In one embodiment, the synthetic super-enhancer is activated by SOX2, the synthetic super-enhancer includes four enhancer sequences derived from different genomic loci, and each of the enhancer sequences includes at least one SOX2 motif. In one embodiment, the synthetic super-enhancer is activated by SOX2, the synthetic super-enhancer includes four enhancer sequences derived from different genomic loci, and each of the enhancer sequences includes at least one SOX dimer motif.

[0080] In one embodiment, one or more enhancer sequences include a sequence having at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 4 to 63 (Table 1 (Table 7)). In a further embodiment, one or more enhancer sequences include a sequence having at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 4 to 13 and SEQ ID NOs: 56 to 63. In yet a further embodiment, one or more enhancer sequences have at least 85%, 90%, 95%, 97%, 99% sequence identity to, or are 100% identical to, a sequence selected from the group consisting of SEQ ID NOs: 4 to 63 (e.g., SEQ ID NOs: 4 to 13 and SEQ ID NOs: 56 to 63). The enhancer sequences described herein (i.e., Table 1 (Table 7)) may be modified by substitution, deletion, or addition of at least one (e.g., less than 20, less than 10, less than 5) nucleotide, and this variant enhancer sequence substantially retains the functional properties of this sequence. It is understood that variants of the enhancer sequences provided herein are still intended to retain the functional activity of the enhancer sequences described herein. For example, Example 4 shows that when tested using the Nanoglo DLR assay, the enhancer sequences described herein were increased by more than 10-fold over mCMV. Thus, variant enhancer sequences within the scope of the present invention should not have an activity less than 75% of the activity of the starting sequence.

[0081] In one embodiment, the synthetic super enhancer comprises one or more enhancer sequences presented in Table 1 (Table 7). Thus, the SSE may comprise one or more enhancer sequences selected from the group consisting of SEQ ID NOs: 4 to 63. Data showing the arrangement of such enhancer sequences by an array of multiple parts, which can increase transcriptional activity without impairing cell selectivity, is presented herein. In a further embodiment, the synthetic super enhancer comprises 1 to 8, 2 to 6, or 3 to 5 enhancer sequences selected from the sequences presented in Table 1 (Table 7). In yet a further embodiment, the synthetic super enhancer comprises 4 enhancer sequences selected from the sequences presented in Table 1 (Table 7).

[0082] In one embodiment, the SSE comprises one or more enhancer sequences selected from the group consisting of SEQ ID NOs: 4 to 36. In an alternative embodiment, the SSE comprises one or more enhancer sequences selected from the group consisting of SEQ ID NOs: 37 to 63, more preferably, SEQ ID NOs: 54 to 63.

[0083] In one embodiment, the SSE comprises one or more enhancer sequences selected from the group consisting of SEQ ID NOs: 4 to 13 and SEQ ID NOs: 54 to 63.

[0084] In a further embodiment, the SSE comprises 4 enhancer sequences selected from the group consisting of SEQ ID NOs: 4 to 36, for example, SEQ ID NOs: 4 to 13, particularly, SEQ ID NOs: 4 to 8. In an alternative embodiment, the SSE comprises 4 enhancer sequences selected from the group consisting of SEQ ID NOs: 37 to 63, preferably, 4 enhancer sequences selected from the group consisting of SEQ ID NOs: 54 to 63, more preferably, 4 enhancer sequences selected from the group consisting of SEQ ID NOs: 56 to 63.

[0085] The SSE may include the sequences set forth in Table 2 (Table 8). In one embodiment, the synthetic super enhancer includes a sequence having at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 64-80. In a further embodiment, the sequence has at least 85%, 90%, 95%, 97%, 99% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 64-80, or is 100% identical. In yet a further embodiment, the SSE includes SEQ ID NO: 70. Variants of the sequences provided herein are still intended to retain the functional activity of the SSE described herein, i.e., the expression of the transgene upon activation of the SSE by a transcription factor.

[0086] For the purpose of comparing two polynucleotide sequences that are similar, the “% sequence identity” between the first nucleotide sequence and the second nucleotide sequence can be calculated using NCBI BLAST v2.0 with the standard settings for nucleotide sequences (BLASTN). For the purpose of comparing two polypeptide sequences that are similar, the “% sequence identity” between the first polypeptide sequence and the second polypeptide sequence can be calculated using NCBI BLAST v2.0 with the standard settings for polypeptide sequences (BLASTP).

[0087] A polypeptide or polynucleotide sequence is said to be the same or “identical” to another polypeptide or polynucleotide sequence if they share 100% sequence identity over their entire lengths. Residues within the sequence are numbered from left to right, i.e., in the case of a polypeptide, from the N-terminus to the C-terminus, and in the case of a polynucleotide, from the 5′-terminus to the 3′-terminus.

[0088] "Difference" between arrays refers to the insertion, deletion, or substitution of a single amino acid residue or nucleotide at a position in the second array compared to the first array. Two polynucleotide sequences can contain one, two, or more such nucleotide differences. Two polypeptide sequences can contain one, two, or more such amino acid differences. Otherwise, an insertion, deletion, or substitution in the second sequence that is identical (100% sequence identity) to the first sequence results in a decrease in % sequence identity.

[0089] Alternatively, for the purpose of comparing a first reference sequence with a second comparison sequence, the number of additions, substitutions, and / or deletions made to the first sequence to generate the second sequence can be determined. "Addition" is the addition of one amino acid residue / nucleotide to the first sequence (including additions at either end of the first sequence). "Substitution" is the substitution of one amino acid residue / nucleotide in the first sequence with a different one amino acid residue / nucleotide. For polypeptide sequences, the substitution can be either conservative or non-conservative. In the context of polynucleotide sequences, the substitution can be either synonymous or non-synonymous. "Deletion" is the deletion of one amino acid residue / nucleotide from the first sequence (including deletions at either end of the first sequence).

[0090] In one embodiment, the synthetic super enhancer includes the addition of a transcriptional regulator, i.e., a sequence that controls the expression of a gene (or a sequence encoding a protein).

[0091] According to another aspect, there is provided a synthetic super enhancer activated by one or more SOX family transcription factors, the synthetic super enhancer including two or more enhancer sequences and at least one SOX motif and / or SOX dimer motif. It is understood that the embodiments described herein applicable to the synthetic super enhancer of the functional nucleic acid molecule are also applicable to this aspect.

[0092] According to a further aspect, there is provided a synthetic super-enhancer that includes four enhancer sequences derived from different genomic loci, each of the enhancer sequences including at least one SOX motif and / or SOX dimer motif and being activated by SOX2.

[0093] Functional nucleic acid molecule The SSE can be present in a construct that includes additional elements that form a functional nucleic acid molecule. As described, such a functional nucleic acid molecule can be used as presented herein to treat recurrent / secondary cancer / tumor in a subject and / or to induce, stimulate or generate an anti-recurrent / secondary cancer / tumor immune response. Thus, according to a further aspect, there is provided the use of a functional nucleic acid molecule that includes the synthetic super-enhancer described herein, operably linked to a transgene.

[0094] According to a further aspect, there is provided the use of a functional nucleic acid molecule that includes a synthetic super-enhancer activated by one or more transcription factors expressed in a target abnormal cell and a payload, wherein the payload is expressed upon activation of the synthetic super-enhancer by the transcription factor.

[0095] The functional nucleic acid molecule can include additional components in addition to the SSE and the transgene. For example, the synthetic super-enhancer can be operably linked to a promoter. Suitable promoters are known in the art. In one embodiment, the promoter is a minimal CMV promoter. In one teaching, the SSE may not be operably linked to a promoter. In an alternative embodiment, the promoter operably linked to the super-enhancer is not a minimal CMV promoter.

[0096] Transgene As described herein, a construct for use may include an SSE that serves to enhance the expression of a transgene within a target cell (e.g., a cancer cell or a primary cancer cell), which has the advantage of ensuring stable treatment for recurrent / secondary cancer and / or establishing, stimulating, or generating a stable anti-recurrent / secondary tumor / cancer immune response. In one embodiment, the transgene may be an exogenous sequence for intracellular expression. In one teaching, the transgene is a sequence encoding a product useful in biology and medicine, such as a prophylactic or therapeutic transgene, e.g., an oligonucleotide encoding a protein or a non-protein. Thus, the transgene may encode a therapeutic payload. For example, the therapeutic payload may be a therapeutic protein, such as an antigen, an antibody, a cytokine (e.g., inducing an immunogenic response), a tumor suppressor protein (e.g., natural / unmutated p53), a differentiation factor (i.e., regulating or reprogramming the fate or identity of a cell), or a protein having nucleic acid editing or gene activity regulatory functions (e.g., DNA or RNA editing or transcriptional regulation), or alternatively changing the cell phenotype (e.g., motility, ECM production, cellular senescence or quiescence). In an alternative embodiment, the transgene encodes an oligonucleotide that does not encode a protein, e.g., RNA (other than mRNA), e.g., miRNA, siRNA, shRNA, or IncRNA. It is understood that a transgene encoding a non-coding RNA can be used in methods of gene expression inhibition.

[0097] In one embodiment, the construct for use takes the form of a functional nucleic acid molecule comprising one or more transgenes. Thus, it is understood by those skilled in the art that different combinations of transgenes may be included.

[0098] According to a further aspect, there is provided a method of expressing a transgene within a target cell, the method comprising the step of expressing a transgene operably linked to a synthetic super enhancer described herein, wherein one or more of the transcription factors are expressed within the target cell.

[0099] Harmful payload As described herein, any transgene of a construct for use as described herein may be operably linked to an SSE and may encode a harmful payload. The term "harmful payload" as used herein refers to a payload that has a negative impact on target cells. The negative impact can be, for example, a negative effect on the health or viability of the cells and / or the ability of the cells to divide. The harmful effect can be direct (e.g., a transgene encoding a substance that harms the cells, such as a pro-apoptotic gene or a suicide gene) or indirect (e.g., a transgene encoding a substance that mobilizes an exogenous factor that causes an effect harmful to the cells).

[0100] The harmful payload can be used to stimulate an immune response that has a negative impact on target cells. Such a response can, for example, alter the local immune microenvironment. In one embodiment, the harmful payload encodes a protein that stimulates an immune response that causes the activation of cytotoxic immune cells. In one embodiment, the harmful payload is selected from chemokines, cytokines, antibodies, or other immunomodulatory proteins. In a further embodiment, the harmful payload is a cytokine selected from pro-inflammatory cytokines such as IL-12, IL-10, IL-2, IFN-α, and GM-CSF. For example, the harmful payload can be a cytokine such as IL-12 that has been found to have anti-cancer activity. IL-12 induces IFN-γ production by resting and activated CD4+ T cells, CD8+ T cells, and natural killer (NK) cells, and enhances the proliferation of activated T cells and NK cells, increases the lytic activity of NK / lymphokine-activated killer cells, and promotes the specific response of cytotoxic T lymphocytes (CTLs). However, since IL-12 cannot be administered as a systemic treatment due to excessive toxicity, targeted therapy in which expression occurs only within the target cells enables the delivery of this payload.

[0101] "Immune response" is a measurable change in at least one cell or one cell type or one endocrine pathway or one exocrine pathway of the immune system (including, but not limited to, cellular response, humoral response, cytokine response, chemokine response).

[0102] "Immune cell" is defined as a cell of the immune system and includes, but is not limited to, CD34+ cells, B cells, CD45+ (lymphocyte common antigen) cells, alpha-beta T cells, cytotoxic T cells, helper T cells, plasma cells, neutrophils, monocytes, macrophages, dendritic cells, phagocytes, granulocytes, natural lymphocyte cells, natural killer (NK) cells, and gamma delta T cells. Typically, immune cells are classified by cell surface molecule analysis by a combination for identification (e.g., by flow cytometry) or by utilizing groups or clusters for sorting immune cells into subpopulations. In the molecules and methods of the present disclosure where the payload is a harmful payload, stimulation of the immune response is intended to detrimentally affect the target cells. When referring to "cytotoxic immune cells", it refers to immune cells in which cell death occurs, particularly cytotoxic T cells (also known as killer T cells).

[0103] In one embodiment, the harmful payload encodes a cytotoxic substance (i.e., a cytotoxic payload).

[0104] In one embodiment, the harmful payload is a suicide gene. A suicide gene is a gene that expresses a protein that causes cell death. Alternatively, a suicide gene may require an externally supplied cofactor or adjuvant drug (e.g., a prodrug) to act. The cofactor or adjuvant drug can then be converted by the product of the suicide gene into a cytotoxic entity. In one embodiment, the suicide gene encodes a protein that can convert an inactive prodrug into a cytotoxic drug. The inactive prodrug can be administered simultaneously or sequentially with the functional nucleic acid molecule.

[0105] In one embodiment, the suicide gene is herpes simplex virus thymidine kinase (HSV-TK). In particular, the HSV-TK gene is used in combination with a prodrug, ganciclovir (GCV) or an analog thereof, such as acyclovir and valacyclovir. In an alternative embodiment, the suicide gene is cytosine deaminase (CD). In particular, the CD gene is used in combination with a prodrug, 5-fluorocytosine (5FC).

[0106] Target cells The constructs / functional nucleic acid molecules described herein can be used to target a variety of cell types (also known as "target cells"). In particular, the cells are mammalian cells, such as human cells. The present disclosure may include the presence of a harmful payload and is thus suitable for targeting abnormal cells (i.e., abnormal cells, such as diseased cells).

[0107] Abnormal cells can be hyperproliferative cells, i.e., cells that proliferate excessively and abnormally. In one embodiment, the abnormal cells are cancer cells or neoplastic cells. In this embodiment, the harmful payload can be an anti-cancer payload.

[0108] Also, since SOX2 has been identified as an oncogene, it can be amplified in other abnormal cells, particularly cancer cells. Examples of other abnormal cells that can be targeted using the methods of the present disclosure include squamous cell carcinomas of the lung and esophagus, among many others.

[0109] The target cells can be primary cancer cells. Primary cancer cells can give rise to recurrent / secondary cancers (via any of the mechanisms described herein).

[0110] In one embodiment, the target cells are cancer cells selected from glioblastoma stem cells, glioma cells, lung cancer cells (particularly squamous cell lung cancer cells), esophageal cancer cells, ovarian cancer cells, breast cancer cells, oral cancer cells (e.g., mouth, tongue, pharynx), gastric cancer cells, small intestine cancer cells, colorectal cancer cells, rectal cancer cells, liver cancer cells, cholangiocarcinoma cells, gallbladder cancer cells, pancreatic cancer cells, bone cancer cells (e.g., osteosarcoma), skin cancer cells, uterine cancer cells, prostate cancer cells, testicular cancer cells, bladder cancer cells, kidney cancer cells, retinal cancer cells, thyroid cancer cells, lymphoma cells, myeloma cells or leukemia cells.

[0111] In certain embodiments, the target cells are glioblastoma stem cells. Glioblastoma is a highly malignant form of glioma and is driven by an increase in the level / activity of transcription factors associated with major regulatory neural stem cells. Glioblastoma stem cells (GSCs) generally have increased expression or activity of key neurogenic transcription factors, including SOX2, which is a key major regulatory factor and a reprogramming factor required for neural stem cell identity (Bulstrode et al. 2017, Gangemi et al. 2009, Guerra-Rebollo et al. 2019, Lopez-Bertoni et al. 2015, and MacLeod et al. 2019). Knockdown of SOX2 in GSCs reduces tumorigenic ability (Gangemi et al. 2009), while its ectopic expression with POU3F2, OLIG2, and SALL2 enhances the GSC state (Suva et al. 2014). In the examples presented herein, it is shown that the GSC-selective SSE retains activity in adeno-associated virus (AAV) and is used to drive the expression of a cytotoxic payload to target the killing of GSCs. Moreover, the activity of the SSE is lost during GSC differentiation and is not present in HEK or fibroblasts. Thus, GSC-selective expression is retained despite a large increase in promoter strength.

[0112] Vector The construct for use can be provided in the form of a vector comprising a synthetic super enhancer or a functional nucleic acid molecule as described herein.

[0113] As used herein, the term "vector" is intended to refer to a molecule capable of transporting a functional nucleic acid molecule. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop that can ligate additional DNA segments. Another type of vector is a viral vector, which can ligate additional DNA segments into this viral genome. Certain vectors are capable of autonomous replication within the introduced host cell (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian and yeast vectors). Other vectors (e.g., non-episomal mammalian vectors) can integrate into the host cell genome upon introduction into the host cell and are thus replicated with the host genome. Moreover, certain vectors are capable of directing the expression of operably linked genes. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). Generally, expression vectors useful in recombinant DNA technology are most often in the form of plasmids.

[0114] Exemplary expression vectors are known in the art and can include, for example, plasmid vectors, viral vectors (e.g., adenovirus, adeno-associated virus, retrovirus or lentivirus vectors), phage vectors, cosmid vectors, and the like. The choice of expression vector can depend on the host cell type used and the purpose of use.

[0115] In one embodiment, the vector is a viral vector. In particular, viral vectors used in the application of gene therapy are well known in the art. Such viruses can be RNA viruses and DNA viruses having either single-stranded (ss) or double-stranded (ds) genomes. For example, viral vectors can include, but are not limited to, adenoviruses, adeno-associated viruses (AAV), alphaviruses, flaviviruses, herpes simplex viruses (HSV), measles viruses, rhabdoviruses, retroviruses, lentiviruses, Newcastle disease viruses (NDV), poxviruses, and picornaviruses. Since the insertion ability and tropism can vary, the viral vector can be selected based on the intended application.

[0116] AAV vectors are increasingly supported as optimal gene therapy viral vectors due to improved safety. However, the cargo size is more restricted to a smaller size. The four-part SSE assembly described herein is ideally suitable for AAV. Thus, in one embodiment, the vector is AAV. Since different AAV serotypes have different tropisms, the type of AAV vector to be used can be selected according to the type of target tissue. The AAV vector can be selected from any one of a number of serotypes known in the art, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11, or can be a non-natural variant. In one embodiment, AAV is selected from AAV1, AAV2, or AAV5.

[0117] Constructs (or functional nucleic acids) for any of the uses or methods described herein can be provided (or administered thereby) by an AAV vector.

[0118] In one teaching, constructs (or functional nucleic acids) for any of the uses or methods described herein can be provided (or administered using) by an AAV1 vector.

[0119] Thus, in the present disclosure, (i) For use in the treatment, prevention, elimination, and / or cure of recurrent / secondary cancer, (ii) For use in the treatment, prevention, elimination, and / or cure of recurrent / secondary cancer in a subject undergoing primary cancer treatment, (iii) For use in generating, stimulating, or inducing an anti-recurrent / secondary cancer immune response, (iv) For use in generating, stimulating, or inducing an anti-recurrent / secondary cancer immune response in a subject undergoing primary cancer treatment, (v) For use in a method of treating, preventing, eliminating, and / or curing recurrent / secondary cancer, (vi) For use in a method of treating, preventing, eliminating, and / or curing recurrent / secondary cancer in a subject undergoing primary cancer treatment, (vii) For use in a method of generating, stimulating, or inducing an anti-recurrent / secondary cancer immune response, (viii) For use in a method of generating, stimulating, or inducing an anti-recurrent / secondary cancer immune response in a subject undergoing primary cancer treatment, (ix) For use in the manufacture of a medicament for treating, preventing, eliminating, and / or curing recurrent / secondary cancer, (x) For use in the manufacture of a medicament for treating, preventing, eliminating, and / or curing recurrent / secondary cancer in a subject undergoing primary cancer treatment, (xi) For use in the manufacture of a medicament for generating, stimulating, or inducing an anti-recurrent / secondary cancer immune response, (xii) For use in the manufacture of a medicament for generating, stimulating, or inducing an anti-recurrent / secondary cancer immune response in a subject undergoing primary cancer treatment An AAV1 (or AAV2, 5, or 8) vector comprising any of the constructs described herein, wherein this construct has the following (cancer cell) - harmful payload: (i) A suicide gene (e.g., a gene that expresses or encodes a protein that causes cell death), (ii) A suicide gene encoding a protein capable of converting an inactive prodrug into a cytotoxic drug, (iii) An element that stimulates an immune response having a negative impact on the target cell, (iv) A protein that stimulates an immune response causing activation of cytotoxic immune cells, (v) Chemokines, (vi) Cytokines (including pro-inflammatory cytokines), (vii) Antibodies or antigen-binding fragments thereof, (viii) Immunomodulatory proteins, (ix) IL-12, (x) IL-10, (xi) IL-2, (xii) IFN-α, (xiii) GM-CSF, (xiv) Cytotoxic substances, (xv) Herpes simplex virus thymidine kinase (HSV-TK), and (xvi) Cytosine deaminase (CD) Provided is an AAV1 (or AAV2, 5 or 8) vector comprising one, two or more of the following:

[0120] According to a further aspect, provided is a vector comprising a synthetic super-enhancer described herein and a transgene (i.e., a payload, e.g., a therapeutic payload) for any of the uses and methods described herein. As described herein, the synthetic super-enhancer can be operably linked to other expression elements, e.g., a promoter.

[0121] Compositions and Kits The disclosure also relates to the use of a composition comprising a construct, functional nucleic acid molecule or vector described herein. The composition can include components that enable delivery of the above construct or functional nucleic acid molecule by viral vectors (such as AAV, lentivirus, etc.) and non-viral vectors (such as nanoparticles, lipid particles, etc.).

[0122] In a further aspect, there is provided the use of a composition comprising a synthetic super enhancer, a functional nucleic acid molecule, a construct or vector described herein, and an acceptable carrier. Suitable carriers are known in the art. In certain embodiments, the carrier is selected based on its ability to facilitate transfection of target cells by one or more functional nucleic acid molecules.

[0123] In a further aspect, the present disclosure provides a composition comprising a functional nucleic acid molecule or vector described herein for use in the treatment or prevention of recurrent / secondary cancer / tumor and / or for use in the generation, stimulation, and / or induction of an anti-recurrent / secondary cancer / tumor immune response.

[0124] In a further aspect, there is provided the use of a construct, a functional nucleic acid molecule (or vector or composition) described herein for the manufacture of a medicament for the treatment or prevention of recurrent / secondary cancer / tumor and / or for use in the generation, stimulation, and / or induction of an anti-recurrent / secondary cancer / tumor immune response.

[0125] Treatment method In a further aspect, there is provided a method for (i) treating or preventing recurrent / secondary cancer / tumor or (ii) generating, inducing or stimulating an anti-secondary cancer / tumor immune response, comprising the step of administering to a patient a molecular construct (or functional nucleic acid molecule) (or vector comprising the same) of the present disclosure. The patient is a human or animal patient suffering from primary cancer and at risk of developing a related recurrent / secondary cancer.

[0126] References to "subject", "patient" or "individual" refer to the subject being treated, particularly a mammalian subject. Mammalian subjects include humans, non-human primates, livestock (e.g., cows), sport animals or pets, such as dogs, cats, guinea pigs, rabbits, rats or mice. In some embodiments, the subject is a human. In alternative embodiments, the subject is a non-human mammal, such as a mouse.

[0127] As used herein, "treatment" of a disease or disorder means a reduction in the frequency and / or severity of at least one sign or symptom of the disease or disorder experienced by a patient.

[0128] "Cancer", as used herein, refers to abnormal growth or division of cells (primary or secondary or recurrent / secondary (as above)). Generally, the growth and / or lifespan of cancer cells exceeds that of normal cells and surrounding tissues and does not cooperate with them. Cancer can be benign, pre-malignant or malignant. Also, cancer can be primary cancer or secondary cancer. Primary cancer is the first organ or tissue where the cancer originated, while secondary cancer is the result of the spread (or metastasis) of primary cancer to another part of the body. As described, in the present disclosure, constructs are provided that can be used to treat primary cancer and that have been found to stimulate, generate or induce an effective immune response against any recurrent / secondary cancer (or primary cancer) that can arise from primary cancer.

[0129] Cancer (primary, recurrent / secondary or secondary) occurs in a variety of cells and tissues, including the oral cavity (e.g., mouth, tongue, pharynx), digestive system (e.g., esophagus, stomach, small intestine, colon, rectum, liver, bile duct, gallbladder, pancreas), respiratory system (e.g., larynx, lung, bronchus), bone, joint, skin (e.g., basal cell, squamous cell, meningioma), breast, genital system (e.g., uterus, ovary, prostate, testis), urinary system (e.g., bladder, kidney, ureter), eye, nervous system (e.g., brain), endocrine system (e.g., thyroid), and hematopoietic system (e.g., lymphoma, myeloma, leukemia, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, acute myeloid leukemia, chronic myeloid leukemia).

[0130] In a further embodiment, the cancer (primary, recurrent / secondary or secondary) is a cancer located in the brain or spinal cord (i.e., the central nervous system), e.g., a primary or recurrent / secondary or secondary cancer located in the central nervous system. In the present disclosure, specific use is found in the treatment of glioblastoma, particularly recurrent / secondary glioblastoma arising from primary glioblastoma.

[0131] Functional nucleic acid molecules (or vectors or compositions) can be administered by any suitable delivery method for the intended treatment, e.g., intravenously, intraarterially, intracardially, intradermally, subcutaneously, intraperitoneally, intramuscularly, or orally. In one embodiment, the functional nucleic acid molecule, vector, or composition is administered systemically. Also, the administration can be direct administration to the site of the lesion. Thus, in one embodiment, the functional nucleic acid molecule, vector, or composition is administered locally, e.g., directly to an organ or tissue, e.g., into a tumor.

[0132] The constructs or functional nucleic acids of the present disclosure can be administered to a subject suffering from a primary cancer (e.g., by direct injection into the tumor) to shrink or reduce the size of any primary tumor. The treated tumor / cancer can then be surgically removed. Without wishing to be bound by theory, the resection is associated with a risk of recurrence (because residual portions of the tumor and / or residual cancer cells / cancer stem cells are left in situ). In particular, recurrence after resection is most often driven by cancer stem cells at the resection cavity margin. As described, the constructs or functional nucleic acids of the present disclosure not only aid in the treatment of primary cancer but also establish in the subject a level of protection against any recurrent cancer that occurs after resection. Again, without wishing to be bound by theory, this protection is suggested to be the result of an immune response that is stimulated and induced upon the appearance of recurrent / secondary cancer / tumor. As described herein, the construct or functional nucleic acid can include one, two, or more harmful payloads, which can include suicide genes, e.g., suicide genes encoding proteins that can convert an inactive prodrug into a cytotoxic drug, and the prodrug can be administered before, during, or after treatment with the construct or functional nucleic acid.

[0133] As described, the constructs or functionality of the present disclosure can be packaged and administered within a vector, e.g., a viral vector. In one teaching, the constructs or functionality of the present disclosure can be packaged and administered within an adenoviral vector, e.g., an AAV1 vector, an AAV2 vector, an AAV5 vector, or an AAV8 vector.

[0134] In one teaching, the primary cancer is glioblastoma. Glioblastoma can be treated with the functional nucleic acids or constructs of the present disclosure to shrink the size of the glioblastoma. The glioblastoma can then be surgically resected. Treatment of the primary cancer with the functional nucleic acids or constructs of the present disclosure establishes in the subject a level of protection against any recurrent / secondary glioblastoma that may result, for example, from cancer (glioblastoma) stem cells remaining at the resection cavity stump.

[0135] According to a further aspect, a method for treating or preventing recurrent / secondary glioblastoma that may arise from primary glioblastoma, comprising the step of administering to a patient suffering from primary glioblastoma the constructs / functional nucleic acid molecules and inactive prodrugs disclosed herein, wherein the construct or functional nucleic acid comprises a synthetic super enhancer activated by a SOX transcription factor and a suicide gene encoding a protein capable of converting the inactive prodrug into a cytotoxic drug, and the suicide gene is expressed in glioblastoma cells upon activation of the synthetic super enhancer by the SOX transcription factor, provides a method.

[0136] In one embodiment, the inactive prodrug is administered simultaneously or sequentially with the functional nucleic acid molecule.

[0137] It is understood that the treatment method includes administration of a therapeutically effective amount. The term "therapeutically effective amount" is effective to restore or treat the symptoms of a disease or disorder and is an amount sufficient to generate, stimulate, and / or induce an effective immune response against any recurrent / secondary cancer that may result from the primary cancer. The therapeutically effective amount can be a "preventively effective amount" if the preventive method can be determined to be a therapy.

[0138] It is understood that the embodiments described herein can be applied to all aspects of the present disclosure. That is, the embodiments described for use can be equally applied to the claimed methods and others.

[0139] Clause A set of clauses defining the present disclosure and its preferred embodiments are as follows.

[0140] 1. A construct for use in (i) the treatment or prevention of recurrent / secondary cancer / tumor, and / or (ii) the generation, stimulation or induction of an anti-recurrent / secondary cancer / tumor immune response, the construct comprising a gene or transgene encoding a harmful payload.

[0141] 2. The construct according to clause 1, wherein the gene or transgene is for expression in target cells and the payload is harmful to cancer cells.

[0142] 3. The construct according to clause 1 or 2, wherein the target cells are primary cancer cells, for example, primary glioblastoma cancer cells.

[0143] 4. The construct according to any one of clauses 1 to 3, wherein the recurrent / secondary cancer is associated with the primary cancer treated using the construct according to clause 1 or 2.

[0144] 5. The construct for use according to any one of clauses 1 to 4, wherein the primary and recurrent / secondary cancers are primary or recurrent / secondary glioblastoma.

[0145] 6. The construct for use according to any one of clauses 1 to 5, wherein the harmful payload encodes a protein that stimulates an immune response that causes the recruitment of cytotoxic immune cells.

[0146] 7. The construct for use according to any one of clauses 1 to 6, wherein the harmful payload encodes a cytotoxic substance.

[0147] 8. The construct for use according to any one of clauses 1 to 7, wherein the harmful payload comprises a suicide gene encoding a protein capable of converting an inactive prodrug into a cytotoxic drug.

[0148] 9. A construct for use as defined in clause 8, wherein the suicide gene is the herpes simplex virus thymidine kinase (HSV-TK) gene and the prodrug is ganciclovir, acyclovir or valacyclovir.

[0149] 10. A construct for use as defined in clauses 1 to 5, wherein the harmful payload comprises the IL-12 gene.

[0150] 11. A construct for use in (i) the treatment or prevention of recurrent / secondary cancer / tumor, and / or (ii) the generation, stimulation or induction of an anti-recurrent / secondary cancer / tumor immune response, comprising a gene or transgene encoding IL-12 and herpes simplex virus thymidine kinase (HSV-TK).

[0151] 12. A construct for use in a subject having primary cancer, wherein the primary cancer is treated with a construct defined by any one of clauses 1 to 11, and the treatment of the primary cancer with this construct induces, stimulates or generates an anti-recurrent / secondary cancer / tumor immune response that prevents or reduces the risk of recurrent / secondary cancer arising from this primary cancer, a construct for use as defined by any one of clauses 1 to 11.

[0152] 13. Use of a construct as defined in clauses 1 to 11 for the manufacture of a medicament for (i) treating or preventing recurrent / secondary cancer / tumor, and / or (ii) establishing, generating or stimulating an anti-recurrent / secondary cancer immune response or an anti-recurrent / secondary tumor immune response.

[0153] 14. The use as described in clause 13, wherein the recurrent / secondary cancer is associated with primary cancer treated using a construct as defined by any one of clauses 1 to 11.

[0154] 15. The use as described in clause 13 or 14, wherein the use of a medicament for treating primary cancer induces, stimulates or generates an anti-recurrent / secondary cancer / tumor immune response that prevents or reduces the risk of recurrent / secondary cancer arising from this primary cancer.

[0155] 16. A method for generating, stimulating or establishing an anti-recurrent / metastatic cancer / tumor immune response that prevents or reduces the risk of recurrent / metastatic cancer arising from primary cancer, the method comprising treating primary cancer / tumor with a construct defined in any one of clauses 1 to 11, and inducing, establishing or stimulating an immune response effective against any recurrent / metastatic cancer / tumor that may arise from primary cancer (already treated) by the treatment of primary cancer / tumor with the construct of the present disclosure.

[0156] 17. The method according to clause 16, wherein the primary cancer is glioblastoma.

[0157] 18. The method according to clause 17, wherein the metastatic cancer / tumor is a recurrent / metastatic glioblastoma arising from primary glioblastoma.

[0158] The construct defined in any one of clauses 1 to 11 may further comprise a synthetic super-enhancer defined by clauses 19 to 30.

[0159] 19. A synthetic super-enhancer activated by one or more transcription factors, comprising two or more enhancer sequences derived from different genomic loci, each enhancer sequence comprising a binding site for a transcription factor and 20 to 400 nucleotides upstream and / or downstream of the binding site for the transcription factor present at that genomic locus.

[0160] 20. The synthetic super-enhancer according to clause 19, wherein each of the two or more enhancer sequences within the synthetic super-enhancer has a length of less than 300 nucleotides, particularly a length of 160 nucleotides.

[0161] 21. The synthetic super-enhancer according to clause 19 or 20, having a length of less than 1200 nucleotides, particularly a length of less than 700 nucleotides.

[0162] 22. The synthetic super-enhancer according to any one of clauses 19 to 21, comprising four enhancer arrays.

[0163] 23. The synthetic super-enhancer according to any one of clauses 19 to 22, wherein the transcription factor is a SOX family transcription factor.

[0164] 24. The synthetic super-enhancer according to any one of clauses 19 to 23, comprising at least one SOX dimer motif.

[0165] 25. The synthetic super-enhancer according to clause 24, wherein the SOX dimer motif comprises SEQ ID NO: 1.

[0166] 26. The synthetic super-enhancer according to any one of clauses 19 to 23, comprising at least one SOX motif.

[0167] 27. The synthetic super-enhancer according to clause 26, wherein the SOX motif is a SOX2 motif.

[0168] 28. The synthetic super-enhancer according to clause 26 or 27, wherein the SOX2 motif comprises SEQ ID NO: 3.

[0169] 29. The synthetic super-enhancer according to any one of clauses 19 to 28, comprising one or more enhancer arrays presented in Table 1 (Table 7).

[0170] 30. The synthetic super-enhancer according to any one of clauses 19 to 29, comprising a sequence having at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 64 to 80.

[0171] 30. The synthetic super-enhancer according to any one of clauses 19 to 30, further comprising a transcriptional regulatory factor.

[0172] The constructs for use in any of the therapeutic uses, methods or medicaments described herein may be further defined as described in any one of clauses 31 to 62.

[0173] 31. A functional nucleic acid molecule comprising a synthetic super-enhancer as described in any one of clauses 19 to 30, operably linked to a transgene.

[0174] 32. The functional nucleic acid molecule according to clause 31, wherein the transgene encodes a therapeutic payload.

[0175] 33. The functional nucleic acid molecule according to clause 31, wherein the transgene encodes a harmful payload.

[0176] 34. The functional nucleic acid molecule according to clause 33, wherein the harmful payload encodes a protein that stimulates an immune response that causes mobilization of cytotoxic immune cells.

[0177] 35. The functional nucleic acid molecule according to clause 33, wherein the harmful payload encodes a cytotoxic substance.

[0178] 36. The functional nucleic acid molecule according to any one of clauses 31 to 35, wherein the harmful payload is a suicide gene encoding a protein capable of converting an inactive prodrug into a cytotoxic drug.

[0179] 37. The functional nucleic acid molecule according to any one of clauses 31 to 36, wherein the synthetic super-enhancer is operably linked to a promoter.

[0180] 38. A functional nucleic acid molecule comprising a synthetic super-enhancer activated by one or more transcription factors expressed in a target abnormal cell and a harmful payload, wherein the payload is expressed upon activation of the synthetic super-enhancer by the transcription factor.

[0181] 39. The functional nucleic acid molecule according to clause 38, wherein the transcription factor is specifically expressed in the target abnormal cell.

[0182] 40. The functional nucleic acid molecule according to clause 38 or 39, wherein the transcription factor is a developmental or stem cell-related transcription factor.

[0183] 41. The functional nucleic acid molecule according to any one of clauses 38 to 40, wherein the transcription factor is a SOX family transcription factor.

[0184] 42. The functional nucleic acid molecule according to any one of clauses 38 to 41, which is activated by SOX2.

[0185] 43. The functional nucleic acid molecule according to any one of clauses 38 to 42, wherein the synthetic super enhancer contains at least one SOX dimer motif.

[0186] 44. The functional nucleic acid molecule according to clause 43, wherein the SOX dimer motif contains SEQ ID NO: 1.

[0187] 45. The functional nucleic acid molecule according to any one of clauses 38 to 42, wherein the synthetic super enhancer contains at least one SOX motif.

[0188] 46. The functional nucleic acid molecule according to clause 45, wherein the SOX motif is a SOX2 motif.

[0189] 47. The functional nucleic acid molecule according to clause 45 or 46, wherein the SOX2 motif contains SEQ ID NO: 3.

[0190] 48. The functional nucleic acid molecule according to any one of clauses 38 to 47, wherein the synthetic super enhancer contains two or more enhancer sequences.

[0191] 49. The functional nucleic acid molecule according to any one of clauses 38 to 48, wherein the synthetic super enhancer contains four enhancer sequences.

[0192] 50. The functional nucleic acid molecule according to clause 48 or 49, wherein each of the two or more enhancer sequences has a different sequence.

[0193] 51. The functional nucleic acid molecule according to any one of clauses 48 to 50, wherein each of the two or more enhancer sequences is derived from a different genomic locus.

[0194] 52. The functional nucleic acid molecule according to any one of clauses 48 to 51, wherein each of the two or more enhancer sequences within the synthetic super-enhancer has a length of less than 300 nucleotides, particularly about 160 nucleotides in length.

[0195] 53. The functional nucleic acid molecule according to any one of clauses 48 to 52, wherein each of the two or more enhancer sequences within the synthetic super-enhancer contains a sequence selected from the SOX motif and / or the SOX dimer motif.

[0196] 54. The functional nucleic acid molecule according to any one of clauses 38 to 53, wherein the synthetic super-enhancer contains one or more enhancer sequences presented in Table 1 (Table 7).

[0197] 55. The functional nucleic acid molecule according to any one of clauses 38 to 54, wherein the synthetic super-enhancer contains a sequence having at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 64 to 80.

[0198] 56. The functional nucleic acid according to any one of clauses 38 to 55, wherein the synthetic super-enhancer is operably linked to a promoter.

[0199] 57. The functional nucleic acid molecule according to any one of clauses 38 to 56, wherein the harmful payload encodes a protein that stimulates an immune response that causes the recruitment of cytotoxic immune cells.

[0200] 58. The functional nucleic acid molecule according to any one of clauses 38 to 56, wherein the harmful payload encodes a cytotoxic substance.

[0201] 59. The functional nucleic acid molecule according to any one of clauses 19 to 58, wherein the harmful payload is a suicide gene encoding a protein capable of converting an inactive prodrug into a cytotoxic drug.

[0202] 60. The functional nucleic acid molecule according to clause 59, wherein the suicide gene is a herpes simplex virus thymidine kinase (HSV-TK) gene, and the prodrug is ganciclovir, acyclovir or valacyclovir.

[0203] 61. The functional nucleic acid molecule according to any one of clauses 38 to 60, wherein the abnormal cell is a cancer cell or a newborn cell, and the harmful payload is an anti-cancer payload.

[0204] 62. The functional nucleic acid molecule according to any one of clauses 38 to 61, wherein the abnormal cell is a glioblastoma stem cell.

[0205] Constructs for any use or method described herein may include a synthetic super enhancer according to any one of clauses 63 to 75.

[0206] 63. A synthetic super enhancer activated by one or more SOX family transcription factors, comprising two or more enhancer sequences, and at least one SOX motif and / or SOX dimer motif.

[0207] 64. The synthetic super enhancer according to clause 63, wherein each of the two or more enhancer sequences has a different sequence.

[0208] 65. The synthetic super enhancer according to clause 63 or 64, wherein each of the two or more enhancer sequences is derived from a different genomic locus.

[0209] 66. The synthetic super enhancer according to any one of clauses 63 to 65, comprising four enhancer sequences.

[0210] 67. The synthetic super enhancer according to any one of clauses 63 to 66, wherein each of two or more enhancer sequences within the synthetic super enhancer has a length of less than 300 nucleotides, particularly a length of 160 nucleotides.

[0211] 68. The synthetic super enhancer according to any one of clauses 63 to 50, wherein each of two or more enhancer sequences within the synthetic super enhancer contains a sequence selected from the SOX motif and / or the SOX dimer motif.

[0212] 69. The synthetic super enhancer according to any one of clauses 63 to 51, which is activated by SOX2.

[0213] 70. The synthetic super enhancer according to clause 69, wherein the SOX dimer motif contains SEQ ID NO: 1.

[0214] 71. The synthetic super enhancer according to any one of clauses 63 to 68, wherein the SOX motif is the SOX2 motif.

[0215] 72. The synthetic super enhancer according to clause 71, wherein the SOX2 motif contains SEQ ID NO: 3.

[0216] 73. The synthetic super enhancer according to any one of clauses 63 to 72, which contains one or more enhancer sequences presented in Table 1 (Table 7).

[0217] 74. The synthetic super enhancer according to any one of clauses 63 to 73, which contains a sequence having at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 64 to 80.

[0218] 75. A synthetic super enhancer that contains four enhancer sequences derived from different genomic loci, and each of the enhancer sequences contains at least one SOX dimer motif and is activated by SOX2.

[0219] Constructs or functional nucleic acids for any of the uses or methods described herein may be provided in the form of vectors described in clauses 76 to 78.

[0220] 76. A vector comprising a synthetic super enhancer described in any one of clauses 19 to 30 or 63 to 75 or a functional nucleic acid molecule described in any one of clauses 31 to 62.

[0221] 77. The vector described in clause 59, which is a viral vector.

[0222] 78. The vector described in clause 59 or 60, which is an adeno-associated virus (AAV).

[0223] Constructs or functional nucleic acids for any of the uses or methods described herein may be provided in the form of compositions described in clauses 79 to 81.

[0224] 79. A composition comprising a synthetic super enhancer described in any one of clauses 19 to 30 or 63 to 75, a functional nucleic acid molecule described in any one of clauses 31 to 62 or a vector described in any one of clauses 76 to 78, and an acceptable carrier.

[0225] 80. A composition comprising a functional nucleic acid molecule described in any one of clauses 31 to 62 for use in therapy.

[0226] 81. A composition comprising a functional nucleic acid molecule described in any one of clauses 31 to 62 for use in the treatment of cancer.

[0227] Also disclosed are teachings that are consistent with the following clauses.

[0228] A method for treating or preventing recurrent / secondary cancer resulting from primary cancer, comprising administering to a patient suffering from primary cancer a functional nucleic acid molecule described in any one of clauses 31 to 62 or a composition described in clause 80 or 81, wherein treatment of the primary cancer with this functional nucleic acid or composition generates, stimulates, or induces an immune response that defends against recurrent / secondary cancer that may arise from the primary cancer.

[0229] 83. The method according to clause 82, wherein the primary and / or recurrent / secondary cancer is a brain cancer, such as glioblastoma.

[0230] 84. The method according to clause 82 or 83, wherein the functional nucleic acid molecule or composition is administered systemically.

[0231] 85. The method according to clause 82 or 83, wherein the functional nucleic acid molecule or composition is administered locally.

[0232] 86. A method for preventing recurrent / secondary glioblastoma, comprising administering to a patient suffering from primary glioblastoma a functional nucleic acid molecule and an inactive prodrug, wherein the functional nucleic acid comprises a synthetic super-enhancer activated by a SOX transcription factor and a suicide gene encoding a protein capable of converting the inactive prodrug into a cytotoxic drug, and the suicide gene is expressed in glioblastoma cells upon activation of the synthetic super-enhancer by the SOX transcription factor.

[0233] 87. The method according to clause 86, wherein the inactive prodrug is administered simultaneously or sequentially with the functional nucleic acid molecule.

[0234] 88. An AAV1 vector comprising a nucleic acid construct for use in (i) the treatment or prevention of recurrent / secondary cancer / tumor, and / or (ii) the generation, stimulation or induction of an anti-recurrent / secondary cancer / tumor immune response, wherein the construct comprises a gene or transgene encoding IL-12 and herpes simplex virus thymidine kinase (HSV-TK).

[0235] 89. A method of (i) treating or preventing recurrent / secondary cancer / tumor, and / or (ii) generating, stimulating or inducing an anti-recurrent / secondary cancer / tumor immune response, comprising administering to a patient suffering from primary cancer an AAV1 vector comprising a nucleic acid construct comprising a gene or transgene for expression in primary cancer cells, wherein the gene or transgene encodes IL-12 and herpes simplex virus thymidine kinase (HSV-TK), whereby treating or preventing the primary cancer treats or prevents any recurrent / secondary cancer / tumor that may arise from the treated primary cancer and / or generates, stimulates or induces an anti-recurrent / secondary cancer / tumor immune response effective for the elimination, treatment or prevention of any recurrent / secondary cancer / tumor that may arise from the treated primary cancer.

[0236] 90. Use of an AAV1 vector comprising a nucleic acid construct in the manufacture of a medicament for (i) treating or preventing recurrent / secondary cancer / tumor, and / or (ii) generating, stimulating or inducing an anti-recurrent / secondary cancer / tumor immune response, wherein the construct comprises a gene or transgene encoding IL-12 and herpes simplex virus thymidine kinase (HSV-TK).

[0237] Here, the present disclosure is illustrated with reference to the following non-limiting examples.

Example

[0238] Materials and Methods Target Vector Cloning To ensure high cloning efficiency for both enhancer cloning and super-enhancer assembly, we constructed a newly designed destination vector to enable efficient Golden Gate cloning, which also reduced the reaction setup time / cost. The design of this destination vector included the reporter gene cassette Nanoluc-Ires-mNGreen-pA and the minimal CMV promoter (mCMV). Bacterial ccdB suicide cassette was used for selection and efficient cloning. CDV2 containing the ccdB cassette spanning its position enabled the assembly of up to four enhancers and spacers. In contrast to CDV1, CDV2 also included the PiggyBac transposase recognition site adjacent to the entire cassette. We used Gibson assembly to generate the Scar-free specially designed destination vector (Gibson et al. 2009).

[0239] Bioinformatics for the design of the SOX2 enhancer oligonucleotide pool Suva et al. published the technical replicates by one cell line for SOX2 binding in GSCs (SOX2r1 and SOX2r2 by the proneural subtype MGG8 cell line), and H3K27ac by three cell lines MGG4 (proneural subtype), MGG6 (classical subtype), and MGG8 (Suva et al. 2014). These also included data from differentiated MGG8 cells (here called differentiated glioblastoma cells (DGC)). For clarity, in contrast to DGC (obtained from GSCs by adding serum that stimulates astrocyte differentiation), GSC lines are also called tumor-propagating cells due to their tumorigenic properties.

[0240] To determine GSC-specific SOX2 peaks, GSC SOX2r1 and SOX2r2 were overlapped with DGC SOX2_H3K27ac (H3K27ac Chip-Seq in highly expressed SOX2 DGC). In addition, shared H3K27ac peaks were also identified among GSC cell lines to define putative enhancer sequences. Such shared peaks were individually overlapped with GSC-specific SOX2r1 and GSC-specific SOX2r2. This protected against the loss of any potentially important regions. Subsequently, the resulting shared peaks were combined into one file, and peaks closer than 100 base pairs (bp) were merged, also removing duplicates. The peak lengths ranged from 15 to 5300 bp, with an average length of 411 bp. Then, the pool was manually curated to remove repetitive and non-enhancer sequences, such as centromeres. This resulted in a curated preliminary pool consisting of 1721 peaks ranging from 15 to 3366 bp with an average length of 402 bp.

[0241] To avoid PCR bias for small fragments, we also discarded all regions less than 100 bp, resulting in 1710 peaks ranging from 115 to 3366 bp with an average length of 404 bp. These peaks were then split into 160-bp fragments. Any fragments less than 100 bp were removed again to avoid PCR bias. 20-bp adapters were ligated to both sides.

[0242] Cell line GSCs were generated using the NSC culture conditions previously reported by Pollard et al. Briefly, cells were grown under feeder-free conditions in serum, as well as in neural basal medium supplemented with N2 and B27 supplements. The cells were grown by supplementing the culture medium with laminin 1 for adhesion. GSC7 has been characterized by Stricker et al. HEK293 cells were obtained from the ATCC cell bank. All other GSC lines and huFb170 were generated in Pollard's laboratory (unpublished). These have been characterized as part of the Glioma Cellular Genetics Resource (www.gcgr.org.uk).

[0243] Construction of a plasmid library array of enhancers We selected a library array format with 160 bp enhancer fragments that were individually cloned into separate plasmids and placed in 96-well plates. These 160 bp were synthesized as a single oligo pool, then randomly cloned into plasmids and isolated. Since 20 bp × 2 was required for PCR amplification of the pool, a library of 200 bp fragments was synthesized (Twist Biosciences). ChIP-seq peaks expressed by GSC were integrated into a single oligo to conform to our oligo synthesis strategy. This oligo nucleotide pool was PCR amplified (using a limited number of cycles to reduce "jackpot" products, which means products with bias and amplification exceeding the oligo nucleotide pool) and cloned into an expression vector by an efficient Golden Gate reaction. Then, 4579 individual plasmids were randomly selected, isolated, and seeded onto 96-well plates as a plasmid DNA library array (×48). Bacterial colony selection and plasmid miniprep generation were assisted by the Edinburgh Genome Foundry (S. Rosser). This library was then screened in 384-well format using an optimized Nanoglo DLR assay (Nano-Glo Dual-Luciferase Reporter Assay, Promega). In each plate, PGK-FFLuc was transiently transfected into GSC7 cells at a ratio of 1:10. Two days later, the Nanoglo DLR assay was performed and quantified on a plate reader.

[0244] PCR amplification and cleanup for library generation We developed highly efficient library amplification and plasmid cloning to avoid inappropriate products, empty arrays, and library redundancy by using Golden Gate cloning and selection against empty vectors. PCR conditions were optimized to limit / minimize background and amplify oligonucleotide pools without amplification bias.

[0245] We used KAPA HiFi Hotstart Polymerase and GC buffer (Roche, KK2501) with an annealing temperature of 68 °C and an extension time of 5 s at 72 °C. The oligonucleotide pool was first amplified for 10 cycles with 0.25 μl (input 2.5 ng), and 0.5 μl of this reaction was used for the next 15 cycles of amplification to reduce PCR bias.

[0246] Magnetic bead-based solid-phase reversible immobilization (SPRI) purification was used to minimize loss and clean up the DNA, followed by Golden Gate cloning. Sanger sequencing of all inserts confirmed that they were diverse, and all sequences could be reverse mapped to the fragments of the pool and adapter-ligated.

[0247] Screening platform for 384-well plates Cells were seeded into 384-well plates using a multi-drop, and transfected the next day using CyBio Felix. Two days later, the experiments were terminated by Nanoglo DLR assay, and Sanger sequencing was performed on all hits with changes greater than 10-fold of mCMV and reverse mapped to the genome.

[0248] Genomic features of validated enhancers Using the online tool GREAT, the basic regulatory domain of each gene was defined as 1 kb downstream and 5 kb upstream of the transcription start site (TSS), which means that neighboring genes are part of the same regulatory domain. To account for distal regulatory phenomena, the gene basic regulatory domain was extended up to 1000 kb towards the nearest upstream and downstream basic regulatory domains. Then, GREAT uses gene annotation ontology and query sequences along with a binomial test to predict target genes by examining the enrichment of gene ontology (Mclean et al. 2010). We investigated the expression patterns of the predicted target genes in human adult tissues (https: / / gtexportal.org / home / ).

[0249] Oligonucleotide pull-down and mass spectrometry Oligos were PCR amplified using biotinylated primers. The PCR products were then SPRI purified to remove biotinylated primer dimers. Such dsDNA fragments were incubated with nuclear extracts of GSC7 and GSC328. They were then bound to streptavidin magnetic beads, washed three times, and the frozen beads were sent for analysis by mass spectrometry.

[0250] Cell culture procedures Cell lines were cultured at 37 °C with 5% CO2. Cells were grown on uncoated cell culture plastic dishes. Glioblastoma stem cell lines were grown under serum-free conditions using conditions reported previously (Pollard et al., 2009). For passaging, cell lines were rinsed with PBS and detached using Accutase for GSC or 0.5% trypsin / EDTA for huFb170 and HEK293, respectively. Cells were collected in wash medium and centrifuged at 300×g for 3 minutes. They were resuspended in their respective growth media and seeded again. For cryopreservation, cells were resuspended in growth medium supplemented with 10% DMSO and stored at -80 °C for short-term storage. For long-term storage, vials were transferred to liquid nitrogen containers.

[0251] Transfection of GSC, huFb170, and HEK293 cells Cells were seeded at the required density. The next day, transfection was performed. Here, Plus reagent and Lipofectamine LTX (Life Technologies, 15338030) were each diluted to half the volume of Opti-MEM I Reduced-Serum Medium (hereinafter referred to as Optimem) (Life Technologies, 31985062). After this step, the Plus reagent / Optimem pre-mixture was added to all DNA samples, followed by the addition of the Lipofectamine LTX / Optimem pre-mixture. The transfection mixture was incubated at room temperature for 5 minutes and then carefully dropped onto the cells. Generally, the medium was not changed. Cells were analyzed 2 days after transfection.

[0252] HEK293 cells were seeded in each plate format at a specific density. The next day, GMEM, DNA, and PEI (self-made) were mixed and incubated at room temperature for 15 minutes to form a complex. Then, the transfection mixture was dropped onto the cells. Analysis was performed 2 days later.

[0253] Nano-Glo Dual-Luciferase Reporter Assay System (Promega) This assay consists of two steps. Transfection was performed using the normalization plasmid PGK firefly luciferase (Promega, E5011), which was transfected at a ratio of 1 / 10 to the plasmid of interest. First, the firefly luciferase activity was measured, which enabled normalization against the transfection efficiency. In the second step, the activity of Nanoluc was determined, which enabled reading of the plasmid of interest. The cells were washed 3× with PBS, and 20 μl was left in a 96-well plate after the final wash (25 μl in a 384-well). Oneglo buffer was added, and the plate was shaken at 480 rpm for 5 minutes to lyse the cells. Then, 20 μl of the cell lysate in 96-well format or 25 μl of the cell lysate in 384-well format was transferred to the corresponding opaque white plate, and luminescence was measured for 0.1 second / well using an Ensight multimode plate reader. In the next Nanoluc reaction in 96-well format, 2 μl of the cell lysate was transferred to an opaque white plate containing 40 μl of PBS, and 20 μl of Stopglo buffer supplemented with substrate was added to each well. In 384-well format, 20 μl of Stopglo buffer containing substrate was added on top of the undiluted cell lysate. The plate was shaken again at 480 rpm for 5 minutes to quench the firefly luciferase reaction and ensure good mixing. The Ensight multimode plate reader was used again to measure Nanoluc activity for 0.1 second / well.

[0254] To account for variability in transfection efficiency between wells, the data obtained from the Nanoluc reaction was normalized against the firefly reaction. The normalized data was used to calculate the fold change relative to the empty vector control mCMV.

[0255] Immunocytochemistry The cells were washed twice with PBS and fixed in 4% PFA for 10 minutes. The cells were permeabilized with 0.1% Triton X-100 (hereinafter referred to as PBST) in PBS. These were blocked with a blocking solution (1% BSA in PBST supplemented with 3% goat serum) and incubated overnight at 4°C with the primary antibody (Table 3). The next day, the cells were washed 3× in PBST, each secondary antibody was applied to the blocking solution, and incubated at room temperature for 45 - 60 minutes. The cells were washed with PBS and incubated for 5 minutes with DAPI nuclear counterstaining at a final concentration of 1 μg / ml. Images were acquired using a Nikon TiE microscope and NIS elements software (Nikon).

[0256]

Table 1

[0257] Flow cytometry For flow cytometry, the cells were detached, pelleted, and resuspended in an appropriate volume of flow cytometry buffer (1% BSA in PBS, v / v). The cells were stained with Draq7 (Abcam, ab109202, f.c., 0.1 μM) for live / dead staining and analyzed using a BD LSRFortessa cell analyzer (4 lasers, BD Bioscience). Analysis of flow cytometry data was performed using FlowJo analysis software (FlowJo, version 10.6.2).

[0258] Quantitative real-time polymerase chain reaction (qRT-PCR) In qRT-PCR, TaqMan Universal PCR Master Mix (Applied Biosystems) and TaqMan gene expression assays (Life Technologies) were used on a Quant Studio7 Flex Real-Time qPCR instrument. RNA samples that did not undergo reverse transcription were utilized on each plate to evaluate DNA contamination and water controls. In addition, qRT-PCR was performed with technical replicates. Data analysis was carried out using the ddCt method, assuming 100% PCR efficiency, which is guaranteed by the TaqMan assay. Briefly, the average value of technical replicates was calculated and normalized against the housekeeping gene GAPDH, thereby obtaining the ddCt value. Such values were further normalized against the calibration sample (GSC7) to obtain the ddCt.

[0259] Generation of entry vectors and final vectors using the Extensible Modular Mammalian Assembly (EMMA) Toolkit Vectors were designed and generated according to Martella et al. 2017. Briefly, BsaI and BsmBI sites were removed to accommodate new parts. The new parts were PCR amplified using primers that generate overhangs that become fusion sites and BsaI sites. This ensured simple and efficient cloning into the partial entry vectors. All partial entry vectors contain the red fluorescent protein (RFP), which is recombined with the part to be cloned, so that only white colonies can contain this part, improving the cloning efficiency. To assemble the expression vectors, all parts were mixed with the recipient vector in an equimolar ratio. To improve the cloning efficiency, the recipient vector contains the bacterial suicide cassette ccdB, which is recombined with the parts of the expression vector. Thus, bacteria that take up the unmodified recipient vector cannot generate colonies.

[0260] Western blot The cells were scraped into PBS and pelleted twice at 0.3 g to remove all the liquid. The cell pellet was resuspended in 70 μl of RIPA buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% NP-40, 0.5% deoxycholic acid, 0.1% SDS, and protease inhibitor (Complete, Roche, 11697498001)) and incubated on ice for 5 minutes. The lysate was centrifuged at 13,000 rpm for 10 minutes at 4°C (centrifuge 5415D, Eppendorf). Further, the supernatant was collected into a new tube. The protein extract was quantified using the Pierce BCA Protein Assay Kit (Thermo Scientific, Cat: 23225) according to the manufacturer's instructions.

[0261] 5% of the total volume of 4× lithium dodecyl sulfate (LDS) buffer containing 50 mM DTT was added to the cell extract, and the sample was denatured at 95 °C for 10 minutes. The sample was added to a 4–12% polyacrylamide gel prepared using Spectra Multicolour Broad Range Protein Ladder (Thermo Scientific, Cat: 26634) or BioRad Precision Plus Protein Dual Colour standards (Cat: 1610377) by Dr. Carla Blin. Transfer of protein bands to an Immobilon PVDF membrane (Millipore, IPVH00010) was performed using a wet electroblot or semi-dry blot using the Biorad Trans-blot turbo system according to the manufacturer's instructions. After membrane blocking for 1 hour at room temperature using 5% milk in TBS-T (TBS + 0.1% Tween 20), it was incubated overnight with rocking in 5% milk in TBS-T with the primary antibody (Table 4). The next morning, the membrane was washed 3 times by washing for 5 minutes at room temperature in TBS-T and incubated for 1 hour at room temperature with 5% milk in TBS-T with the secondary antibody conjugated to horseradish peroxidase (Table 4). Again, the membrane was washed 3 times for 5 minutes in TBS-T and developed using a homemade enhanced chemiluminescence (ECL) solution or Clarity ECL Western Blotting substrate (Bio-Rad, Cat: 170-5061), and images were acquired using X-ray film or a Bio-Rad ChemiDoc™ Imager.

[0262]

Table 2

[0263] Nuclear extract All buffers were prepared the day before, passed through a 22-μm filter (except for the dialysis buffer), and left overnight at 4°C. DTT and protease inhibitors were added just before use. Cells were scraped onto the medium, collected onto a 50-ml Falcon, and centrifuged at 1350 rcf for 5 minutes at 4°C. The pellet was resuspended in 50 ml of ice-cold PBS, a fixed volume was taken to count the cells, and centrifuged again at 1350 rcf for 5 minutes at 4°C. The pellet was then resuspended in 5 ml of ice-cold buffer A (10 mM HEPES pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, protease inhibitor (Complete, Roche, 11697498001)) per 40 million cells and incubated on ice for 10 minutes. The cell suspension was transferred to a glass Dounce homogenizer and disrupted on ice 40 times. The cell suspension was transferred to a Falcon and centrifuged at 1350 rcf for 10 minutes at 4°C. The supernatant was discarded (cytoplasmic extract), and the pellet was resuspended in 100 μl of ice-cold buffer B (20 mM HEPES pH 7.9, 5% glycerol, 1 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA pH 8.0, 0.5 mM DTT, protease inhibitor (Complete, Roche, 11697498001)) per 10 million cells. The suspension was rotated at 4°C for 30 minutes and transferred into a dialysis membrane (SnakeSkin, Thermo Scientific, Cat 68100). Dialysis was carried out by rotating in 500 ml of dialysis buffer (20 mM HEPES pH 7.9, 5% glycerol, 100 mM KCl, 0.83 mM EDTA pH 8.0, 1.66 mM DTT, protease inhibitor (Complete, Roche, 11697498001)) at 4°C for 2 hours. The dialysis buffer was exchanged (500 ml), and dialysis was continued overnight at 4°C. The extract was collected into a 1.5-ml tube and centrifuged at the maximum speed for 15 minutes at 4°C (centrifuge 5415D Eppendorf). The supernatant was collected into a new 1.5-ml tube and quantified (Pierce BCA Protein Assay Kit, Thermo Scientific, Cat 23225). A fixed volume of 100 μg was rapidly frozen in liquid nitrogen and stored at -80°C.

[0264] PCR Amplification and Purification with Biotinylated Primers The enhancer was amplified using PrimeStar Max (Takara) according to the manufacturer's instructions with a 5'-biotinylated primer (Table 5).

[0265]

Table 3

[0266] PCR purification using Agencourt AMPure XP magnetic beads (Beckman Coulter) was performed according to the manufacturer's instructions. Quantification of the PCR products using Tapestation and reagents was performed according to the manufacturer's instructions.

[0267] Precipitation of Interaction Proteins Streptavidin magnetic beads (New England Biolabs, S1420S) were used according to the manufacturer's instructions. Briefly, 10 μl of beads were aliquoted into LoBind tubes and washed three times on a magnetic stand with binding buffer (20 mM Tris-HCl pH 7.5, 0.5 M NaCl, 1 mM EDTA). 20 μl of biotinylated DNA (∼20 pmol) was mixed with 200 μl of binding buffer and added to the beads. The suspension was rotated at room temperature for 2 h. The beads were washed three times again with binding buffer, and 50 μg of nuclear extract was added (total volume 50 μl). 150 μl of dialysis buffer (20 mM HEPES pH 7.9, 5% glycerol, 100 mM KCl, 0.83 mM EDTA pH 8.0, 1.66 mM DTT, protease inhibitor (Complete, Roche, 11697498001)) was added, and the beads, DNA, and nuclear extract were rotated overnight at 4 °C. The next morning, the beads were washed three times with wash buffer (20 mM HEPES pH 7.9, 5% glycerol, 250 mM NaCl, 0.83 mM EDTA pH 8.0, 1.66 mM DTT). For Western blot, proteins were eluted in 20 μl of loading buffer (sodium dodecyl sulfate (LDS) buffer containing 50 mM DTT) and boiled for 5 min to denature the proteins. For mass spectrometry, the beads were sent to the facility for drying.

[0268] Zebrafish experiment All embryos were obtained by natural spawning and collected in conditioned aquarium water containing 0.00001% methylene blue. From 6 h post-fertilization (hpf) until the experimental period, embryos were treated with 200 μM N-phenylthiourea (PTU) (Sigma) to inhibit pigmentation (Karlsson, Von Hofsten, and Olsson 2001).

[0269] Zygotes were injected during the one-cell stage. Eggs were maintained in 1.5% agarose (Sigma) as described for microinjection by Nusslein-Volhard and Dahm 2002. The microinjection needle was pulled from a glass capillary (Harvard Apparatus, USA) using a P-97 Flaming / Brown Micropipette Puller (Sutter Instruments, USA). The needle pulling parameters were as follows: Heat: 550; Pull: 200; Velocity: 55; Time: 150. Microinjections were performed on a PV820 Pneumatic PicoPump (World Precision Instruments [WPI], USA) system. DNA constructs were generated using the Tol2Kit system (Kawakami 2007, Kwan et al. 2007). Plasmid DNA (30 ng / μL) containing Tol2 capping mRNA (20 ng / μL) supplemented with approximately 2 nL of 0.2% w / v phenol red (Sigma) to facilitate visualization of the injection volume was injected. After anesthesia with tricaine, images of embryos at 24 - 48 hpf were acquired live on a wide-field fluorescence microscope.

[0270] Adeno-associated virus (AAV) transduction assay The transfection confluence on the day was set to 60 - 70% by performing transfection of 6-well plates the day after seeding HEK293 cells. The medium was changed before transfection of HEK293 cells with PEI to condition the medium with the virus for 2 days. A few days later, HEK293 or GSC7 cells were seeded at low density (10 - 20%) in the same or smaller plate formats (6 or 12-well plates). The AAV conditioned medium was centrifuged at 1300 rpm for 4 minutes to remove contaminating cells, and the supernatant was transferred to HEK293 cells seeded at low density. The cells were analyzed on a microscope or by flow cytometry.

[0271] Seeding and transduction of cells for the killing assay On the day of cell seeding, cells were detached using the method described above. Cells were counted using a hemocytometer and seeded into either 96-well (1,000 cells / well in 50 μL), 24-well (30,000 cells / well in 500 μL), or 6-well (60,000 cells / well in 2 mL) Corning plates and placed in a 37°C / 5% CO2 incubator. The next day, AAV virus stocks were thawed at room temperature, and an appropriate amount of virus stock was added to the required amount of culture medium to achieve a final multiplicity of infection (MOI) of 5 × 10. 5 The culture medium containing the viral particles was added to the cells without replacing the existing medium. The cells were returned to 37°C / 5% CO2.

[0272] Drug Treatment Lyophilized GCV was diluted in DMSO to obtain a stock concentration of 100 mM. The GCV stock was aliquoted and stored at -20°C for up to one month. To generate a working stock concentration, GCV was diluted 1:100 with the appropriate culture medium, and then 20 μL of such working stock was added to wells already containing 80 μL of culture medium (yielding a further 1:5 dilution). The final concentration of GCV on cells was 200 μM.

[0273] As a negative control, DMSO was diluted 1:100 with the appropriate culture medium to obtain a working stock. 20 μL of the working stock was added to wells already containing 80 μL of culture medium. As a positive control, 20 μL of DMSO was added to wells with 80 μL of culture medium to obtain a final concentration of 20% DMSO.

[0274] Incucyte Live Cell Imaging To track cell proliferation and morphological changes during treatment, cells were monitored using an Incucyte live-cell imaging system. Images of the entire well of a Corning 96-well plate were acquired every 4 hours. Confluence was estimated using confluence scoring basic analysis software (Incucyte). Images at specific time points were extracted to verify cell confluence and morphology.

[0275] MTT assay On the day of the assay, the culture medium was replaced with a 0.3 mg / mL MTT solution (diluted with a culture medium appropriate for the cell line). The cells were left standing in an incubator at 37 °C / 5% CO2 for 3 hours. After incubation, the medium was removed and 70 μL of DMSO was added to each well. Each plate was maintained in the dark at 37 °C for 20 minutes with occasional shaking. Before reading the plate, each well was visually inspected to confirm that all the crystals (formazan) had dissolved. The plate was read with a plate reader at an absorbance of 560 nm.

[0276] Data analysis Most of the data analysis was performed using Microsoft Excel version 16.23 for Mac and GraphPad Prism 7. Error bars were shown as the standard deviation of the mean. Some of the data analysis was performed using RStudio version 1.1.456 (RStudio Team 2015). Figures were created using biorender.com and Adobe Illustrator 22.0.1. In addition, open-source programs such as bedtools (Quinlan and Hall 2010), GREAT (Mclean et al. 2010), fastasplitter (Stothard 2000), and MEME (Bailey et al. 2009) were used for various analyses.

[0277] (Example 1) Identification of functional SOX2 enhancer To initially establish a proof of concept for whether it is possible to obtain synergistic activity when combining individually active enhancers to form clusters of arrays of multiple parts, we first identified a small set of candidate enhancers. These were selected based on proximal genes (POU3F2, POU3F3, CHD7, ASCL1, SOX6, ETV1) with known roles in neural stem cell (NSC) self-renewal and using available differential expression data of NSCs versus primary human fibroblasts. It also included five SOX2 candidate self-regulatory enhancers. Such enhancers (typically about 500 - 800 bp) were cloned into plasmid expression vectors containing mNeonGreen and luciferase reporter cassettes, and individual enhancer activities were examined using two independent patient-derived glioblastoma stem cell (GSC) cell lines (G7 and G328) (Figure 1). Six of these enhancers were functional, and NanoLuc expression increased by more than 10-fold over mCMV (254, 270, 282, 292, 312, and 316; in the range of 10 - 300-fold). None of these were active in HEK293 cells (negative control).

[0278]

Table 4

[0279] (Example 2) Synergistic increase in transcriptional activity occurs by the association of four different enhancers To investigate whether it was possible to increase activity by combining multiple enhancers, we first constructed tandem versions (i.e., multiple copies of the same enhancer) of either two (2×), four (4×), or eight (8×) enhancers (concatemers). We selected three different individual enhancers with variable strengths: 270 (≈100-fold), 254 (≈20-fold), and 312 (≈10-fold). At 270, we observed only modest synergy in both the two- and four-part enhancers compared to individual enhancer activity. In the four-part enhancer, ≈500-fold activation occurred compared to ≈100-fold in the individual enhancer (Figure 1F). Surprisingly, we did not observe further increases by increasing the copies to an eight-part concatemer. A similar trend was observed in the independent patient strain GSC328. Only background levels of luciferase activation comparable to the mCMV negative control were seen in HEK293 cells for all constructs.

[0280] Next, we examined whether synergistic enhancement could be achieved by mixing enhancers from different genes (Figure 1K). Thus, we combined the top four individual enhancers (270, 282, 292, and 316) identified in G7 to form a new synthetic enhancer (named T4). Indeed, we observed a significant increase in the expression of T4, which exceeded 5000-fold that of mCMV (Figure 1L), using the NanoLuc reporter assay (both the Nano-Glo Luciferase Assay and the Nano-Glo Dual-Luciferase Reporter Assay, Promega). Further verification using flow cytometry to score mNGreen in G7 cells (Figure 1I) showed that the level of T4 was approximately 50 - 60% of the levels observed for full-length CMV in G7 cells. Thus, combining enhancers from different genes results in a significant increase in synergistic effects and enhancer activity in GSCs, but no increase in background expression in HEK293 cells. Henceforth, such cell-type-specific synthetic clusters of enhancers are referred to as "synthetic super-enhancers" (SSEs).

[0281] (Example 3) The activity of the T4 synthetic super-enhancer in GSCs decreases after astrocyte differentiation Next, we examined whether the SSE-T4 synthetic super enhancer is specific to the GSC state and thus lost during the differentiation into astrocytes where SOX2 is downregulated. G7 cells can be efficiently differentiated into astrocyte-like cells after exposure to fetal calf serum (FCS) for a period of 10 - 15 days. Therefore, the SSE-T4 expression cassette was stably transfected into GSC (G7) (using the PiggyBac transposase system), and mNGreen+ cells were sorted using fluorescence-activated cell sorting (FACS). The next day, these were seeded in 5% FCS and mNGreen expression was measured over time every 5 days for 15 days using flow cytometry (Figures 1H - 1J). The expression of mNGreen decreased significantly by day 5 based on live cell fluorescence imaging. This was confirmed by flow cytometry, which showed that by day 5, approximately 50% of the cells had lost mNGreen, and by day 15, approximately 80% of the cells had lost mNGreen. Such data indicate that SSE-T4 is highly active in GSCs but not in astrocyte-like differentiated progeny, and thus the SSE is cell type specific.

[0282] (Example 4) Systematic screening of a plasmid library array of SOX2 enhancers The above data provided proof of principle that by combining enhancers to form a four-part array, transcriptional activity can be increased without compromising background expression in non-GSC cells. We used a full set of candidate genome-wide SOX2 enhancers in a high-throughput luciferase assay based on 384-well plates to systematically identify functional enhancers with optimal performance and a smaller size when used in SSE, and thus determined that functional screening of enhancer fragments (160 bp) was feasible. By performing such screening in patient-derived GSCs, we can overcome the limitations of existing serum-propagated cancer cell lines that do not have the appropriate identity and associated enhancers.

[0283] We selected 160 bp because it provides a size sufficient for nucleosome binding and may have important properties in the development of grammar in functional enhancers (Soufi et al., 2015). Moreover, 160 bp is a convenient size for the synthesis of oligonucleotide library pools that incorporate 20-bp ends for subsequent plasmid library array construction. We supported the array of plasmid libraries constructed from synthetic oligonucleotides over strategies that screen pools of other libraries, such as STARR-seq (Muerdter et al., 2018; Arnold et al., 2013), to ensure an improvement in signal-to-noise and straightforward validation of hits.

[0284] Therefore, we designed a set of oligonucleotides that can be synthesized as a pool. We re-analyzed the dataset of Suva et al., 2014 and defined a set of GSC-specific SOX2 binding peaks that overlap with H3K27ac, thereby identifying 1710 different enhancers. Such enhancers were on average about 400 bp (range: 115 - 3366 bp). We split such sequences in silico into 160-bp elements with 100-bp overlaps. Then, 9523 different oligonucleotide sequences were synthesized as a pool, PCR amplified, and cloned into an array of plasmid libraries in 48 × 96-well plates (a total of 4579 individual plasmids). Sanger sequencing of a subset of the resulting plasmids confirmed that the majority (about 70%) of such plasmids contained appropriate enhancers.

[0285] The SOX2 enhancer library was screened using an optimized luciferase assay in a 384-well format. 135 plasmids were identified as functional and showed more than a 10-fold increase over mCMV. Next, 52 out of such 135 initial hits were verified using a triplicate of an independent Nanoglo DLR assay (Figure 2). 16 out of such 52 were found more than twice. The hits were Sanger sequenced to confirm the inclusion of the enhancer and reverse mapped to the human genome by the predicted fragments from the initially designed set.

[0286] Next, we verified the top 17 fragments (more than a 30-fold increase over mCMV) using an independent flow cytometry assay for multiple cell lines (Figure 2). These were active in an independent patient-derived GSC line (GSC328), but inactive in the non-neuronal cells tested (HEK293 cells and human primary fibroblasts huFb170). We found that the majority of the top hits were sequences evolutionarily conserved across vertebrates. The top 5 enhancers exceeded 100-fold each (range: 100 - 260-fold) compared to mCMV based on NanoLuc. These were verified by flow cytometry.

[0287] ID1101 is located in the intron of PRCP, ID2904 is located in the intron region of the MYO18 gene and proximal to SEZ6L, ID0109 is located in a gene-rich region between TPK1 and CNTNAP2, and ID4328 is located in the intron region of the zinc finger transcription factor ZNF438. ID0876 is located in the intron region of NWD2.

[0288]

Table 5

[0289] Thus, through our functional screening, many 160bp enhancer fragments were identified that, despite being only one-fourth to one-fifth the size, had significantly improved activity compared to the full-length enhancer (above).

[0290] (Example 5) Generation of a Potent and Selective Synthetic Super-Enhancer Using the Newly Identified SOX2 Enhancer We constructed synthetic super-enhancers by clustering the most active fragments individually from the screening into four-part arrays and confirmed whether their strengths were synergistically improved while maintaining cell-type selectivity.

[0291] To generate the most potent possible synthetic super-enhancers, we designed four new sets of four-part arrays using sequences from the top 17 enhancers. These were assembled upstream of mCMV. The fragments were ranked according to the median fold change relative to mCMV. The four most active enhancers (ID1101, ID2904, ID0109, ID4328, represented as "1") were clustered into a four-part array to form a synthetic super-enhancer. Enhancers containing the second group (represented as "2") were clustered to form a synthetic super-enhancer, and so on. We assumed that the four most potent enhancers would show the highest level of expression, the next group would show the second-highest expression, and so on. Additionally, by testing the synthetic super-enhancers in different cell types (GSC7, GSC328, HEK293, and huFb170), we also investigated their cell-type selectivity.

[0292] All four constructs examined (C1, C3, C5, and C7) induced significant and selective expression in GSC7 and GSC328, but only background expression in HEK293. No expression was detected in huFb170. Each of these synthetic super-enhancers (C1, C3, C5, and C7) showed higher fluorescence intensity and a higher percentage of mNGreen+ cells in GSC7 and GSC328 than the most active synthetic super-enhancer, SSE-T4 (the first-generation SSE discussed above), when using the candidate enhancer. Notably, C1 and C7 induced higher expression levels than the positive control CMV, which is itself one of the most powerful viral promoters. Thus, we observed a clear synergistic effect of the four-part enhancer array that results in extremely potent and cell-type-selective synthetic super-enhancers.

[0293] We further reduced the size of the synthetic super-enhancer by removing the adapter and spacer (e.g., in C1, from 974 bp to 640 bp). Such synthetic super-enhancers, since they are seven constructs and their design follows the same principle as C1, C3, C5, and C7, were named SSE 1 - 7. All constructs showed a similar level of activity. This level was comparable to that of full CMV and, in some cases, higher than CMV (SSE1, SSE2, SSE3, SSE5 by Nanoglo DLR assay and SSE-1, SSE-2, SSE-3, SSE-5, SSE-7 by flow cytometry in GSC7). Also, SSE-1, SSE-2, SSE-5, and SSE-7 showed higher intensity than CMV in GSC7. Analysis of mNGreen expression by flow cytometry was also performed for GSC328, HEK293, and huFb170. The trend was similar in GSC328. The mean fluorescence intensity (MFI) was comparable to that of CMV, and the intensity of SSE-6 was clearly less than that of CMV. In HEK293 cells, all synthetic super-enhancers induced transcription to a level similar to that of mCMV. mCMV showed 32.5% mNGreen+ cells compared to non-transfected cells. SSE demonstrated a slight increase in expression compared to mCMV in HEK293. The activity of SSE1 - SSE7 in huFb170 was undetectable (above 1%). In both reporter gene assays (Nanoglo DLR assay and flow cytometry), a trend was observed where the smaller the synthetic super-enhancer, the higher its activity, as seen in each construct C1 vs. SSE-1, C3 vs. SSE-3, etc. (Figure 3B, Figure 3C, Figure 3D). This was confirmed by live cell imaging (Figure 3E). Each of the synthetic super-enhancers SSE-1, SSE-3, SSE-5, and SSE-7 examined had activity similar to that of CMV.

[0294] (Example 6) Synthetic super-enhancers have low activity in differentiated GSCs To investigate the activities of SSE-2 to SSE-7 under differentiating conditions, GSC7 cells were stably transfected using the Piggybac transposase system and sorted for mNGreen+ cells. A similar number of live cells were sorted for the mCMV and non-transfected controls. Cells were then seeded in medium containing 5% FCS but no growth factors to induce astrocyte differentiation.

[0295] mNGreen expression during differentiation was measured on days 0, 1, 5, 10, and 15 (Figures 4E and 4G). We observed a faint signal over time, suggesting that the synthetic super enhancer is most highly expressed in GSCs and its activity decreases during differentiation.

[0296] (Example 7) Highly selective region-specific expression in the forebrain is revealed by zebrafish embryos Zebrafish are well-suited as an experimental model for exploring the tissue specificity of enhancers. We identified expression in tissues shared among all constructs, such as the ventral spinal cord, otic placode, and in the midbrain / hindbrain and anterior forebrain. Some fragments, such as the anterior / posterior small intestine, had additional activity. We generally observed low expression at 24 hpf, which maintained an increase until the last time point measured at 48 hpf maximum. For the controls, we observed widespread non-specific EGFP expression in the CMV control and no expression in the mCMV using the same assay (Figure 4). The mCMV control showed green fluorescent eyes, probably due to false transcription of the crystal eye promoter (Figure 4K).

[0297] (Example 8) SSEs can be used in adeno-associated viruses to induce selective killing of GSCs To examine whether SSE is suitable for driving a cytotoxic payload and selectively killing GSCs, we transduced the GSC7 cell line and the huFB170 cell line with adeno-associated virus 2 (AAV2) containing the suicide gene thymidine kinase (TK) derived from herpes simplex virus (HSV) in a polycistronic mRNA with the reporter gene mCherry linked by p2A. This cassette was driven by either the constitutively active CMV promoter (CMV_HSV-TK-v5_P2A_mCherry) as a positive control or the promoter of interest SSE-7 (SSE-7-HSV-TK-v5-P2A-mCherry). HSV-TK is a well-established prodrug that metabolizes the non-cytotoxic prodrug ganciclovir (GCV) to a cytotoxic product by phosphorylation. Phosphorylated GCV causes interference with DNA synthesis resulting in premature termination and cytotoxicity by competing with dGTP as a substrate for polymerase.

[0298] By flow cytometry and Western blot, it was confirmed that SSE-7 drives the expression of both mCherry and HSV-TKv5 in GSC7, but not in fibroblasts (huFb170) (Figure 5). Cell confluence was recorded through cytotoxicity experiments. The decrease in cell proliferation and confluence was specifically observed in cells transduced with CMV or SSE-7-driven TKv5-P2A-mCherry and treated with 200 μM GCV (Figure 6A). A significant negative effect on cell confluence was not observed in cells treated with virus or ganciclovir alone. MTT assay data demonstrated the loss (or absence) of metabolic activity, which serves as an approximate reading of the cell viability of GSC7 cells treated with CMV or SSE-7-driven TKv5-P2A-mCherry and GCV (Figure 6B). There was no statistically significant difference between CMV and SSE-7 (13% vs. 12% viability) (p-value = 0.7792). The effect of ganciclovir alone on GSC7 cell viability was minor (89% viability). Live cell imaging confirmed that such findings showing low cell density and morphological changes were associated with stress, damage, or death (round cells) of the cells after treatment (Figure 6C). In short, both CMV and SSE-7 can drive the expression of the HSV-TK-v5-P2A-mCherry construct to a sufficient level in GSC7 cells to induce cell death in the presence of ganciclovir.

[0299] Based on live cell imaging data at the end of the experiment (day 17), the majority of huFB170 cells transduced with the CMV-driven virus rather than the SSE-7-driven virus were killed (Figure 6D). This was confirmed by MTT data, demonstrating complete viability in cells transduced with SSE-7-driven TKv5-P2A-mCherry compared to 6.3% viability in cells transduced with the CMV-driven virus (Figure 6E, n = 1).

[0300] (Example 9) SSE shows activity in various GSCs In the proposed gene therapy, it is desirable that SSE is highly expressed across diverse patient-derived glioblastoma cell lines but is inactive in other cell types. The experiments described in Example 8 were repeated in five patient-derived glioblastoma cell lines (E17, E21, E28, E31, E34). Such strains were selected for this experiment because they covered the spectrum of genetic and transcriptional GBM subtypes and were shown to be tumorigenic in the mouse brain. Overall, SSE7 is active in all cell lines, but to varying degrees, highly expressed in the positive control cell line GSC7 (Figure 7C) as well as E17 and E28 (Figure 7C) which both belong to the classical subtype, comparable to full-length CMV, but with low expression frequency in E21, E31, and E34 (Figure 7C).

[0301] (Example 10) De novo motif analysis identifies SOX dimer motifs enriched in the top 32 hits We used the MEME tool (Bailey et al. 2009) capable of identifying de novo motifs. We used the top 32 hits as input. Unexpectedly, the prominent motif that emerged was the SOX motif with a partial palindromic structure (Figure 8A). SOX2 does not form homo- or hetero-dimers with other SOX factors, but dimerization of members of the SOXE group (SOX8, SOX9, SOX10) is well established. Interestingly, SOX9 is a well-known regulator of NSC and GSC but has not been studied as deeply as SOX2 (Bulstrode et al. 2017, Huang et al. 2015, Mateo et al. 2015, and D. K. Singh et al. 2017). This motif immediately suggested that SOX2 and SOX9 could operate in shared enhancers (Figure 8C).

[0302] Next, we investigated whether this dimer motif is a general feature of the oligonucleotide pool of the sequences, and mapped it to the top 52 hits (all sequences having a 10-fold fold change relative to mCMV) compared to unconfirmed hits by validation, and to 10 random sequences of the first oligonucleotide pool. The motif is present at high frequency in the top 32 hits and at low frequency in the remaining top 33 to top 52 hits.

[0303] (Example 11) The SOX dimer motif is essential for the activity of ID2904 in GSCs, suggesting cooperation between SOX2 and SOX9 in the key GSC functional enhancer. Next, we explored whether the SOX dimer is essential for enhancer function. We generated a series of variants of the motif for ID2904. The synthesized variants had 1) reduction of the interval between the half-sites (-2n), 2) expansion of the interval between the motif half-sites, 3) and 4) deletion of one half-site, 5) substitution with random sequences of the flanking sequences, 6) inversion of the motif, 7) mutation of the core nucleotides, 8) substitution with high-affinity motifs as reported by Jolma et al. 2013, 9) random sequences as negative controls, and 10) tetramer formation of the motif. Such different variants enabled us to functionally analyze the key sequences within the dimer motif (Figure 8).

[0304] We observed that the activity of ID2904 was almost completely lost by almost all variations of the SOX dimer motif, thereby confirming the importance of this dimer motif for enhancer function. The exception was the change in the distance between the two half-sites, which caused an increase in activity. This is interesting because it is consistent with the idea that the interval can be "partially optimized" and that a 2-bp reduction can drive stronger binding of the SOX9 dimer (Farley et al. 2015). We conclude that the SOX dimer motif is essential for the enhancer activity of ID2904. This is also consistent with Huang et al. 2015, who defined the optimal interval for the SOXE factor motif.

[0305] Next, we aimed to confirm whether SOX2 and SOX9 actually bind to the enhancer and the SOX dimer motif. For this purpose, a pull-down assay was performed, where we biotinylated ID1101 (the top hit in the screening) and ID2901 and incubated them with nuclear extracts. Western blot and mass spectrometry were performed to identify the key transcription factors that interact with these fragments (Figure 8). In data analysis, ID1101 was normalized against the average value of the random sequence 599 (in each cell line). The cutoff was set at a 2-fold enrichment relative to 599 in both cell lines. To screen for transcription factors, a list of all transcription factors was obtained from UniProt and overlapped with the proteins detected in both cell lines.

[0306] In the co-immunoprecipitation experiment, SOX2 was specifically pulled down by ID1101 in GSC7 and GSC328, demonstrating the binding of SOX2 to the enhancer (Figure 8). This was confirmed by mass spectrometry, showing that SOX2 was enriched 23-fold in GSC328 and 4.4-fold in G7, thereby confirming the binding of this transcription factor.

[0307] In addition, the data presented above and in Figure 8J demonstrate that the sequence relationship (grammar) surrounding the motif is functionally important, as the activity was completely lost upon substitution of the wild-type (WT) sequences 20 bp upstream and downstream.

[0308] (Example 12) The SOX dimer motif helps to screen for active enhancers in silico We hypothesized that the presence of motifs might have the potential to improve our predictive power regarding which enhancers are active. To test this hypothesis, we mapped the motifs to all full-length enhancers of the original SOX2 ChIP-seq dataset. Based on predictive target genes with known functions in GBM or neural stem cells and clustering within the genome that implies regulation of the same predictive target genes, 70 regions were selected for the initial screening. These were examined in the Nanoglo DLR assay. These were named "Clustered SOX dimer motif enhancers" (CSE). Thus, using the frequency and rational design of the SOX dimer motif, we improved the success rate of identifying positive enhancers approximately 11-fold, i.e., from approximately 2% (52 / 2610) in the original screening to approximately 22.8% (16 / 70) in our rational selection. Positive hits were further validated by flow cytometry in GSC7, GSC328, HEK293, and huFb170 (Figure 9).

[0309] We performed further screening with 209 enhancers to identify more active enhancers. The enhancers were reduced to 160 bp based on the predicted motif (named minCSE, see Table 1 (Table 7)). To generate additional SSEs for screening, based on data from the human protein atlas (https: / / www.proteinatlas.org / ), 160-bp enhancer fragments from different target genes or predicted target genes enriched in the brain / brain-specific or glioblastoma-enriched / glioblastoma-specific, covering pathways such as proliferation or stemness, were mixed.

[0310]

Table 6

[0311] Conclusion Mapping of transcription factor binding sites, chromatin proximity, or enhancer-related histone marks are widely used to search for important transcription factors (TFs) and associated enhancers. However, the main limitation is that TFs bind rather promiscuously and functional enhancers represent only a small subset. Here we demonstrate that it is possible to cause the formation of synthetic super-enhancers by combining arrays of functional enhancer sequences. Utilizing the natural “grammar” of endogenous functional enhancer sequences, but combining them in optimal sizes and clusters, we were able to obtain SSEs with synergistically increased transcriptional activity without sacrificing cell-type selectivity. Thus, our data support that combining individual enhancers can generate synthetic super-enhancers that are extremely powerful in driving transcription while retaining selectivity.

[0312] References Arnold et al. 2013 “Genome-wide quantitative enhancer activity maps identified by STARR-seq.” Science 339(6123): 1074-1077 Bailey et al. 2009. “MEME Suite: Tools for Motif Discovery and Searching.” Nucleic Acids Research 37(Suppl. 2): 202-8. Boumahdi et al. 2014. “SOX2 controls tumour initiation and cancer stem-cell functions in squamous-cell carcinoma.” Nature 511(7508): 246-50. Bulstrode et al. 2017. “Elevated FOXG1 and SOX2 in Glioblastoma Enforces Neural Stem Cell Identity through Transcriptional Control of Cell Cycle and Epigenetic Regulators.” Genes & Development 31(8): 757-73. Corish and Tyler-Smith, 1999. “Attenuation of Green Fluorescent Protein Half-Life in Mammalian Cells.” Protein Engineering 12(12): 1035-40. Farley et al. 2015 “Suboptimization of Developmental Enhancers.” Science (New York, N.Y.) 350(6258): 325-28. Gangemi et al. 2009 “SOX2 Silencing in Glioblastoma Tumor-Initiating Cells Causes Stop of Proliferation and Loss of Tumorigenicity.” Stem Cells 27(1): 40-48. Gibson et al. 2009 “Enzymatic Assembly of DNA Molecules up to Several Hundred Kilobases.” Nature Methods 6(5): 343-45. Guerra-Rebollo et al. 2019 “Targeting of Replicating CD133 and OCT4 / SOX2 Expressing Glioma Stem Cells Selects a Cell Population That Reinitiates Tumors upon Release of Therapeutic Pressure.” Scientific Reports 9(1): 9549. Huang et al. 2015 “SOXE Transcription Factors Form Selective Dimers on Non-Compact DNA Motifs through Multifaceted Interactions between Dimerization and High-Mobility Group Domains.” Scientific Reports 5(January): 1-12. Jolma et al. 2013 “Multiplexed Massively Parallel SELEX for Characterization of Human Transcription Factor Binding Specificities.” Genome Research 20(6): 861-73. Karlsson, Von Hofsten, and Olsson, 2001 “Generating Transparent Zebrafish: A Refined Method to Improve Detection of Gene Expression during Embryonic Development.” Marine Biotechnology 3(6): 522-27. Kawakami, 2007 “Tol2: A Versatile Gene Transfer Vector in Vertebrates.” Genome Biology 8(Suppl. 1): 1-10. Kwan et al. 2007 “The Tol2kit: A Multisite Gateway-Based Construction Kit for Tol2 Transposon Transgenesis Constructs.” Developmental Dynamics 236(11): 3088-99. Lopez-Bertoni et al. 2015 “DNMT-Dependent Suppression of MicroRNA Regulates the Induction of GBM Tumor-Propagating Phenotype by Oct4 and Sox2.” Oncogene 34(30): 3994-4004. Lujan et al. 2012 “Direct Conversion of Mouse Fibroblasts to Self-Renewing, Tripotent Neural Precursor Cells.” PNAS 109(7): 2527-32. MacLeod et al. 2019 “Genome-Wide CRISPR-Cas9 Screens Expose Genetic Vulnerabilities and Mechanisms of Temozolomide Sensitivity in Glioblastoma Stem Cells.” Cell Reports 27(3): 971-986.e9. Martella et al. 2017 “EMMA: An Extensible Mammalian Modular Assembly Toolkit for the Rapid Design and Production of Diverse Expression Vectors.” ACS Synthetic Biology 6(7): 1380-92. Mclean et al. 2010 “GREAT Improves Functional Interpretation of Cis-Regulatory Regions.” Nature Biotechnology 28(5): 495-501. Muerdter et al. 2018 “Resolving Systematic Errors in Widely Used Enhancer Activity Assays in Human Cells.” Nature Methods 15(2): 141-49. Nusslein-Volhard and Dahm, 2002 “Zebrafish: a practical approach” New York: Oxford University Press: 303 pages. Pollard et al. 2009 “Glioma stem cell lines expanded in adherent culture have tumor-specific phenotypes and are suitable for chemical and genetic screens” Cell Stem Cell 4(6): 568-80. Quinlan and Hall, 2010 “BEDTools: A Flexible Suite of Utilities for Comparing Genomic Features.” Bioinformatics 26(6): 841-42. D. K. Singh et al. 2017 “Oncogenes Activate an Autonomous Transcriptional Regulatory Circuit That Drives Glioblastoma.” Cell Reports 18(4): 961-76. S. K. Singh et al. 2004 “Identification of Human Brain Tumour Initiating Cells.” Nature 432(7015): 396-401. Soufi et al. 2015 “Pioneer Transcription Factors Target Partial DNA Motifs on Nucleosomes to Initiate Reprogramming.” Cell 161(3): 555-68. Stothard, 2000 “Internet On-Ramp.” BioTechniques 28(6): 1102-4. Stricker et al. 2013 “Widespread resetting of DNA methylation in glioblastoma-initiating cells suppresses malignant cellular behavior in a lineage-dependent manner” Genes Dev. 27(6): 654-69. Suva et al. 2014 “Reconstructing and Reprogramming the Tumor-Propagating Potential of Glioblastoma Stem-like Cells.” Cell 157(3): 580-94.

[0313]

Table 7A

[0314]

Table 7B

[0315]

Table 7C

[0316]

Table 7D

[0317]

Table 7E

[0318]

Table 7F

[0319]

Table 7G

[0320]

Table 7H

[0321]

Table 7I

[0322]

Table 7J

[0323]

Table 7K

[0324]

Table 7L

[0325]

Table 7M

[0326]

Table 7N

[0327]

Table 7O

[0328]

Table 8A

[0329]

Table 8B

[0330]

Table 8C

[0331]

Table 8D

[0332]

Table 8E

[0333]

Table 8F

[0334]

Table 8G

[0335]

Table 8H

[0336]

Table 8I

Claims

1. (i) for the treatment or prevention of recurrent / secondary cancer / tumor, and / or (ii) for the generation, stimulation or induction of an anti-recurrent / secondary cancer / tumor immune response, wherein the construct or functional nucleic acid comprises a gene or transgene encoding a harmful payload.

2. The pharmaceutical composition according to claim 1, wherein the recurrent / secondary cancer is associated with a primary cancer treated with the construct or functional nucleic acid defined in claim 1.

3. The pharmaceutical composition according to claim 1, wherein the gene or introduced gene is for expression in target primary cancer cells, and the payload is harmful to primary cancer cells.

4. The pharmaceutical composition according to claim 3, wherein the target primary cancer cells are primary glioblastoma cancer cells, and the recurrent / secondary cancer is recurrent / secondary glioblastoma.

5. The pharmaceutical composition according to claim 1, wherein the toxic payload comprises or encodes a protein that stimulates an immune response that causes the recruitment of cytotoxic immune cells.

6. The pharmaceutical composition according to claim 1, wherein the gene or transgene is operably bound to a synthetic super-enhancer, and the synthetic super-enhancer comprises two or more enhancer sequences derived from different genomic loci.

7. The pharmaceutical composition according to claim 6, wherein each enhancer sequence comprises a binding site for a transcription factor and, optionally, 20 to 400 nucleotides upstream and / or downstream of the binding site for the transcription factor.

8. The pharmaceutical composition according to claim 6, wherein the enhancer sequences derived from different genomic loci are activated by a common transcription factor or a set of cell type-associated transcription factors.

9. The pharmaceutical composition according to claim 6, wherein the synthetic superenhancer comprises at least one SOX motif and / or a SOX dimer motif.

10. The pharmaceutical composition according to claim 9, wherein the SOX dimer motif comprises or consists of SEQ ID NO: 1, SEQ ID NO: 2, and / or SEQ ID NO:

3.

11. The pharmaceutical composition according to claim 6, wherein the synthetic super-enhancer is activated by one or more SOX family transcription factors.

12. The pharmaceutical composition according to claim 6, wherein each enhancer sequence comprises or comprises a sequence having at least 80% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 4 to 63.

13. The pharmaceutical composition according to claim 6, wherein the synthetic super-enhancer comprises or comprises a sequence having at least 80% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 64 to 80.

14. The pharmaceutical composition according to claim 1, wherein the toxic payload contains or encodes a cytotoxic substance, or further contains or further encodes a cytotoxic substance.

15. The pharmaceutical composition according to claim 1, wherein the toxic payload includes or encodes a suicide gene that encodes a protein capable of converting an inactive prodrug into a cytotoxic drug.

16. The pharmaceutical composition according to claim 15, wherein the suicide gene is a herpes simplex virus thymidine kinase (HSV-TK) gene, and the prodrug is ganciclovir, acyclovir, or valacyclovir.

17. The pharmaceutical composition according to claim 1, wherein the toxic payload comprises or encodes the IL-12 gene.

18. The pharmaceutical composition according to claim 1, wherein the construct or functional nucleic acid is administered to a subject having primary cancer and having been treated for the primary cancer with the construct or functional nucleic acid, and the treatment of the primary cancer with the construct or functional nucleic acid induces, stimulates or generates an anti-recurrent / secondary cancer / tumor immune response that prevents or reduces the risk of recurrent / secondary cancer arising from the primary cancer.

19. A pharmaceutical composition comprising a construct or functional nucleic acid for (i) the treatment or prevention of recurrent / secondary cancer / tumor, and / or (ii) the generation, stimulation or induction of an anti-recurrent / secondary cancer / tumor immune response, wherein the construct or functional nucleic acid comprises a gene or transgene encoding IL-12 and herpes simplex virus thymidine kinase (HSV-TK).

20. The pharmaceutical composition according to claim 19, wherein the recurrent / secondary cancer / tumor is recurrent / secondary glioblastoma.

21. The pharmaceutical composition according to any one of claims 1 to 20, wherein the construct or functional nucleic acid is packaged within an AAV vector.

22. The pharmaceutical composition according to claim 21, wherein the construct or functional nucleic acid is packaged within an AAV1 vector.

Images