Functional nucleic acid molecules and methods

Abstract

Disclosed are constructs configured to express an adverse (e.g., cytotoxic and immunomodulatory) payload in a cell, which are useful in the treatment or prevention of recurrent / secondary cancers / tumors, such as cancer cells. And eliciting, inducing, enhancing, or promoting an immune response effective against a recurrent / secondary cancer / tumor that may be caused by the primary cancer / tumor.

Description

Technical Field

[0001] The present disclosure relates to constructs for treating or preventing recurrent / secondary cancers / tumors. The constructs may include an unfavorable payload for killing target (cancer) cells. Those constructs may include synthetic super-enhancers activated by one or more transcription factors, including synthetic super-enhancers activated by one or more transcription factors of the SOX family. Background Art

[0002] There is great interest in the identification of cell type-specific enhancers and promoters that can be used for gene therapy, particularly where therapeutic protein cargo needs to be expressed in a highly cell-selective manner to ensure maximum dosing and minimize off-target effects. However, current strategies have had only limited success and must often compromise on size, selectivity, or strength.

[0003] Two different but complementary strategies can be used to identify cell type-specific enhancers. First, strong and selective natural enhancers for the cell type of interest may come from systematic functional genetic dissection of regulatory networks for the cell type or lineage of interest. Second, fully synthetic enhancers can be constructed based on artificially synthesized DNA binding motifs of known key transcription factors (TFs). Databases of such motifs—usually about 5-10 base pairs in length—have been defined based on biochemical analysis of the strongest binding, such as JASPAR (Jolma et al., 2013). These transcription factor binding motifs can be synthesized and screened in high throughput using pooled libraries (Jolma et al., 2013). However, each approach has disadvantages. The strength of a single natural enhancer is often insufficient for phenotypic assays, or is limited by size constraints or inappropriate selectivity. Artificial promoters constructed based on concatemers of transcription factor binding motifs can induce expression, but do not have the appropriate “grammar”—spacing, order, and appropriate affinity—that is present in natural enhancer sequences and is required to recruit cooperating TFs and cofactors (Farley et al., 2015). These will therefore lack selectivity and robustness.

[0004] The art describes promoter sequences, enhancer sequences and methods for identifying these sequences, but these all have the above-mentioned disadvantages. WO2014066848 and US2014 / 0296218 describe natural super-enhancers claimed to be useful for regulating the expression of cell type-specific genes and methods for identifying the super-enhancers. WO2017155973 describes synthetic enhancers for recombinant parvoviruses, particularly enhancers with reduced size to enable delivery of large amounts of genetic payloads. WO2012101191 describes the design of promoters for selective expression of genes by using transcription factor binding site motif predictions to produce minimized promoters based on these motifs.

[0005] US5952221 describes an adeno-associated viral vector comprising a first and a second nucleic acid sequence.

[0006] US6218180 describes gene therapy for the treatment of solid tumors using recombinant adeno-associated viral vectors.

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

[0008] Zhang et al. described how pre-existing anti-tumor immunity enhances the anti-tumor effect of chemotherapy (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, there remains a need in the art to provide options for treating recurrent / secondary cancers, as well as to provide enhancers with suitable selectivity and activity for use in therapeutic approaches such as gene therapy. Summary of the invention

[0011] The present disclosure is based on the discovery that constructs designed and configured to express adverse (e.g., cytotoxic and immunomodulatory) payloads in cells can be used to treat or prevent recurrent / secondary cancers / tumors.

[0012] In one teaching, any disclosed construct can be used not only to treat, eliminate or prevent primary cancer / tumor, but also to elicit, induce, enhance or promote an effective immune response against recurrent / secondary cancer / tumor that may arise from the primary cancer / tumor.

[0013] It should be noted that while the present disclosure is presented in the context of constructs for use, the present disclosure further extends to:

[0014] (i) methods of using the disclosed constructs to treat recurrent / secondary tumors / cancers or to elicit, induce, enhance or promote an immune response effective against recurrent / secondary cancers / tumors; and

[0015] (ii) Use of the disclosed constructs in the preparation of a medicament for treating recurrent / secondary tumors / cancers or for eliciting, inducing, enhancing or promoting an effective immune response against recurrent / secondary cancers / tumors.

[0016] In the context of the present disclosure, the terms (primary and / or recurrent / secondary) cancer / (primary and / or recurrent / secondary) tumor are interchangeable—cancer is a disease characterized by abnormally proliferating (cancerous) cells, and a tumor is a collection or mass of these cells. Therefore, the term "recurrent / secondary cancer" or "recurrent / secondary tumor" includes cancers (or tumors) associated with primary cancers (or tumors). Recurrent / secondary cancers may be the result of regrowth of primary cancers, or the recurrence of cancer stem cells. In this regard, recurrent / secondary cancers may represent the same cancer type as primary cancers, and may include the same cancer cells / same cancer cell types (e.g., cells of the same type, morphology, and / or with the same tumor markers). Primary and recurrent / secondary cancers may be located in the same or different tissues. Recurrent / secondary cancers may occur at a certain time after the occurrence of primary cancers, or at a certain time after the primary cancers are treated, prevented, or cured.

[0017] An immune response that is "effective against recurrent / secondary tumors (or cancers)" includes antibodies or cell-mediated responses that promote the clearance or removal of recurrent / secondary cancer cells and / or recurrent / secondary tumors from tissues. Without being bound by theory, it is suggested that when a recurrent / secondary cancer / tumor occurs in a subject who has been treated for a primary cancer / tumor, an immune response can be induced (via immunological memory) using the constructs of the present disclosure. The immune response may include measurable changes (including but not limited to cell-mediated responses, humoral responses, cytokine responses, chemokine responses) in at least one cell of the immune system, or at least one cell type, or at least one endocrine pathway, or at least one exocrine pathway. Thus, the constructs of the present disclosure provide an option to treat primary cancers without the need to re-administer the construct to the subject (or any other treatment option (e.g., chemotherapy)), but rely on an induced or elicited immune response (induced / elicited due to initial treatment with the construct) to promote treatment, cure, and / or clearance of any recurrent / secondary tumors / cancers that may be caused by the primary tumor / cancer.

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

[0019] Thus, 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 having or being predisposed / susceptible to a primary cancer / tumor.

[0020] In addition, the present disclosure provides a method of establishing an anti-recurrent / secondary cancer immune response or an anti-recurrent / secondary tumor immune response in a subject, wherein the recurrent / secondary cancer / tumor may be caused by a primary cancer, the method comprising treating the primary cancer / tumor with a construct of the present disclosure, wherein treating the primary cancer / tumor with the construct of the present disclosure induces, establishes or elicits an effective immune response against any recurrent / secondary cancer / tumor that may be caused by the (treated) primary cancer. In one teaching, the methods of the present disclosure provide methods for (i) treating or preventing recurrent / secondary cancers / tumors and / or (ii) enhancing, eliciting or inducing an immune response against recurrent / secondary cancers / tumors, the methods comprising administering a construct of the present disclosure (optionally packaged in a viral vector) to a patient suffering from a primary cancer to treat or prevent the primary cancer, and thereby prevent or treat any recurrent / secondary cancers / tumors that may arise from the primary cancer being treated, and / or enhancing, eliciting or inducing an immune response against recurrent / secondary cancers / tumors that may effectively eliminate, treat or prevent any recurrent / secondary cancers / tumors that may arise from the primary cancer being treated.

[0021] The present disclosure further provides the use of the construct of the present disclosure in the preparation of a medicament for establishing, enhancing or eliciting an anti-recurrent / secondary cancer immune response or an anti-recurrent / secondary tumor immune response.

[0022] The subject to be administered the construct of the present disclosure may include a human or animal subject suffering from or having a tendency to suffer from or be susceptible to primary cancer. In these subjects, any of the disclosed constructs can be used to treat, remove or prevent primary cancer. As mentioned above, it has been shown that initial treatment (using the construct) establishes or triggers an immune response so that if recurrent / secondary cancer occurs, a protective anti-tumor / cancer immune response is induced, wherein the immune response cures or removes recurrent / secondary cancer / tumor. This phenomenon is described herein as immune memory, and (without wishing to be bound by theory) it is suggested to use the construct to treat or prevent primary cancer, induce or trigger immune memory (for the cancer), so that if recurrent / secondary cancer or tumor occurs, the immune response is activated to promote the removal and / or treatment of the recurrent / secondary cancer / tumor.

[0023] The remainder of this disclosure will describe the features of the constructs - it should be understood that any of these constructs are used in the various uses, compositions of use, methods and medicaments described herein. Therefore, when the specification refers to some product (e.g., construct or functional nucleic acid) "for use" - this should be interpreted as not only related products for use in treating or preventing recurrent / secondary cancer (by enhancing, inducing or eliciting anti-recurrent / secondary cancer / tumor immune responses), but also related products for use in the methods of this disclosure or for use in the manufacture of medicaments described in this disclosure.

[0024] The construct of the present disclosure may include an unfavorable payload. In the context of the present disclosure, the term "unfavorable payload" refers to an element (e.g., gene or transgenic) that is expressible in a target cell and has a negative impact on the cell. The negative impact may be, for example, a negative impact on cell health or vigor and / or cell division, proliferation and / or differentiation ability. The adverse impact may be achieved directly (e.g., a gene or transgenic encoding a substance that is harmful to the cell, such as a pro-apoptotic gene or suicide gene) or indirectly (e.g., a gene or transgenic encoding a substance that recruits external factors that cause a harmful effect on the cell).

[0025] In view of the above, constructs for the various uses, methods and medicaments described herein may comprise one or more (i.e., one, two, three, four or more) of any of the following:

[0026] (i) suicide genes (e.g., genes that express or encode proteins that cause cell death;

[0027] (ii) a suicide gene encoding a protein capable of converting an inactive prodrug into a cytotoxic drug;

[0028] (iii) elements that stimulate immune responses that have negative effects on target cells;

[0029] (iv) proteins that stimulate an immune response leading to activation of cytotoxic immune cells;

[0030] (v) chemokines;

[0031] (vi) cytokines (including pro-inflammatory cytokines);

[0032] (vii) antibodies or antigen-binding fragments thereof;

[0033] (viii) immunomodulatory proteins;

[0034] (ix) IL-12;

[0035] (x) IL-10;

[0036] (xi) IL-2;

[0037] (xii) IFN-α;

[0038] (xiii) GM-CSF;

[0039] (xiv) cytotoxic substances;

[0040] (xv) herpes simplex virus thymidine kinase (HSV-TK); and

[0041] (xvi) Cytosine deaminase (CD).

[0042] In one teaching, the adverse payload can include IL-12.

[0043] Without wishing to be bound by theory, IL-12 has anti-cancer activity and can induce IFN-γ production by resting and / or activated CD4+ T cells, CD8+ T cells, and natural killer (NK) cells, as well as enhance the proliferation of activated T and NK cells, increase the lytic activity of NK / lymphokine-activated killer cells, and promote specific cytotoxic T lymphocyte (CTL) responses. Although IL-12 is toxic, this particular payload can be used as a targeted therapy in which expression is controlled to occur only in specific target cells, such as specific cancer cells or primary cancer cells.

[0044] Thus, the present disclosure provides a construct comprising an element capable of expressing IL-12 in a cell for initiating, inducing, enhancing or promoting an immune response effective against a recurrent / secondary cancer / tumor. The cell may be a cancer cell. The cell may be a primary cancer cell, wherein the primary cancer cell is associated with a recurrent / secondary cancer against which the immune response is effective.

[0045] In further teachings, the undesirable payload can include or further include a suicide gene.

[0046] The undesirable payload may include or further include herpes simplex virus thymidine kinase (HSV-TK).

[0047] The adverse payloads may include any two of the adverse payloads listed / described above.

[0048] In one example, constructs for various uses described herein may include elements capable of expressing cytokines (e.g., IL12) and suicide genes (e.g., HSV-TK) in target cells. Likewise, the cell may be a cancer cell. The cell may be a primary cancer cell, wherein the primary cancer cell is associated with a recurrent / secondary cancer for which an immune response is effective.

[0049] In view of the above, the present disclosure provides a construct comprising elements capable of expressing IL-12 and HSV-TK in cells for initiating, inducing, enhancing or promoting an immune response effective against recurrent / secondary cancers / tumors. The cell may be a cancer cell. The cell may be a primary cancer cell, wherein the primary cancer cell is associated with a recurrent / secondary cancer against which the immune response is effective.

[0050] When the construct comprises a suicide gene, the construct can be used in combination with a prodrug. For example, when the suicide gene comprises HSV-TK, the construct can be administered in combination with ganciclovir (and / or its analogs, such as acyclovir and valacyclovir). When the suicide gene comprises cytosine deaminase, the construct can be administered in combination with 5-fluorocytosine (5FC).

[0051] The construct for use in any use, method or medicament of the present disclosure may further comprise a super-enhancer element.

[0052] Super-enhancer elements can be activated by one or more transcription factors and can comprise two or more enhancer sequences from different genomic loci, each of which comprises a binding site for the transcription factor.

[0053] In another teaching, the synthetic super enhancer used in the construct of the present invention can be activated by one or more transcription factors, wherein the synthetic super enhancer comprises two or more enhancer sequences from different genomic loci, wherein each enhancer sequence comprises a binding site for the transcription factor and 20 to 400 nucleotides upstream and / or downstream of the transcription factor binding site present in its genomic locus.

[0054] The term "synthetic" may include super-enhancer constructs in which two or more enhancer sequences (usually located at different genomic loci) are separated and combined in a new construct. Thus, synthetic super-enhancers may not exist in nature and may be described as artificial or "manufactured". Thus, disclosed herein is the use of constructs activated by one or more transcription factors, wherein in addition to any adverse payload, the construct also comprises two or more enhancer sequences from different genomic loci, wherein each enhancer sequence comprises a binding site for a transcription factor. For convenience, the term "synthetic super-enhancer" will be used to cover any super-enhancer / construct described herein.

[0055] Further provided are functional nucleic acids for use encoding any construct described herein. Therefore, the term "construct" as used herein refers to a product comprising multiple elements, which are configured to be expressed in cells (e.g., cancer cells, including, e.g., primary cancer cells). Therefore, the present disclosure provides functional nucleic acid molecules comprising transgenics, which themselves comprise the unfavorable payloads described herein. The transgenics may be operably linked to a promoter designed to ensure that the transgenics are expressed in target cells. Therefore, in one teaching, the transgenics may be operably linked to a synthetic super enhancer of the type described herein.

[0056] In one teaching, the present disclosure provides a functional nucleic acid molecule comprising a super enhancer construct as described herein, which is operably connected to a transgene encoding an unfavorable payload for inducing an anti-tumor immune response. Similarly, the payload encoded by the transgene may include a cytokine (e.g., IL12) and a suicide gene (e.g., HSK-TK). The functional nucleic acid may be used in combination with a prodrug as a substrate of the suicide gene product.

[0057] According to another aspect, a functional nucleic acid molecule for use is provided, the 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 an unfavorable payload, wherein the payload is expressed when the synthetic super enhancer is activated by the transcription factor. In a further teaching, the use of a functional nucleic acid molecule is provided, the 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 an unfavorable payload, wherein the payload is expressed when the super enhancer construct is activated by the transcription factor.

[0058] According to another aspect, the construct for the purposes described herein may comprise a synthetic super enhancer activated by one or more transcription factors of the SOX family, wherein the synthetic super enhancer comprises two or more enhancer sequences and at least one SOX motif and / or SOX dimer motif. In one teaching, a super enhancer construct activated by one or more transcription factors of the SOX family is provided, wherein the super enhancer construct comprises two or more enhancer sequences and at least one SOX motif and / or SOX dimer motif.

[0059] According to another aspect, a construct for any use described in the present disclosure may comprise a synthetic super enhancer activated by SOX2, wherein the synthetic super enhancer comprises four enhancer sequences from different genomic loci, and wherein each enhancer sequence comprises at least one SOX dimer motif. In one teaching, a construct of the present disclosure may comprise a super enhancer construct activated by SOX2, wherein the super enhancer construct comprises four enhancer sequences from different genomic loci, and wherein each enhancer sequence comprises at least one SOX dimer motif.

[0060] According to another aspect, the construct of the present disclosure may be provided in the form of a vector.

[0061] According to another aspect, the present disclosure provides a composition for use, such as a pharmaceutically acceptable composition (optionally comprising a pharmaceutically acceptable excipient), wherein the composition comprises any construct described herein.

[0062] In one teaching, the present disclosure provides a method for treating recurrent / secondary glioblastoma, the method comprising administering a functional nucleic acid molecule and an inactive prodrug to a patient suffering from or predisposed to / susceptible to a primary glioblastoma to treat the primary glioblastoma and induce or elicit an effective immune response against any recurrent / secondary glioblastoma that may arise from the primary glioblastoma,

[0063] 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 an inactive prodrug into a cytotoxic drug, and

[0064] The suicide gene is expressed in glioblastoma cells upon activation of a synthetic super-enhancer by the SOX transcription factor.

[0065] In one teaching, a method of treating recurrent / secondary glioblastoma is provided, the method comprising administering a functional nucleic acid molecule and an inactive prodrug to a patient suffering from or predisposed to / susceptible to a primary glioblastoma to treat the primary glioblastoma and induce or elicit an effective immune response against any recurrent / secondary glioblastoma that may arise from the primary glioblastoma,

[0066] wherein the functional nucleic acid comprises a super enhancer construct activated by a SOX transcription factor and a suicide gene encoding a protein capable of converting an inactive prodrug into a cytotoxic drug, and

[0067] Wherein the suicide gene is expressed in glioblastoma cells upon activation of the super-enhancer construct by the SOX transcription factor. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] Figure 1 : Candidate full-length enhancers identified from SOX2 ChIP-Seq peaks can act synergistically and have selective activity. (A) Schematic diagram of constructs driven by mCMV and candidate enhancers, illustrated with candidate enhancer 270. Activity of 14 candidate enhancers in GSC7 (B) or G328 (C) when located upstream of mCMV; full-length CMV promoter is provided as a positive reference control. (D) Activity of candidate enhancers in HEK293 cells. (E) Schematic diagram of enhancer 270 clustering. (F) Enhancer activity is highest when 4x enhancers 270 are clustered together. (G) Activity of clustered enhancer constructs in HEK293 cells. (H) Overview of selectivity in differentiating GSCs. (I) Flow cytometry of SSE-T4 activity (denoted as "4xTop4" in Figure K). (J) Live cell images of activity in SSE-T4 (also referred to herein as SSE-4) in cells scored in Figure (I). (K) Schematic diagram of construct design for testing various enhancer combinations. (L) Relative activity of constructs from panel K in luciferase assay in GSC7 (not to scale). Paired two-way ANOVA, * = p value ≤ 0.05, ** = p value ≤ 0.01. Each point represents one biological replicate.

[0069] Figure 2 : Functional enhancer screening of arrayed plasmid libraries (~4000 plasmids) using a plate-based luciferase reporter assay. (A) Experimental overview of enhancer screening. (B) Results of enhancer screening. (C) Validation of 135 hits initially screened in the Nanoluc DLR assay. (D) Most hits (>10 greater than mCMV) are located within intronic regions. (E) Percentage of mNGreen+ cells for the top 17 hits in GSC7, (F) GSC328, (G) HEK293, (H) huFb710. (I) MFI of mNGreen+ cells in GSC7, (J) GSC328, (K) HEK293, and (L) huFb170.

[0070] Figure 3: Comparison of synthetic super-enhancers with restriction enzyme adapter cloning sites removed. (A) Design outline of second generation synthetic super-enhancers—C1 represents 2a generation (974 bp) and Gb1 represents 2b generation (640 bp); A = 20 bp adapter, adjacent white box = 90 bp spacer; n = 3 biological replicates. Note: The apparent difference in size is due to the 6×4 bp cloning sites, which were not considered in this protocol for simplicity. (B) Nanoglo DLR assay comparing 2a and 2b generation synthetic super-enhancers (with and without cloning site / adaptor removal, respectively). (C) Percentage of mNGreen+ cells for 2a and 2b generation synthetic super-enhancers. (D) MFI of mNGreen+ cells in 2a and 2b generation synthetic super-enhancers. (E) Representative live cell images of 2a and 2b generation synthetic super-enhancers; 20× magnification, scale bar = 50 μm.

[0071] Figure 4 : SSE-7 is selective for GSCs and forebrain neural stem cells (NSCs). (A) Schematic design of the four-part combination including SSEs (SSE-1 to SSE-7; numbers on the left). (B) Flow cytometry confirmed that these all have similar levels of activity as CMV. (C) In HEK293 cells, all SSEs are inactive. (D) In ​​human fibroblasts (huFb170), all SSEs are inactive. (E) Live cell images of SSE-7 and controls during astrocyte differentiation using 5% FCS. (F) Percentage of mNGreen+ cells for SSE-7 and controls in 5% FCS. (G) MFI of mNGreen+ cells normalized to CMV-expressed SSE-7 at day 0. (H) Overview of the experiment to evaluate SSE-7 in zebrafish embryos and larvae. (J) Injection controls. (I) Schematic diagram of the constructs used in this experiment. (K) Activity of 2a generation plasmids C1, C3, C5 and C7 at 24 and 48 hours post fertilization (hpf) (Note: these correspond to the latter derivatives: SSE-1, 3, 5 and 7, respectively, and contain adapter sequences).

[0072] Figure 5: Flow cytometry and western blot results of SSE-7 driving expression of multiple proteins (a) Schematic diagram of the expression cassette, indicating that SSE-7 expresses both mCherry and HSV-TKv5 in a polycistronic transcript (including the P2A self-cleaving peptide sequence). (b) Flow cytometry data showing mCherry expression levels after delivery of the nucleic acid using viral delivery (AAV). (c) Quantitative flow cytometry results to score the percentage of cells expressing positive mCherry, and (d, e, and f) show the gating strategy. (g) Western blot confirms HSV-TK protein expression (detected using a fused v5 C-terminal epitope tag). (h) Quantitative western blot results in (g).

[0073] Figure 6 : Cytotoxicity results of MTT assay and live Incucyte imaging. (a) Changes in the confluence of human GSC7 cells transduced or not transduced with CMV or SSE-7 driven TKv5-P2A-mCherry over time in the presence of 200 μM GCV, 0.2% DMSO (vector control) or 20% DMSO (positive control). (b) Summary of MTT assay data in GSC7 cells under the described conditions. Each point represents a biological replicate. MTT values ​​are normalized to vector control. Error bars represent SD (N=3). (c) Live images of GSC7 cells on days 3 and 8, showing cell number and morphological changes in cells transduced with constructs in the presence of 200 μM GCV. (d) Live images of huFB170 cells on day 17, showing cell number and morphological changes in cells transduced with constructs in the presence of 200 μM GCV. (e) Summary of MTT assay data in huFB170 cells under the described conditions. Each point represents a biological replicate. MTT values ​​were normalized to vehicle control (N=1).

[0074] Figure 7 : Activity of SSE-7 in a range of GSCs. (A) Schematic representation of the expression cassette. (B) Experimental summary, (C) Results of percentage of mCherry expression in individual cells in various cell lines tested. GSC7 is the positive control cell line tested (from which the fragments were initially screened). E17, E28 (formerly G328), E21, E31, E34 refer to five additional patient-derived glioblastoma cell lines.

[0075] Figure 8: The SOX dimer motif is a major contributor to activity and is bound by SOX2 and SOX9. (A) SOX dimer motifs identified by MEME in our dataset enriched in the top 32 active fragments. (B) Dimeric SOXE motif from the TRANSFAC database (Huang et al., 2015). (C) The strongest motif found among known motifs by HOMER. (D) SOX dimer motifs found in our dataset were mapped to putative enhancers that lack transcriptional activity. (E) SOX dimer motifs from (A) were mapped to the top 51 positive hits from the first generation screen, confirming that this motif occurs at a higher frequency. The activity of the fragments is shown on the left. (F) DNA was PCR amplified with biotinylated primers and incubated with nuclear extracts. The pulled down eluate was analyzed by mass spectrometry to identify bound proteins. (G) Summary of proteins pulled down by ID1101 (top circles represent all human transcription factors). SOX2 is a common target for interaction with ID1101 in both cell lines. Among the 11 common hits across GSC7 and GSC328, there are three proteins of interest (NOC2L and DMAP1, both of which are components of the histone acetyltransferase complex; TFIID, a key component of the basal transcription machinery); several SOX family member proteins were also isolated. SOX2 selectively interacts with ID1101 in (H) GSC7 and (I) GSC328. (J) SOX dimer motif variants (S9D1-10) of the wild-type (WT) sequence used to study activity. The WT sequence is included as SEQ ID NO:83, S9 D1-S9D2 are included as SEQ ID NO:84-85, and S9D6-S9D9 are included as SEQ ID NO:86-89. (K) Activity of SOX dimer motif mutants in GSC7 and (L) GSC328. (M) SOX9 selectively interacts with ID1101 and ID2904. Sequence 599 is a random DNA negative control.

[0076] Fig. 9 : A second generation screen was performed for enhancers enriched in the SOX dimer motif with putative target genes expressed in association with brain or glioma. (A) Selection of candidate enhancers for the second generation screen. (B) SOX dimer motif. (C) First and (D) second screens to identify active enhancers based on the SOX dimer motif and candidate target genes. (E) Schematic diagram of second generation SSE assembly and subsequent measurement using reporter gene assays. (F) Screening of 209 second generation SSEs in (F) GSC7 and (G) huFb170.

[0077] Fig.10: Significant survival and tumor clearance were observed with an exemplary SSE-7 with dual payloads. Proof of concept for selective clearance of tumor cells in vivo was demonstrated by selective expression of cytotoxic and immunomodulatory payloads by GBM stem cells. (A) Schematic diagram of an AAV design in which SSE-7 drives dual expression of a cytotoxic enzyme / prodrug payload (HSV-TK) and the immunostimulatory cytokine IL-12. These were produced on a single cistron controlled by SSE-7 (via P2A), ensuring combined expression. (B) Kaplan-Meier survival curves demonstrate the significant survival advantage of gene therapy using SSE-7 and the long-term viability and lack of toxicity in treated mice. (C and D) Bioluminescent imaging of live mice enables tracking of tumor growth. (C) In an immunocompetent GBM mouse model, tumors grow rapidly and mice are sacrificed within 3-4 weeks. (D) If tumors were treated by direct injection of AAV with an SSE-driven payload, tumors completely disappeared within a few weeks, with all untreated control mice shown in (C) succumbing to large tumor masses with no detectable tumor cells >5 weeks later.

[0078] Fig.11 : SSE activity does not require the mCMV promoter. (A) Schematic representation of the vector design used to explore the requirement for the mCMV promoter linked to SSE, where the mCMV was replaced by a random DNA sequence. (B) Flow cytometric analysis of SSE with and without mCMV confirmed that the SSE remained functional and highly active. (C) Schematic representation of the vector design used to explore the requirement for a random DNA sequence, used as a spacer to position the SSE upstream of the transcription start site. (D) Flow cytometric activity analysis confirmed that the SSE still functioned without the additional random sequence for positioning, but at reduced levels of activity.

[0079] Fig.12 : The variability of SSE-7 activity between patient GSCs is correlated with the levels of neuronal differentiation markers such as NEUROG2. The SSE constructs disclosed herein are highly specific for immature GBM stem-like cells. As cells begin to enter alternative differentiation pathways, such as NEUROG2-driven neuronal differentiation or SOX10-driven oligodendrocyte differentiation, the activity of SSE decreases. (A) Volcano plot shows differentially expressed genes identified by RNA-seq profiles of SSE-7-mCherry reporter high and low cells. Lower expressing cells have abundant expression of key transcription factors that mark the transition from a stem cell state to differentiation. In contrast, the highest expression has increased levels of SOX1, which is most closely associated with SOX2. (B) Heat map of RNA-seq gene expression patterns of the top 50 differentially expressed genes from (A). Arrows indicate NEUROG2.

[0080] Fig.13 A series of second-generation SSEs were shown to be active in mouse GSCs. Flow cytometry assessment of the activity of SSEs containing enhancer fragments rich in SOX motifs and SOX dimer motifs confirmed that these could drive high levels of expression in GSCs—comparable to full-length CMV.

[0081] Fig.14 : Exemplary SSE-7 also has good activity when used with lentivirus. Flow cytometry quantifies the levels of the mCherry reporter in GSCs transduced with lentivirus carrying the SSE-7-mCherry reporter. The high levels of expression achieved with lentivirus and SSE-7 demonstrate that a wide range of viral vectors can be used to deliver transgenes. Two different GSC patient cell lines, E31 and E55, were used in (A) and (B), respectively.

[0082] Fig.15 : Exemplary constructs for treating or preventing recurrent / secondary cancers packaged in AAV1 vectors for administration / delivery.

[0083] Fig.16 : Rechallenging tumor cells in treated mice reveals immune memory.

[0084] Fig.17 : Evaluation of the ability of a panel of AAV serotypes to transduce human GSC models (seven different patients) gave priority to AAV-1. AAV-2 or AAV-5 represented a suitable alternative contingency. In vitro transduction efficiency was determined by determining the percentage of cells expressing GFP 4 days after AAV transduction. Mean values ​​of 3 biological replicates (*2 biological replicates). DETAILED DESCRIPTION

[0085] The present disclosure provides constructs:

[0086] (i) for the treatment, prevention, elimination and / or cure of recurrent / secondary cancer;

[0087] (ii) for the treatment, prevention, elimination and / or cure of recurrent / secondary cancer in subjects undergoing treatment for primary cancer;

[0088] (iii) for enhancing, eliciting or inducing immune responses against recurrent / secondary cancers;

[0089] (iv) for enhancing, inducing or inducing resistance to recurrence in subjects undergoing treatment for primary cancer

[0090] / Secondary cancer immune response;

[0091] (v) methods for treating, preventing, eliminating and / or curing recurrent / secondary cancers;

[0092] (vi) methods for treating, preventing, eliminating and / or curing recurrent / secondary cancer in a subject who is undergoing treatment for a primary cancer;

[0093] (vii) methods for enhancing, eliciting or inducing immune responses against recurrent / secondary cancers;

[0094] (viii) for enhancing, inducing or inducing resistance to recurrence in a subject undergoing treatment for primary cancer

[0095] / Methods for secondary cancer immune responses;

[0096] (ix) for the manufacture of a medicament for treating, preventing, eliminating and / or curing recurrent / secondary cancer;

[0097] (x) for the manufacture of a medicament for treating, preventing, eliminating and / or curing recurrent / secondary cancer in a subject who is undergoing treatment for a primary cancer;

[0098] (xi) for the manufacture of a medicament for enhancing, eliciting or inducing an immune response against recurrent / secondary cancer;

[0099] (xii) for the manufacture of a medicament for enhancing, eliciting or inducing an immune response against recurrent / secondary cancer in a subject being treated for primary cancer.

[0100] The present disclosure provides for use new (synthetic) super enhancers (SSE) and transgene delivery, such as therapeutic and adverse payloads. Based on advances in knowledge of cell type-specific gene regulation, the inventors believe that it may be possible to generate SSEs by artificially (or synthetically) assembling enhancers that retain core functional elements and transcription factor motifs within full-length enhancers. This may include utilizing natural and functional flanking sequences around direct transcription factor motifs in these enhancers. This strategy aims to retain the natural local transcription factor (TF) binding environment (low affinity and appropriate spacing) of key cell type-specific TFs, but promote high concentrations of TF binding to trigger strong transcription, i.e., capture the appropriate syntax (spacing, affinity, and direction of co-binding proteins) of TF motifs within cell type-specific enhancers, but enhance transcriptional activity by combining into multi-part arrays. As described herein, it was found that the use of combinatorial assembly of enhancers with functional annotations to construct SSEs produced strong and highly cell type-selective regulatory elements that will have many applications, most notably in gene therapy.

[0101] Synthetic super-enhancer

[0102] The present disclosure provides a construct for use comprising a super enhancer activated by one or more transcription factors, wherein the super enhancer comprises two or more enhancer sequences from different genomic loci, wherein each enhancer sequence comprises a binding site for a transcription factor. In another teaching, the present disclosure provides the purposes 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 from different genomic loci, wherein each enhancer sequence comprises a binding site for the transcription factor and 20 to 400 nucleotides upstream and / or downstream of the transcription factor binding site present in its genomic locus. In further teaching, the present disclosure provides the purposes of a super enhancer construct activated by one or more transcription factors, wherein the super enhancer construct comprises two or more enhancer sequences from different genomic loci, wherein each enhancer sequence comprises a binding site for a transcription factor.

[0103] As used herein, "enhancer" refers to a DNA sequence (e.g., "enhancer sequence") that binds to a protein (e.g., a transcription factor) to enhance the transcription of a gene. The term "enhancer" may include a specific short region (usually 50-1500 bp) of DNA that binds to, for example, a transcription factor. However, an enhancer sequence may further include a sequence portion that is located on the flanking or surrounding flanks of the direct transcription factor motif. Enhancers typically act as cis-regulatory elements, but may also act in trans and may activate more than one target gene. Due to DNA folding and conformation or by the promotion of multi-molecular compartments (called transcription hubs or condensates, which may be formed by phase separation), they may be located at the distal end of the transcription start site (TSS) of the gene being regulated. In contrast, a promoter is located upstream of the TSS on the same DNA sequence near the transcription start site. The promoter will be bound by RNA polymerase II to initiate transcription.

[0104] H3K27Ac is a histone modification commonly found in enhancers and can be used to predict regions of enhancer activity. Whether a sequence enhances gene transcription (and thus can be referred to as an "enhancer") can be determined using methods known in the art, most typically using a plasmid-based reporter gene assay.

[0105] Super enhancers are also involved in the activation or regulation of transcription. In nature, the term "super enhancer" refers to a sequence comprising multiple enhancer clusters, typically across a region of 5-15kb. Compared with common enhancers, these "super enhancers" are highly occupied by transcriptional coactivators (such as mediator complexes). They show greater activity than common enhancers, and often participate in driving the expression of cell type-specific genes related to maintaining cell recognition or disease states (see, for example, US2014 / 0296218 and US2014 / 0287932, which are incorporated herein by reference in their entirety). It should be understood that "super enhancers", "synthetic super enhancers" (or "SSE") or super enhancer constructs mentioned herein refer to super enhancers not present in nature, that is, artificially constructed super enhancers designed for purposes described herein, which can show features associated with natural super enhancers. Specifically, SSE is an artificial construct comprising two or more enhancer sequences (possibly from different genomic loci). Likewise, for the avoidance of doubt, the term "SSE" will be used to include any super enhancer, synthetic super enhancer, and / or super enhancer construct described herein. The SSE may be highly enriched in transcription factor motifs to stimulate transcriptional activation. This allows SSE sequences to be shorter than natural super enhancers because the high concentration of transcription factors bound to the SSE promotes transcriptional activity.

[0106] In one embodiment, the present disclosure provides one or more transgenes for use by coupling one or more transgenes to an SSE, the SSE being activated by a transcription factor (or a unique combination of transcription factors) expressed in a target cell (e.g., a cancer cell or a primary cancer cell). Thus, endogenous expression of the transcription factor in the target cell is used to initiate expression of an exogenous transgene in a highly selective manner. Preferably, the transcription factor has a cell type or lineage-specific expression pattern and is therefore minimally expressed in cells other than the target cell, i.e., it is not ubiquitously expressed. This enables cell type or cell state selective activation of the SSE and expression of the transgene, thereby minimizing off-target toxicity. In one embodiment, the transcription factor is differentially expressed in the target cell, i.e., compared to other cell types. The use of SSEs also provides high expression of the transgene, preferably approaching or equivalent to transgene expression when a strong promoter such as the full-length cytomegalovirus (CMV) promoter is used. SSEs are highly selective, strong, and can be small in size (preferably less than 500 base pairs). These provide a "switch" in gene expression for the associated open reading frame / transgene, ranging from minimal expression to very strong expression depending on the cell type identity or state.

[0107] Enhancer and super-enhancer function may involve the presence of a promoter, such as a minimal promoter (e.g., minimal CMV), which contains key regions that facilitate recruitment of the transcriptional machinery and typically contains a TSS. In an alternative embodiment, the SSE of the present disclosure may not contain a promoter and / or a minimal CMV promoter.

[0108] As used herein, the term "transcription factor" refers to a protein that binds to a regulatory element of a target gene to regulate (e.g., increase or decrease) target gene expression. Transcription factors (TFs) are generally grouped according to the type of DNA binding domain present in the TF. In eukaryotes, the most common DNA binding domains are zinc fingers, homeodomains, basic leucine zippers, and basic helix-loop-helix domains. TF paralogous genes with the same DNA binding domain generally recognize similar DNA sequences. The synthetic super enhancers described herein can be combined with one or more transcription factors, i.e., one or more transcription factors bind to SSE, resulting in activation. Activation occurs through the recruitment of RNA polymerase II, thereby activating the expression of related genes (i.e., the transgene), and enhancing transcription to a level above that expressed only by the promoter. The combination of the transcription factor with SSE may include related transcription and chromatin regulatory mechanisms.

[0109] In one embodiment, the transcription factor is a developmental or stem cell-related transcription factor. These types of transcription factors are associated with high levels of activation in stem cells or cell development processes.

[0110] In one embodiment, the transcription factor is a transcription factor of the SOX family. The SOX (SRY-related HMG box) gene family of transcription factors includes 20 members in the human and mouse genomes, and all share a high mobility group (HMG) box domain. The SOX family members with high homology are divided into the following groups: (SoxA, SoxB1, SoxB2, SoxC, SoxD, SoxE, SoxF, SoxG and SoxH). In one embodiment, the transcription factor is SoxB1 (i.e., SOX1, SOX2, SOX3) or SoxE (i.e., SOX8, SOX9, SOX10) transcription factor.

[0111] SOX2 has emerged as a key regulator of cancer stem cells (Bulstrode et al., 2017, Gangemi et al., 2009, Guerra-Rebollo et al., 2019, Lujan et al., 2012, DK Singh et al., 2017, SK Singh et al., 2004, and Boumahdi et al., 2014). Suvà et al., 2014 reported a ChIP-seq dataset of glioblastoma stem cells (GSCs) and their differentiated progeny, and they classified the histone H3 enhancer marker (H3K27ac) and SOX2 binding that are unique to GSCs. In particular, the inventors of the present application have identified a set of candidate SOX2-regulated enhancers that function in GSCs across cell lines from different patient sources. Therefore, in one embodiment, the transcription factor is SOX2.

[0112] The transcription factor binds to a specific motif (i.e., transcription factor binding site) in the target enhancer through the DNA binding domain of the transcription factor. Typically, the transcription factor binding site sequence is 5-15 bp long.

[0113] In one embodiment, the synthetic super enhancer (SSE) comprises at least one SOX dimer motif. This motif may be important for the function of the SSE in the target cell, for example, many enhancer sequences described herein are shown to contain SOX dimer sites necessary for maintaining activity in glioblastoma stem cells. The enhancer element may contain a SOX binding site (sometimes a palindromic sequence) where the SOX transcription factor can homologously or heterodimerize. Typically, the spacing between each monomer binding motif is about 8-12 bp.

[0114] In one embodiment, the SOX dimer motif comprises SEQ ID NO: 1:

[0115] ACAAAGRGSVBYTKK

[0116] in:

[0117] 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; K represents G or T.

[0118] In another embodiment, the SOX dimer motif comprises SEQ ID NO: 2:

[0119] RRRRASARAGRRRBBHDDBWH

[0120] in:

[0121] 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; W represents A or T.

[0122] In one embodiment, the synthetic super enhancer comprises at least one SOX motif. In another embodiment, the SOX motif is a SOX2 motif.

[0123] In one embodiment, the SOX2 motif comprises SEQ ID NO:3:

[0124] WSARAGRSMYMHTBB

[0125] in:

[0126] 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; B represents C, G or T.

[0127] In one embodiment, wherein the synthetic super enhancer comprises a sequence selected from the group consisting of a SOX motif and / or a SOX dimer motif.

[0128] As explained 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 another embodiment, the synthetic super enhancer comprises two to eight enhancer sequences. In yet another embodiment, the synthetic super enhancer comprises four enhancer sequences.

[0129] Enhancer elements can be identified using techniques known in the art. For example, the methods described herein use ChIP-seq data sets of specific cell types to identify enhancers bound by the transcription factor of interest, or use chromatin accessibility assays (e.g., ATAC-seq or MNase maps). Publicly available resources facilitate genome-wide identification of active enhancers in various organisms and cell types, such as ENCODE or The Cancer Genome Atlas (TCGA) data sets.

[0130] Preferably, the enhancer sequences present in the SSE do not all have the same sequence (i.e., the SSE is not a concatemer of the same enhancer element). Thus, in one embodiment, the SSE comprises at least two different enhancer sequences. In one embodiment, each enhancer sequence has a different sequence. In one embodiment, each enhancer sequence is activated by the same transcription factor.

[0131] The data provided herein show that assembling enhancers from different genomic loci into a 4-part array (about 640bp) surprisingly leads to an increase in their activity by multiple orders of magnitude without compromising selectivity. Therefore, each enhancer sequence used in SSE may come from different genes. In another embodiment, each enhancer sequence comes from different genomic loci. These enhancer sequences are still preferably activated by a common transcription factor or a group of cell type-related transcription factors. In one embodiment, each enhancer sequence is activated by the transcription factor and comes from different genomic loci. In one embodiment, the synthetic super enhancer comprises two or more (e.g., four) enhancer sequences from different genomic loci.

[0132] In one embodiment, each enhancer sequence comprises the binding site of the transcription factor and 20 to 400 nucleotides upstream and / or downstream of the transcription factor binding site present in its genomic locus. This allows the surrounding environment of the transcription factor binding site (i.e., directly upstream and / or downstream) to be included in the enhancer sequence, thereby retaining the enhancer function. The upstream and / or downstream sequences that are usually present in the natural enhancer sequence and retain the spacing, order, direction and / or affinity may help to recruit synergistic transcription factors and cofactors. In addition, the sequence environment around the transcription factor binding motif may help to bind and synergize to improve transcription efficiency. Those skilled in the art can identify / obtain the sequence of 20 to 400 nucleotides upstream and / or downstream of the target binding site from publicly available genomic databases. The technician can determine the transcription factor activity by functional analysis (such as reporter gene assays as described herein or other methods widely used in the art). In one embodiment, each enhancer sequence comprises the binding site of the transcription factor and 20 to 400 nucleotides upstream and / or downstream of the transcription factor binding site present in its genomic locus, wherein the upstream and / or downstream sequences are sufficient to ensure that the enhancer retains its function. In another embodiment, the enhancer sequence comprises 20 to 400 nucleotides upstream and / or downstream of the transcription factor binding site, such as 20 to 300 nucleotides upstream and / or downstream of the transcription factor binding site, or preferably 20 to 200 nucleotides upstream and / or downstream of the transcription factor binding site. In another embodiment, the enhancer sequence comprises 50 to 400 nucleotides upstream and / or downstream of the transcription factor binding site, such as 50 to 300 nucleotides upstream and / or downstream of the transcription factor binding site, or preferably 50 to 200 nucleotides upstream and / or downstream of the transcription factor binding site. In another embodiment, the enhancer sequence comprises 80 to 400 nucleotides upstream and / or downstream of the transcription factor binding site, such as 80 to 300 nucleotides upstream and / or downstream of the transcription factor binding site, or preferably 80 to 200 nucleotides upstream and / or downstream of the transcription factor binding site.

[0133] In one embodiment, each enhancer sequence in 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 another embodiment, each enhancer sequence in the SSE is less than 300 nucleotides in length. In one embodiment, each enhancer sequence in the synthetic super enhancer is 20 to 500 nucleotides in length, such as 50 to 250 nucleotides in length or 100 to 200 nucleotides in length.

[0134] It should be understood that these embodiments can be combined. Therefore, in another embodiment, the enhancer sequence comprises 20 to 400 nucleotides upstream and / or downstream of the transcription factor binding site, wherein the enhancer sequence length is less than 500 nucleotides. In another embodiment, the enhancer sequence comprises 20 to 200 nucleotides upstream and / or downstream of the transcription factor binding site, wherein the enhancer sequence length is less than 300 nucleotides.

[0135] In another embodiment, the length of each enhancer sequence in the SSE is about 160 nucleotides. In particular, 160 base pairs (bp) are selected because this provides sufficient size for nucleosome binding and is considered an important feature of grammatical evolution within functional enhancers. In addition, 160 bp is a convenient size for synthesizing a pooled oligonucleotide library that incorporates 20 bp ends for subsequent construction of an arrayed plasmid library.

[0136] In one embodiment, the synthetic super enhancer is less than 2000 nucleotides in length. In another 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 another 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 640 nucleotides in length.

[0137] The motifs described herein may be present in one or more enhancers of the SSE (see, e.g., Figure 8 E) Thus, in one embodiment, the one or more enhancers comprise a sequence selected from the group consisting of a SOX motif and / or a SOX dimer motif.

[0138] In one embodiment, each enhancer sequence in the synthetic super enhancer comprises a SOX motif, such as a SOX2 motif. In one embodiment, each enhancer sequence in the synthetic super enhancer comprises a SOX dimer motif.

[0139] In one embodiment, the synthetic super-enhancer is activated by SOX2, wherein the synthetic super-enhancer comprises four enhancer sequences from different genomic loci, and wherein each enhancer sequence comprises at least one SOX dimer motif and / or at least one SOX dimer motif. In one embodiment, the synthetic super-enhancer is activated by SOX2, wherein the synthetic super-enhancer comprises four enhancer sequences from different genomic loci, and wherein each enhancer sequence comprises at least one SOX2 motif. In one embodiment, the synthetic super-enhancer is activated by SOX2, wherein the synthetic super-enhancer comprises four enhancer sequences from different genomic loci, and wherein each enhancer sequence comprises at least one SOX dimer motif.

[0140] In one embodiment, one or more enhancer sequences comprise a sequence having at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 4-63 (Table 1). In another embodiment, one or more enhancer sequences comprise a sequence having at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 4-13 and 56-63. In yet another embodiment, one or more enhancer sequences comprise a sequence having at least 85%, 90%, 95%, 97%, 99% sequence identity or 100% identity to a sequence selected from the group consisting of SEQ ID NO: 4-63 (such as SEQ ID NO: 4-13 and 56-63). The enhancer sequences described herein (i.e., Table 1) may be modified by at least one (such as less than 20, less than 10, less than 5) nucleotide substitution, deletion or addition, wherein the variant enhancer sequence substantially retains the functional characteristics of the sequence. It should be understood that the variants of the enhancer sequences provided herein are still intended to retain the functional activity of the enhancer sequences. For example, Example 4 shows that the enhancer sequence increased activity by more than 10-fold compared to mCMV when tested using the Nanoglo DLR assay. Thus, the activity of variant enhancer sequences included in this range should not be less than 75% of the activity of the starting sequence.

[0141] In one embodiment, the synthetic super enhancer comprises one or more enhancer sequences shown in Table 1. Thus, the SSE may comprise one or more enhancer sequences selected from the group consisting of SEQ ID NO: 4-63. The data provided herein show that arranging these enhancer sequences in a multipart array can increase transcriptional activity without compromising cell specificity. In another embodiment, the synthetic super enhancer comprises 1-8, 2-6 or 3-5 enhancer sequences selected from the sequences shown in Table 1. In another embodiment, the synthetic super enhancer comprises four enhancer sequences selected from the sequences shown in Table 1.

[0142] In one embodiment, the SSE comprises one or more enhancer sequences selected from the group consisting of SEQ ID NOs: 4 to 36. In another 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).

[0143] In one embodiment, the SSE comprises one or more enhancer sequences selected from the group consisting of SEQ ID NOs: 4-13 and 54-63.

[0144] In another embodiment, the SSE comprises four enhancer sequences selected from the group consisting of SEQ ID NOs: 4-36 (such as 4-13, especially 4-8). In another embodiment, the SSE comprises four enhancer sequences selected from the group consisting of SEQ ID NOs: 37-63, preferably four enhancer sequences selected from the group consisting of SEQ ID NOs: 54-63, more preferably four enhancer sequences selected from the group consisting of SEQ ID NOs: 56-63.

[0145] The SSE may comprise a sequence as described in Table 2. In one embodiment, the synthetic super enhancer comprises a sequence having at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 64-80. In another embodiment, the sequence has at least 85%, 90%, 95%, 97%, 99% sequence identity or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 64-80. In another embodiment, the SSE comprises SEQ ID NO: 70. The sequence variants provided herein are still intended to retain the functional activity of the SSE, i.e., transgene expression after transcription factor activation of the SSE.

[0146] To compare two closely related polynucleotide sequences, the "percent sequence identity" between the first nucleotide sequence and the second nucleotide sequence can be calculated using NCBI BLAST version 2.0, using standard settings for nucleotide sequences (BLASTN). To compare two closely related polypeptide sequences, the "percent sequence identity" between the first polypeptide sequence and the second polypeptide sequence can be calculated using NCBI BLAST version 2.0, using standard settings for polypeptide sequences (BLASTN).

[0147] A polypeptide or polynucleotide sequence is said to be identical or "identical" to another polypeptide or polynucleotide sequence if they have 100% sequence identity over their entire length. The residues in the sequence are numbered from left to right, i.e., from the N- to the C-terminus for polypeptides and from the 5' to the 3' terminus for polynucleotides.

[0148] A "difference" between sequences refers to an insertion, deletion or substitution of a single amino acid residue or nucleotide in a position in the second sequence compared to the first sequence. Two polynucleotide sequences may contain one, two or more such nucleotide differences. Two polypeptide sequences may contain one, two or more such amino acid differences. An insertion, deletion or substitution in a second sequence that is identical to the first sequence (100% sequence identity) results in a decrease in the percentage of sequence identity.

[0149] Alternatively, in order to compare a first reference sequence to a second comparison sequence, the number of additions, substitutions and / or deletions made to the first sequence to produce the second sequence can be determined. An "addition" is the addition of an amino acid residue / nucleotide to the first sequence (including additions at either end of the first sequence). A "substitution" is the replacement of an amino acid residue / nucleotide in the first sequence with a different amino acid residue / nucleotide. With respect to polypeptide sequences, substitutions may be conservative or non-conservative. In the context of polynucleotide sequences, substitutions may be synonymous or non-synonymous. A "deletion" is the deletion of an amino acid residue / nucleotide from the first sequence (including deletions at either end of the first sequence).

[0150] In one embodiment, the synthetic super-enhancer additionally comprises transcriptional regulators, ie, sequences that control gene expression (or sequences encoding proteins).

[0151] According to another aspect, a synthetic super enhancer activated by one or more transcription factors of the SOX family is provided, wherein the synthetic super enhancer comprises two or more enhancer sequences and at least one SOX motif and / or SOX dimer motif. It should be understood that the embodiments of the synthetic super enhancer applicable to functional nucleic acid molecules described herein are also applicable to this aspect.

[0152] According to another aspect, a synthetic super enhancer activated by SOX2 is provided, wherein the synthetic super enhancer comprises four enhancer sequences from different genomic loci, and wherein each enhancer sequence comprises at least one SOX motif and / or SOX dimer motif.

[0153] Functional Nucleic Acid Molecules

[0154] The SSE may be present in a construct comprising additional elements that form a functional nucleic acid molecule. As described above, such functional nucleic acid molecules may be used as described herein to treat a recurrent / secondary cancer / tumor in a subject and / or to induce, elicit or enhance an anti-recurrent / secondary cancer / tumor immune response. Thus, according to another aspect, there is provided a use of a functional nucleic acid molecule comprising a synthetic super enhancer as described herein, the synthetic super enhancer being operably linked to a transgene.

[0155] According to another aspect, there is provided a 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 and an unfavorable payload, wherein the payload is expressed when the synthetic super enhancer is activated by the transcription factor.

[0156] The functional nucleic acid molecule may comprise additional components of the SSE and transgene. For example, the synthetic super enhancer may 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 another embodiment, the promoter operably linked to the super enhancer is not a minimal CMV promoter.

[0157] genetically modified

[0158] As described herein, the construct used may include an SSE for the purpose of enhancing the expression of a transgene in a target cell (e.g., a cancer cell or a primary cancer cell) - this has the advantage of ensuring robust treatment of recurrent / secondary cancers and / or establishing, eliciting or improving a robust anti-recurrent / secondary tumor / cancer immune response. In one embodiment, the transgene may be an exogenous sequence for expression in a cell. In one teaching, the transgene is a sequence encoding a product useful in biology and medicine, such as a preventive or therapeutic transgene, such as an oligonucleotide encoding a protein or non-protein. Thus, the transgene may encode a therapeutic payload. For example, a therapeutic payload may include: a therapeutic protein, such as an antigen, an antibody, a cytokine (e.g., inducing an immunogenic response), a tumor suppressor protein (e.g., a native / unmutated p53), a differentiation factor (i.e., regulating or reprogramming cell fate or recognition), or a protein having a nucleic acid editing or gene activity regulating function (e.g., DNA or RNA editing or transcriptional regulation) or otherwise changing a cell phenotype (e.g., mobility, ECM production, cell senescence or quiescence). In another embodiment, the transgene encodes a non-protein coding oligonucleotide, such as an RNA (other than an mRNA), for example, a miRNA, a siRNA, a shRNA or an lncRNA. It should be understood that a transgene encoding a non-coding RNA can be used in a gene silencing method.

[0159] In one embodiment, the construct used takes the form of a functional nucleic acid molecule comprising one or more transgenes. Thus, one skilled in the art will appreciate that different combinations of transgenes may be included.

[0160] According to another aspect, there is provided a method of expressing a transgene in a target cell, the method comprising expressing a transgene operably linked to a synthetic super enhancer as described herein, wherein one or more of the transcription factors are expressed in the target cell.

[0161] Unfavorable payload

[0162] As described herein, the transgene used in any construct for use described herein can be operably linked to the SSE and can encode an adverse payload. As used herein, the term "adverse payload" refers to a payload that has a negative effect on the target cell. The negative effect can be, for example, a negative effect on the health or vitality of the cell and / or the ability of the cell to divide. The adverse effect can be achieved directly (e.g., the transgene encodes a substance that is harmful to the cell, such as a pro-apoptotic gene or a suicide gene) or indirectly (e.g., the transgene encodes a substance that recruits an external factor that causes a deleterious effect on the cell).

[0163] The unfavorable payload can be used to stimulate an immune response that has a negative impact on target cells. For example, this reaction can change the local immune microenvironment. In one embodiment, the unfavorable payload encodes a protein that stimulates an immune response, and the immune response causes the activation of cytotoxic immune cells. In one embodiment, the unfavorable payload is selected from: chemokines, cytokines, antibodies or other immunomodulatory proteins. In another embodiment, the unfavorable payload is a proinflammatory cytokine, such as selected from the following cytokines: IL-12, IL-10, IL-2, IFN-α and GM-CSF. For example, the unfavorable payload can be a cytokine, such as IL-12 that has been shown to have anticancer activity. IL-12 causes induction of static and activated CD4+T cells, CD8+T cells and natural killer (NK) cells to produce IFN-γ, and enhances the proliferation of activated T and NK cells, increases the lytic activity of NK / lymphokine-activated killer cells, and promotes specific cytotoxic T lymphocytes (CTL) reactions. However, IL-12 cannot be administered as a systemic treatment due to excessive toxicity, therefore, targeted therapies that are expressed only within target cells would be able to deliver this payload.

[0164] An "immune response" is a measurable change in at least one cell, or at least one cell type, or at least one endocrine pathway, or at least one exocrine pathway of the immune system (including but not limited to a cell-mediated response, a humoral response, a cytokine response, a chemokine response).

[0165] "Immune cells" are defined as cells of the immune system, including but not limited to CD34+ cells, B cells, CD45+ (lymphocyte common antigen) cells, α-βT cells, cytotoxic T cells, helper T cells, plasma cells, neutrophils, monocytes, macrophages, dendritic cells, phagocytes, granulocytes, innate lymphocytes, natural killer (NK) cells and γδT cells. Typically, immune cells are classified by means of combined cell surface molecule analysis (e.g., by flow cytometry) to identify or group or cluster, thereby distinguishing immune cells into subpopulations. In the molecules and methods of the present disclosure, when the payload is an unfavorable payload, the stimulated immune response is intended to have an adverse effect on target cells. Mention of "cytotoxic immune cells" refers to immune cells that cause cell death, particularly cytotoxic T cells (also referred to as killer T cells).

[0166] In one embodiment, the adverse payload encodes a cytotoxic substance (ie, a cytotoxic payload).

[0167] In one embodiment, the unfavorable 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 provided cofactor or codrug (e.g., a prodrug) to function. The cofactor or codrug can then be converted into a cytotoxic entity by the product of the suicide gene. 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 concomitantly with a functional nucleic acid molecule or administered sequentially with a functional nucleic acid molecule.

[0168] 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 its analogs such as acyclovir and valacyclovir. In another embodiment, the suicide gene is cytosine deaminase (CD). In particular, the CD gene is used in combination with a prodrug 5-fluorocytosine (5FC).

[0169] Target cells

[0170] The constructs / functional nucleic acid molecules described herein can be used to target a variety of cell types (also referred to as "target cells"). In particular, the cells are mammalian cells, such as human cells. The present disclosure may include the presence of an unfavorable payload and is therefore suitable for targeting abnormal cells (i.e., abnormal cells, such as non-healthy cells).

[0171] The abnormal cell may be a hyperproliferative cell, i.e., a cell that proliferates abnormally excessively. In one embodiment, the abnormal cell is a cancer cell or a tumor cell. In this embodiment, the adverse payload may be an anti-cancer payload.

[0172] SOX2 may also be amplified in other abnormal cells, particularly cancer cells, as it has been identified as an oncogene. 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, as well as many other squamous cell carcinomas.

[0173] The target cell may be a primary cancer cell. The primary cancer cell may give rise to a recurrent / secondary cancer (via any of the mechanisms described herein).

[0174] In one embodiment, the target cell is a cancer cell selected from the following: glioblastoma stem cells, glioma cells, lung cancer cells (particularly squamous 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, bile duct cancer 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.

[0175] In a specific embodiment, the target cell is a glioblastoma stem cell. Glioblastoma is an invasive form of glioma, driven by the elevated levels / activity of master regulators of neural stem cell-related transcription factors. Glioblastoma stem cells (GSCs) typically have high expression or activity of key neural developmental transcription factors, including SOX2, which is a key master regulator and reprogramming factor required for neural stem cell recognition (Bulstrode et al., 2017, Gangemi et al., 2009, Guerra-Rebollo et al., 2019, Lopez-Bertoni et al., 2015 and MacLeod et al., 2019). Knockout of SOX2 in GSCs reduces their tumorigenicity (Gangemi et al., 2009), while its ectopic expression enhances the GSC state together with POU3F2, OLIG2 and SALL2 (Suvà et al., 2014). The examples presented herein demonstrate that a GSC-selective SSE remains active in an adeno-associated virus (AAV) and is used to drive expression of a cytotoxic payload to target and kill GSCs. Furthermore, the activity of the SSE is lost during GSC differentiation and is absent in HEK or fibroblasts. Thus, GSC-selective expression is retained even when the strength of the promoter is greatly increased.

[0176] Carrier

[0177] The construct used may be provided in the form of a vector comprising a synthetic super enhancer or a functional nucleic acid molecule as described herein.

[0178] The term "vector" as used herein refers to a molecule capable of transporting the functional nucleic acid molecule. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop to which additional DNA fragments can be connected. Another type of vector is a viral vector, in which additional DNA fragments can be connected to the viral genome. Some vectors can replicate autonomously in the host cell into which they are introduced (e.g., bacterial vectors with bacterial replication origins and free mammalian and yeast vectors). Other vectors (e.g., non-free mammalian vectors) can be integrated into the genome of the host cell after being introduced into the host cell, thereby replicating together with the host genome. In addition, some vectors can guide the expression of genes effectively connected thereto. This vector is referred to as a "recombinant expression vector" (or simply "expression vector") in this article. Generally, expression vectors useful in recombinant DNA technology are usually in the form of plasmids.

[0179] Exemplary expression vectors are known in the art and may include, for example, plasmid vectors, viral vectors (e.g., adenovirus, adeno-associated virus, retrovirus or lentivirus vectors), phage vectors, cosmid vectors, etc. The choice of expression vector depends on the type of host cell used and the purpose of use.

[0180] In one embodiment, the vector is a viral vector. Viral vectors, particularly those used for gene therapy applications, are well known in the art. Such viruses can be RNA and DNA viruses with single-stranded (ss) or double-stranded (ds) genomes. For example, viral vectors include but are not limited to adenoviruses, adeno-associated viruses (AAV), alphaviruses, flaviviruses, herpes simplex viruses (HSV), measles viruses, rhabdoviruses, retroviruses, slow viruses, Newcastle disease virus (NDV), poxviruses, and picornaviruses. Insertion capacity and tropism can vary, so viral vectors can be selected according to the intended application.

[0181] AAV vectors are increasingly favored as optimal gene therapy viral vectors because these vectors have improved safety. However, one limitation is their smaller cargo size. The SSE 4 partial assembly described herein is very suitable for AAV. Therefore, in one embodiment, the vector is AAV. Different AAV serotypes differ in their tropism, so the type of AAV vector used can be selected according to the type of targeted tissue. The AAV vector can be selected from many serotypes known in the art, such as any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 or AAV11, or can be a non-natural variant. In one embodiment, the AAV is selected from: AAV1, AAV2 or AAV5.

[0182] The constructs (or functional nucleic acids) for any of the uses or methods described herein may be provided in (or administered in) an AAV vector.

[0183] In one teaching, a construct (or functional nucleic acid) for any use or method described herein can be provided within (or administered using) an AAV1 vector.

[0184] Thus, the present disclosure provides an AAV1 (or AAV2, 5, or 8) vector comprising any of the constructs described herein:

[0185] (i) for the treatment, prevention, elimination and / or cure of recurrent / secondary cancer;

[0186] (ii) for the treatment, prevention, elimination and / or cure of recurrent / secondary cancer in subjects undergoing treatment for primary cancer;

[0187] (iii) for enhancing, eliciting or inducing immune responses against recurrent / secondary cancers;

[0188] (iv) for enhancing, inducing or inducing resistance to recurrence in subjects undergoing treatment for primary cancer

[0189] / Secondary cancer immune response;

[0190] (v) for use in methods of treating, preventing, eliminating and / or curing recurrent / secondary cancer;

[0191] (vi) for use in a method of treating, preventing, eliminating and / or curing recurrent / secondary cancer in a subject who is undergoing treatment for a primary cancer;

[0192] (vii) for use in methods of enhancing, eliciting or inducing an immune response against recurrent / secondary cancer;

[0193] (viii) for enhancing, inducing or inducing resistance to recurrence in a subject undergoing treatment for primary cancer

[0194] / Methods for secondary cancer immune responses;

[0195] (ix) for the manufacture of a medicament for treating, preventing, eliminating and / or curing recurrent / secondary cancer;

[0196] (x) for the manufacture of a medicament for treating, preventing, eliminating and / or curing recurrent / secondary cancer in a subject who is undergoing treatment for a primary cancer;

[0197] (xi) for the manufacture of a medicament for enhancing, eliciting or inducing an immune response against recurrent / secondary cancer;

[0198] (xii) for the manufacture of a medicament for enhancing, eliciting or inducing an immune response against recurrent / secondary cancer in a subject undergoing treatment for a primary cancer;

[0199] Wherein the construct comprises one, two or more of the following (cancer cell) adverse payloads:

[0200] (i) suicide genes (e.g., genes that express or encode proteins that cause cell death;

[0201] (ii) a suicide gene encoding a protein capable of converting an inactive prodrug into a cytotoxic drug;

[0202] (iii) elements that stimulate immune responses that have negative effects on target cells;

[0203] (iv) proteins that stimulate an immune response leading to activation of cytotoxic immune cells;

[0204] (v) chemokines;

[0205] (vi) cytokines (including pro-inflammatory cytokines);

[0206] (vii) antibodies or antigen-binding fragments thereof;

[0207] (viii) immunomodulatory proteins;

[0208] (ix) IL-12;

[0209] (x) IL-10;

[0210] (xi) IL-2;

[0211] (xii) IFN-α;

[0212] (xiii) GM-CSF;

[0213] (xiv) cytotoxic substances;

[0214] (xv) herpes simplex virus thymidine kinase (HSV-TK); and

[0215] (xvi) Cytosine deaminase (CD).

[0216] According to another aspect, a vector comprising a synthetic super enhancer as described herein, and a transgene (i.e., a payload, such as a therapeutic payload) is provided for use in any of the uses and methods described herein. As described herein, the synthetic super enhancer may be operably linked to other expression elements, such as a promoter.

[0217] Compositions and kits

[0218] The present disclosure also relates to the use of compositions comprising constructs, functional nucleic acid molecules or vectors as described herein. The compositions may include components capable of delivering the constructs or functional nucleic acid molecules by viral vectors (AAV, lentivirus, etc.) and non-viral vectors (nanoparticles, lipid particles, etc.).

[0219] According to another aspect, a use of a composition is provided, the composition comprising a synthetic super enhancer, a functional nucleic acid molecule, a construct or a vector as described herein, and an acceptable carrier. Suitable carriers are known in the art, and in certain embodiments, the selection of a carrier is based on its ability to promote transfection of target cells with one or more functional nucleic acid molecules.

[0220] According to another aspect, the present disclosure provides a composition comprising a functional nucleic acid molecule or vector as described herein for treating or preventing recurrent / secondary cancer / tumor and / or for enhancing, eliciting and / or inducing an anti-recurrent / secondary cancer / tumor immune response.

[0221] According to another aspect, there is provided the use of the construct, functional nucleic acid molecule (or vector or composition) defined herein for the preparation of a medicament for treating or preventing recurrent / secondary cancer / tumor and / or for enhancing, eliciting and / or inducing an anti-recurrent / secondary cancer / tumor immune response.

[0222] Treatment

[0223] According to another aspect, a method for (i) treating or preventing recurrent / secondary cancer / tumor or (ii) enhancing, inducing or eliciting an anti-secondary cancer / tumor immune response is provided, the method comprising administering a molecular construct (or functional nucleic acid molecule) of the present disclosure (or a vector comprising the same) to a patient, wherein the patient is a human or animal patient with a primary cancer and is at risk of developing a related recurrent / secondary cancer.

[0224] Reference to a "subject," "patient," or "individual" refers to a subject to be treated, particularly a mammalian subject. Mammalian subjects include humans, non-human primates, farm animals (such as cattle), sports animals, or pet animals, 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.

[0225] As used herein, "treating" a disease or condition refers to reducing the frequency and / or severity of at least one sign or symptom of the disease or condition experienced by a subject.

[0226] As used herein, "cancer" refers to the abnormal growth or division of cells (primary or secondary or recurrent / secondary (as described above)). Typically, cancer cells grow and / or live longer than, and out of coordination with, the normal cells and tissues around them. Cancer may be benign, pre-malignant, or malignant. Cancer may also be a primary cancer or a secondary cancer. A primary cancer is the original organ or tissue in which the cancer begins, while a secondary cancer is the result of the spread (or metastasis) of the primary cancer to another part of the body. As described above, the present disclosure provides constructs that can be used to treat primary cancers, but the constructs have been found to elicit, enhance, or induce an effective immune response to any recurrent / secondary cancer that may be caused by the primary cancer (or a certain primary cancer).

[0227] Cancer (primary, recurrent / secondary, or recurrent) 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, bronchi), bones, joints, skin (e.g., basal cell, squamous cell, meningioma), breast, reproductive 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 lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia).

[0228] In another embodiment, the (primary, recurrent / secondary or secondary) cancer is a cancer located in the brain or spinal cord (i.e., the central nervous system), such as a primary or recurrent / secondary or secondary cancer located in the central nervous system. The present disclosure has particular use in the treatment of glioblastomas, particularly recurrent / secondary glioblastomas arising from primary glioblastomas.

[0229] The functional nucleic acid molecule (or carrier or composition) can be used by any suitable delivery mode for the intended treatment, such as intravenous, intra-arterial, intracardiac, intradermal, subcutaneous, interperitoneal, intramuscular or oral. In one embodiment, the functional nucleic acid molecule, carrier or composition is systemically administered. Administration can also be directly to the disease site. Therefore, in one embodiment, the functional nucleic acid molecule, carrier or composition is topically administered, for example, directly administered to the organ or tissue, such as intratumoral administration.

[0230] The construct or functional nucleic acid of the present disclosure can be administered to a subject with a primary cancer (e.g., by direct injection into a 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, resection carries a risk of recurrence (due to residual portions of the tumor and / or residual cancer cells / cancer stem cells remaining in situ). In particular, recurrence after resection is usually caused by cancer stem cells at the edge of the resection cavity. As described above, the construct or functional nucleic acid of the present disclosure not only helps treat the primary cancer, but also establishes a level of protection against any recurrent cancer caused after resection in the subject. Again, without being bound by theory, this protection is believed to be the result of an immune response triggered and induced when recurrent / secondary cancer / tumors occur. As described herein, the construct or functional nucleic acid may comprise one, two or more unfavorable payloads; when the unfavorable payload comprises a suicide gene, such as a suicide gene encoding a protein capable of converting an inactive prodrug into a cytotoxic drug, the prodrug may be administered before, during or after treatment with the construct or functional nucleic acid.

[0231] As described above, the construct or functional nucleic acid of the present invention can be packaged in a vector (e.g., a viral vector) and administered. In one teaching, the construct or functional nucleic acid of the present invention can be packaged in an adenoviral vector and administered, such as an AAV1 vector, an AAV2 vector, an AAV5 vector, or an AAV8 vector.

[0232] In one teaching, the primary cancer is a glioblastoma. The glioblastoma can be treated with a functional nucleic acid or construct disclosed herein to reduce the size of the glioblastoma. The glioblastoma can then be surgically removed. Treatment of the primary cancer with a functional nucleic acid or construct disclosed herein establishes a level of protection against any recurrent / secondary glioblastoma in a subject, which may be caused by, for example, cancer (glioblastoma) stem cells remaining in the margin of the resection cavity.

[0233] According to another aspect, there is provided a method for treating or preventing recurrent / secondary glioblastoma that may arise from a primary glioblastoma, comprising administering to a patient suffering from a primary glioblastoma a construct / functional nucleic acid molecule disclosed herein and an inactive prodrug,

[0234] 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 an inactive prodrug into a cytotoxic drug, and

[0235] The suicide gene is expressed in glioblastoma cells upon activation of a synthetic super-enhancer by the SOX transcription factor.

[0236] In one embodiment, the inactive prodrug is administered concomitantly with or sequentially with the functional nucleic acid molecule.

[0237] It will be understood that the method of treatment will include administering a therapeutically effective amount. The term "therapeutically effective amount" refers to an amount effective to improve or treat the symptoms of a disease or condition, as well as an amount sufficient to enhance, elicit and / or induce an effective immune response to any recurrent / secondary cancers that may have arisen from the primary cancer. A therapeutically effective amount may be a "prophylactically effective amount" because prevention can be considered treatment.

[0238] It will be understood that the embodiments described herein are applicable to all aspects of the present disclosure, ie embodiments described with respect to the uses are equally applicable to the claimed methods etc.

[0239] Terms

[0240] A set of clauses defining the present disclosure and its preferred aspects are as follows:

[0241] 1. A construct for (i) treating or preventing recurrent / secondary cancer / tumor and / or (ii) enhancing, eliciting or inducing an anti-recurrent / secondary cancer / tumor immune response, the construct comprising a gene or transgene encoding an adverse payload.

[0242] 2. The construct of clause 1, wherein the gene or transgene is for expression in a target cell and the payload is detrimental to the cancer cell.

[0243] 3. The construct of clause 1 or 2, wherein the target cell is a primary cancer cell, such as a primary glioblastoma cancer cell.

[0244] 4. The construct of any of the preceding clauses, wherein the recurrent / secondary cancer is associated with a primary cancer that has been treated using the construct of claim 1 or 2.

[0245] 5. The construct of any one of the preceding claims, wherein the primary and recurrent / secondary cancer is primary or recurrent / secondary glioblastoma.

[0246] 6. The construct of any of the preceding clauses, wherein the adverse payload encodes a protein that stimulates an immune response that results in the recruitment of cytotoxic immune cells.

[0247] 7. The construct of any of the preceding clauses, wherein the adverse payload encodes a cytotoxic substance.

[0248] 8. The construct of any of the preceding clauses, wherein the unfavorable payload comprises a suicide gene encoding a protein capable of converting an inactive prodrug into a cytotoxic drug.

[0249] 9. The construct of clause 8, wherein the suicide gene is the herpes simplex virus thymidine kinase (HSV-TK) gene and the prodrug is ganciclovir, acyclovir or valacyclovir.

[0250] 10. The construct of clauses 1-5, wherein the unfavorable payload comprises an IL-12 gene.

[0251] 11. A construct for (i) treating or preventing recurrent / secondary cancer / tumor and / or (ii) enhancing, eliciting or inducing an anti-recurrent / secondary cancer / tumor immune response, the construct comprising a gene or transgene encoding IL-12 and herpes simplex virus thymidine kinase (HSV-TK).

[0252] 12. The construct of any preceding claim, wherein the construct is for use in a subject suffering from a primary cancer and wherein the primary cancer is treated with a construct as defined in any of clauses 1 to 11, wherein treatment of the primary cancer with the construct induces, elicits or enhances an anti-recurrent / secondary cancer / tumor immune response which prevents or reduces the risk of a recurrent / secondary cancer arising from the primary cancer.

[0253] 13. Use of a construct as defined in clauses 1 to 11 for the preparation of a medicament for (i) treating or preventing recurrent / secondary cancer / tumours and / or (ii) establishing, enhancing or eliciting an anti-recurrent / secondary cancer immune response or an anti-recurrent / secondary tumour immune response.

[0254] 14. The use according to clause 13, wherein the recurrent / secondary cancer is associated with a primary cancer that has been treated with the construct according to any one of clauses 1 to 11.

[0255] 15. The use of clause 13 or 14, wherein the treatment of the primary cancer with the medicament induces, triggers or enhances an anti-recurrent / secondary cancer / tumor immune response, which prevents or reduces the risk of recurrent / secondary cancer caused by the primary cancer.

[0256] 16. A method of enhancing, eliciting or establishing an anti-recurrent / secondary cancer / tumor immune response in a subject, said immune response preventing or reducing the risk of recurrent / secondary cancer caused by a primary cancer, said method comprising treating the primary cancer / tumor with a construct as defined in any of clauses 1 to 11, wherein treating the primary cancer / tumor with the construct of the present disclosure induces, establishes or elicits an immune response effective against any recurrent / secondary cancer / tumor that may be caused by the (treated) primary cancer.

[0257] 17. The method of clause 16, wherein the primary cancer is glioblastoma.

[0258] 18. The method of clause 17, wherein the secondary cancer / tumor is a recurrent / secondary glioblastoma arising from the primary glioblastoma.

[0259] The construct defined in any one of claims 1 to 11 may further comprise a synthetic super enhancer as defined in clauses 19 to 30:

[0260] 19. A synthetic super enhancer that is activated by one or more transcription factors, wherein the synthetic super enhancer comprises two or more enhancer sequences from different genomic loci, wherein each enhancer sequence comprises a binding site for the transcription factor and 20 to 400 nucleotides upstream and / or downstream of the transcription factor binding site present in its genomic locus.

[0261] 20. The synthetic super enhancer according to clause 19, wherein each of the two or more enhancer sequences within the synthetic super enhancer is less than 300 nucleotides in length, in particular 160 nucleotides in length.

[0262] 21. A synthetic super enhancer according to clause 19 or clause 20, wherein the synthetic super enhancer is less than 1200 nucleotides in length, in particular less than 700 nucleotides in length.

[0263] 22. A synthetic super enhancer according to any one of clauses 19 to 21, wherein the synthetic super enhancer comprises four enhancer sequences.

[0264] 23. A synthetic super enhancer according to any one of clauses 19 to 22, wherein the transcription factor is a transcription factor of the SOX family.

[0265] 24. A synthetic super enhancer according to any one of clauses 19 to 23, wherein the synthetic super enhancer comprises at least one SOX dimer motif.

[0266] 25. A synthetic super enhancer according to clause 24, wherein the SOX dimer motif comprises SEQ ID NO: 1.

[0267] 26. A synthetic super enhancer according to any one of clauses 19 to 23, wherein the synthetic super enhancer comprises at least one SOX motif.

[0268] 27. A synthetic super enhancer according to clause 26, wherein the SOX motif is a SOX2 motif.

[0269] 28. A synthetic super enhancer according to clause 26 or clause 27, wherein the SOX2 motif comprises SEQ ID NO:3.

[0270] 29. A synthetic super enhancer according to any one of clauses 19 to 28, wherein the synthetic super enhancer comprises one or more enhancer sequences shown in Table 1.

[0271] 30. A synthetic super enhancer according to any one of clauses 19 to 29, wherein the synthetic super enhancer comprises a sequence having at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 64-80.

[0272] 30. A synthetic super enhancer according to any one of clauses 19-30, wherein the synthetic super enhancer additionally comprises a transcriptional regulator.

[0273] A construct for use in any therapeutic use, method or medicament described herein may be further defined according to any of clauses 31-62.

[0274] 31. A functional nucleic acid molecule comprising a synthetic super enhancer according to any one of clauses 19-30, said synthetic super enhancer being operably linked to a transgene.

[0275] 32. The functional nucleic acid molecule according to clause 31, wherein the transgene encodes a therapeutic payload.

[0276] 33. The functional nucleic acid molecule according to clause 31, wherein the transgene encodes an unfavorable payload.

[0277] 34. A functional nucleic acid molecule according to clause 33, wherein the adverse payload encodes a protein that stimulates an immune response leading to the recruitment of cytotoxic immune cells.

[0278] 35. The functional nucleic acid molecule according to clause 33, wherein the adverse payload encodes a cytotoxic substance.

[0279] 36. A functional nucleic acid molecule according to any one of clauses 31 to 35, wherein the unfavorable payload is a suicide gene encoding a protein capable of converting an inactive prodrug into a cytotoxic drug.

[0280] 37. A functional nucleic acid molecule according to any one of clauses 31 to 36, wherein the synthetic super enhancer is operably linked to a promoter.

[0281] 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 an unfavorable payload, wherein the payload is expressed when the synthetic super enhancer is activated by the transcription factor.

[0282] 39. The functional nucleic acid molecule according to clause 38, wherein the transcription factor is differentially expressed in the target abnormal cells.

[0283] 40. A functional nucleic acid molecule according to clause 38 or clause 39, wherein the transcription factor is a developmental or stem cell-associated transcription factor.

[0284] 41. A functional nucleic acid molecule according to any one of clauses 38 to 40, wherein the transcription factor is a transcription factor of the SOX family.

[0285] 42. A functional nucleic acid molecule according to any one of clauses 38 to 41, which is activated by SOX2.

[0286] 43. A functional nucleic acid molecule according to any one of clauses 38 to 42, wherein the synthetic super enhancer comprises at least one SOX dimer motif.

[0287] 44. The functional nucleic acid molecule according to clause 43, wherein the SOX dimer motif comprises SEQ ID NO: 1.

[0288] 45. The functional nucleic acid molecule according to any one of clauses 38 to 42, wherein the synthetic super enhancer comprises at least one SOX motif.

[0289] 46. ​​The functional nucleic acid molecule according to clause 45, wherein the SOX motif is a SOX2 motif.

[0290] 47. A functional nucleic acid molecule according to clause 45 or clause 46, wherein the SOX2 motif comprises SEQ ID NO:3.

[0291] 48. A functional nucleic acid molecule according to any one of clauses 38 to 47, wherein the synthetic super enhancer comprises two or more enhancer sequences.

[0292] 49. A functional nucleic acid molecule according to any one of clauses 38 to 48, wherein the synthetic super enhancer comprises four enhancer sequences.

[0293] 50. The functional nucleic acid molecule according to clause 48 or clause 49, wherein each of the two or more enhancer sequences has a different sequence.

[0294] 51. A functional nucleic acid molecule according to any one of clauses 48-50, wherein each of the two or more enhancer sequences is from a different genomic locus.

[0295] 52. A 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 is less than 300 nucleotides in length, in particular about 160 nucleotides in length.

[0296] 53. A functional nucleic acid molecule according to any one of clauses 48 to 52, wherein two or more enhancer sequences within the synthetic super enhancer comprise a sequence selected from a SOX motif and / or a SOX dimer motif.

[0297] 54. A functional nucleic acid molecule according to any one of clauses 38 to 53, wherein the synthetic super enhancer comprises one or more enhancer sequences shown in Table 1.

[0298] 55. A functional nucleic acid molecule according to any one of clauses 38 to 54, wherein the synthetic super enhancer comprises a sequence having at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 64-80.

[0299] 56. A functional nucleic acid according to any one of clauses 38 to 55, wherein the synthetic super enhancer is operably linked to a promoter.

[0300] 57. A functional nucleic acid molecule according to any one of clauses 38-56, wherein the adverse payload encodes a protein that stimulates an immune response leading to the recruitment of cytotoxic immune cells.

[0301] 58. A functional nucleic acid molecule according to any one of clauses 38-56, wherein the adverse payload encodes a cytotoxic substance.

[0302] 59. A functional nucleic acid molecule according to any one of clauses 19-38, wherein the unfavorable payload is a suicide gene encoding a protein capable of converting an inactive prodrug into a cytotoxic drug.

[0303] 60. A functional nucleic acid molecule according to clause 59, wherein the suicide gene is the herpes simplex virus thymidine kinase (HSV-TK) gene, and the prodrug is ganciclovir, acyclovir or valacyclovir.

[0304] 61. A functional nucleic acid molecule according to any one of clauses 38-60, wherein the abnormal cell is a cancer cell or a tumor cell, and the adverse payload is an anti-cancer payload.

[0305] 62. The functional nucleic acid molecule according to any one of clauses 38-61, wherein the abnormal cell is a glioblastoma stem cell.

[0306] A construct for any use or method described herein may comprise a synthetic super enhancer according to any one of clauses 63 to 75

[0307] 63. A synthetic super enhancer activated by one or more transcription factors of the SOX family, wherein the synthetic super enhancer comprises two or more enhancer sequences and at least one SOX motif and / or SOX dimer motif.

[0308] 64. A synthetic super enhancer according to clause 63, wherein each of the two or more enhancer sequences has a different sequence.

[0309] 65. A synthetic super enhancer according to clause 63 or clause 64, wherein each of the two or more enhancer sequences is from a different genomic locus.

[0310] 66. A synthetic super enhancer according to any one of clauses 63 to 65, wherein the synthetic super enhancer comprises four enhancer sequences.

[0311] 67. A synthetic super enhancer according to any one of clauses 63 to 66, wherein each of the two or more enhancer sequences within the synthetic super enhancer is less than 300 nucleotides in length, in particular 160 nucleotides in length.

[0312] 68. A synthetic super enhancer according to any one of clauses 63-50, wherein two or more enhancer sequences within the synthetic super enhancer comprise a sequence selected from a SOX motif and / or a SOX dimer motif.

[0313] 69. A synthetic super enhancer according to any one of clauses 63 to 51 which is activated by SOX2.

[0314] 70. A synthetic super enhancer according to clause 69, wherein the SOX dimer motif comprises SEQ ID NO: 1.

[0315] 71. A synthetic super enhancer according to any one of clauses 63 to 68, wherein the SOX motif is a SOX2 motif.

[0316] 72. A synthetic super enhancer according to clause 71, wherein the SOX2 motif comprises SEQ ID NO:3.

[0317] 73. A synthetic super enhancer according to any one of clauses 63 to 72, wherein the synthetic super enhancer comprises one or more enhancer sequences shown in Table 1.

[0318] 74. A synthetic super enhancer according to any one of clauses 63 to 73, wherein the synthetic super enhancer comprises a sequence having at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 64-80.

[0319] 75. A synthetic super enhancer that is activated by SOX2, wherein the synthetic super enhancer comprises four enhancer sequences from different genomic loci, and wherein each enhancer sequence comprises at least one SOX dimer motif.

[0320] The construct or functional nucleic acid for any use or method described herein may be provided in the form of a vector according to clauses 76-78:

[0321] 76. A vector comprising a synthetic super enhancer according to any one of items 19-30 or 63-75, or a functional nucleic acid molecule according to any one of items 31-62.

[0322] 77. The vector according to clause 59, wherein the vector is a viral vector.

[0323] 78. A vector according to clause 59 or clause 60, wherein the vector is an adeno-associated virus (AAV).

[0324] The construct or functional nucleic acid for any use or method described herein may be provided in the form of a composition according to clauses 79-81: 79. A composition comprising a synthetic super enhancer according to any of clauses 19-30 or 63-75, a functional nucleic acid molecule according to any of clauses 31-62 or a vector according to any of clauses 76-78, and an acceptable carrier.

[0325] 80. A composition comprising a functional nucleic acid molecule according to any one of clauses 31 to 62 for use in therapy.

[0326] 81. A composition comprising a functional nucleic acid molecule according to any one of clauses 31 to 62 for use in treating cancer.

[0327] Also disclosed are teachings consistent with the following clauses: 82. A method for treating or preventing recurrent / secondary cancer caused by a primary cancer, the method comprising administering a functional nucleic acid molecule according to any one of clauses 31 to 62 or a composition according to clause 80 or 81 to a patient suffering from the primary cancer, wherein treatment of the primary cancer with the functional nucleic acid or composition enhances, triggers or induces an immune response, which has a protective effect against recurrent / secondary cancers that may be caused by the primary cancer.

[0328] 83. The method according to clause 82, wherein the primary and / or recurrent / secondary cancer is a brain cancer, such as glioblastoma.

[0329] 84. The method according to clause 82 or clause 83, wherein the functional nucleic acid molecule or composition is administered systemically.

[0330] 85. The method according to clause 82 or clause 83, wherein the functional nucleic acid molecule or composition is administered topically.

[0331] 86. A method for preventing recurrent / secondary glioblastoma comprising administering a functional nucleic acid molecule and an inactive prodrug to a patient suffering from primary glioblastoma,

[0332] 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

[0333] The suicide gene is expressed in glioblastoma cells when the synthetic super enhancer is activated by SOX transcription factor.

[0334] 87. The method according to clause 86, wherein the inactive prodrug is administered concomitantly with or sequentially with the functional nucleic acid molecule.

[0335] 88. An AAV1 vector comprising a nucleic acid construct for (i) treating or preventing recurrent / secondary cancer / tumor and / or (ii) enhancing, eliciting 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).

[0336] 89. A method for (i) treating or preventing recurrent / secondary cancers / tumors and / or (ii) enhancing, eliciting or inducing an immune response against recurrent / secondary cancers / tumors, the method comprising administering to a patient suffering from a primary cancer an AAV1 vector comprising a nucleic acid construct, the nucleic acid construct comprising a gene or transgene for expression in cells of the primary cancer, wherein the gene or transgene encodes IL-12 and herpes simplex virus thymidine kinase (HSV-TK), thereby treating or preventing the primary cancer and treating or preventing any recurrent / secondary cancers / tumors that may be caused by the treated primary cancer and / or enhancing, eliciting or inducing an immune response against recurrent / secondary cancers / tumors, the immune response being effective in clearing, treating or preventing any recurrent / secondary cancers / tumors that may be caused by the treated primary cancer.

[0337] 90. Use of an AAV1 vector comprising a nucleic acid construct in the preparation of a medicament for (i) treating or preventing recurrent / secondary cancers / tumors and / or (ii) enhancing, eliciting 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).

[0338] The present disclosure will now be illustrated with reference to the following non-limiting examples.

[0339] Example

[0340] Materials and Methods

[0341] Destination vector cloning

[0342] To ensure high cloning efficiency for enhancer cloning and super enhancer assembly, we constructed a new custom destination vector for efficient golden gate cloning. This also reduced reaction setup time / cost. The destination vector design includes: reporter gene cassette Nanoluc-Ires-mNGreen-pA and minimal CMV promoter (mCMV). Bacterial suicide ccdB cassette was used for selection and efficient cloning. CDV2 contains ccdB cassettes spanning multiple positions, enabling the assembly of up to four enhancers and spacers. Compared to CDV1, CDV2 also contains PiggyBac transposase recognition sites flanking the entire cassette. We used Gibson assembly to generate scar free custom destination vectors (Gibson et al., 2009).

[0343] Bioinformatics for designing SOX2 enhancer oligonucleotide library

[0344] Suvà and colleagues published technical replicates for SOX2 binding in GSCs in one cell line (SOX2r1 and SOX2r2 in the MGG8 cell line, proneural subtype) and H3K27ac in three cell lines MGG4 (proneural subtype), MGG6 (classical subtype), and MGG8 (Suvà et al., 2014).

[0345] They also include data for differentiated MGG8 cells, referred to herein as differentiated glioma cells (DGCs). For clarity, GSC lines are also referred to as tumor propagating cells due to their tumorigenic properties, in contrast to DGCs, which are derived from GSCs herein by the addition of serum that stimulates astrocyte differentiation.

[0346] To determine the GSC-specific SOX2 peaks, GSC SOX2r1 and SOX2r2 were overlapped with DGC SOX2_H3K27ac (H3K27ac Chip-Seq in SOX2-overexpressing DGCs). In addition, common H3K27ac peaks were also identified between GSC cell lines to determine putative enhancer sequences. These common peaks overlapped with GSC-specific SOX2r1 and GSC-specific SOX2r2, respectively. This prevented the loss of any potential important regions. The resulting common peaks were then merged into one file, and peaks with a distance of less than 100 base pairs (bp) were merged, which also removed duplications. The length of the peaks ranged from 15-5300bp, with an average length of 411bp. Next, the library was manually curated to remove repetitive sequences and non-enhancer sequences, such as centromeres. This produced a selected preliminary library containing 1721 peaks ranging from 15-3366bp with an average length of 402bp.

[0347] To avoid PCR bias towards small fragments, we also discarded all regions smaller than 100 bp, resulting in 1710 peaks ranging from 115-3366 bp with an average length of 404 bp. These peaks were then divided into 160 bp fragments. Any fragments smaller than 100 bp were again removed to avoid PCR bias. 20 bp adapters were attached to each side.

[0348] Cell lines

[0349] GSCs were generated using NSC culture conditions previously reported by Pollard et al. Briefly, cells were expanded in serum- and feeder-free conditions in Neurobasal medium containing N2 and B27 supplements. Cells were allowed to grow adherently by supplementing the culture medium with laminin-1. GSC7 was characterized by Stricker et al. HEK293 cells were obtained from the ATCC cell bank. All other GSC lines and huFb170 were from the Pollard laboratory (unpublished) and were characterized as part of the Glioma Cell Genetics Resource ( www.gcgr.org.uk ).

[0350] Construction of enhancer array plasmid library

[0351] We chose an arrayed library format where 160 bp enhancer fragments were cloned individually into different plasmids and arrayed in 96-well plates. These 160 bp fragments were synthesized as a single oligo pool, which was then randomly cloned into plasmids and isolated. PCR amplification of the library required 20 bp × 2, so a library of 200 bp fragments was synthesized (Twist Biosciences). To meet our oligonucleotide synthesis strategy, ChIP-seq peaks expressed by GSCs were combined into a single oligonucleotide. This oligonucleotide pool was PCR amplified (using limited cycles to reduce bias and "jackpot" products, meaning that products that are more easily amplified will outnumber the oligonucleotide pool) and cloned into an expression vector via an efficient Golden Gate reaction. 4579 individual plasmids were then randomly picked, isolated, and plated as a plasmid DNA library for the array in 96-well plates (x48). Bacterial colony picking and production of small-scale plasmid preps were supported by the Edinburgh Genome Foundry (S. Rosser). The library was then screened in a 384-well format using an optimized Nanoglo DLR assay (Nano-Glo Dual Luciferase Reporter Assay, Promega). Each plate was transiently transfected into GSC7 cells with PGK-FFLuc at a ratio of 1:10. After 2 days, the Nanoglo DLR assay was performed and assayed on a plate reader.

[0352] PCR amplification and cleanup for library production

[0353] We developed very high efficiency library amplification and plasmid cloning by using Golden Gate cloning and selection against empty vector to avoid inappropriate products, empty sequences and library redundancy. PCR conditions were optimized to amplify oligonucleotide pools with limited / no background and no amplification bias.

[0354] 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 with 0.25 μl (2.5 ng input) for 10 cycles, and 0.5 μl of the reaction was used for the subsequent 15 cycles of amplification to reduce PCR bias.

[0355] Prior to Golden Gate cloning, magnetic bead-based solid phase reversible immobilization (SPRI) purification was used to minimize losses and purify the DNA. Sanger sequencing of all inserts confirmed that they were distinct and that all sequences could be mapped back to the library fragments and included adapters.

[0356] 384-well plate screening platform

[0357] Cells were seeded in 384-well plates using multidrop and transfected using CyBio Felix the next day. Two days later, the experiment was terminated using the Nanoglo DLR assay, and all hits with a fold change greater than 10 for mCMV were Sanger sequenced and mapped back to the genome.

[0358] Genomic characterization of validated enhancers

[0359] The online tool GREAT defines the basal regulatory domain of each gene as 1 kb downstream and 5 kb upstream of the transcription start site (TSS), meaning that nearby genes are part of the same regulatory domain. To account for distal regulatory events, the basal gene regulatory domain is extended to the nearest basal upstream and downstream regulatory domains within 1000 kb. GREAT then uses the ontology of gene annotations and query sequences and the binomial test to test the enrichment of gene ontology to predict target genes (Mclean et al., 2010). We investigated the expression patterns of the predicted target genes in human adult tissues ( https: / / gtexportal.org / home / ).

[0360] Oligonucleotide pull-down and mass spectrometry

[0361] The oligonucleotides were PCR amplified using biotinylated primers. The PCR products were then SPRI purified to remove biotinylated primer dimers. These dsDNA fragments were incubated with nuclear extracts from GSC7 and GSC328. They were then bound to streptavidin magnetic beads, washed three times, and the frozen beads were sent for mass spectrometry analysis.

[0362] Cell culture procedures

[0363] Cell lines were cultured at 37°C, 5% CO2. Cells were grown on uncoated cell culture plastic dishes. Glioblastoma stem cell lines were grown in serum-free conditions using previously reported conditions (Pollard et al., 2009). For passaging, cell lines were rinsed with PBS and GSCs were separated with Accutase, or huFb170 and HEK293 were separated with 0.5% trypsin / EDTA. Cells were absorbed in wash medium and centrifuged at 300xg for 3min. They were resuspended in their respective growth medium and re-plated. For cryopreservation, cells were resuspended in growth medium supplemented with 10% DMSO and stored at -80°C for short term. For long term storage, vials were transferred to liquid nitrogen containers.

[0364] Transfection of GSC, huFb170 and HEK293 cells

[0365] Inoculate cells with required density.Transfect the next day.For this reason, Plus reagent and LipofectamineLTX (Life Technologies, 15338030) are diluted separately in the Opti-MEM I serum reduction culture medium (further referred to as Optimem) (Life Technologies, 31985062) of half volume.After this step, Plus reagent / Optimem premix is ​​added in all DNA samples, and Lipofectamine LTX / Optimem premix is ​​then added.The transfection mixture is incubated at room temperature for 5min, and then carefully dripped on the cell.Usually, do not carry out culture medium replacement.Transfection analyzes cells after two days.

[0366] HEK293 cells were seeded at the specified density in the respective plate formats. The next day, GMEM, DNA and PEI (homemade) were mixed and incubated at room temperature for 15 min to form complexes. The transfection mixture was then dropped onto the cells and analyzed two days later.

[0367] Nano-Glo Dual Luciferase Reporter Assay System (Promega)

[0368] The assay consists of two steps. Transfection is performed using the standardized plasmid PGK-firefly-luciferase (Promega, E5011), which is transfected with the target plasmid at a ratio of 1 / 10. First, the firefly luciferase activity is measured, which allows standardization of transfection efficiency. In the second step, the activity of Nanoluc is determined, which is the reading of the target plasmid. The cells are washed 3 times with PBS, leaving 20 μl in a 96-well plate after the last wash (leaving 25 μl in 384 wells). Oneglo buffer is added, and the plate is shaken at 480rpm for 5min to lyse the cells. Then 20 μl of cell lysates in a 96-well format or 25 μl of cell lysates in a 384-well format are transferred to the corresponding opaque white plate, and the Ensight multi-mode plate reader is used to measure luminescence 0.1s in each well. For the following 96-well format Nanoluc reactions, transfer 2 μl of cell lysate to an opaque white plate containing 40 μl PBS and add 20 μl Stopglo buffer supplemented with substrate to each well. In the 384-well format, add 20 μl Stopglo buffer containing substrate on the undiluted cell lysate. Shake the plate again at 480 rpm for 5 min to terminate the firefly luciferase reaction and ensure good mixing. Again using the Ensight multi-mode plate reader, measure the Nanoluc activity for 0.1 s per well.

[0369] Data obtained with Nanoluc reactions were normalized to firefly reactions to account for variability in transfection efficiency between wells. Normalized data were used to calculate fold changes over empty vector control mCMV.

[0370] Immunocytochemistry

[0371] Cells were washed twice with PBS and fixed in 4% PFA for 10 min. Cells were infiltrated with 0.1% Triton-100 in PBS (further referred to as PBST). They were blocked with blocking solution (1% BSA in PBST containing 3% goat serum) and incubated with primary antibodies at 4 ° C overnight (Table 3). The next day, cells were washed 3 times in PBST, and respective secondary antibodies were added to the blocking solution and incubated at room temperature for 45-60 min. Cells were washed with PBS and incubated for 5 min with DAPI nuclear counterstain at a final concentration of 1 μg / ml. Images were collected using a Nikon TiE microscope and NIS element software (Nikon).

[0372] Table 3: List of antibodies used in immunohistochemistry experiments

[0373]

[0374] Flow cytometry

[0375] For flow cytometry, cells were separated, pelleted and resuspended in an appropriate volume of flow cytometry buffer (1% BSA in PBS, v / v). Cells were stained with Draq7 (Abcam, ab109202, fc 0.1 μM) as a live / dead stain and analyzed using a BD LSRFortessa cell analyzer (4 lasers, BD Bioscience). Analysis of flow cytometry data was performed in FlowJo analysis software (FlowJo, version 10.6.2).

[0376] Quantitative real-time polymerase chain reaction (qRT-PCR)

[0377] For qRT-PCR, TaqMan Universal PCRMaster Mix (Applied Biosystems) and TaqMan Gene Expression Assay (Life Technologies) were used on the Quant Studio7 Flex real-time qPCR machine. RNA samples that did not undergo reverse transcription to evaluate DNA contamination and water controls were used on each plate. In addition, qRT-PCR was performed in technical replicates. Data analysis was performed using the ddCt method, which assumes 100% PCR efficiency and is guaranteed by the TaqMan assay. In short, the mean value of technical replicates was calculated and standardized to the housekeeping gene GAPDH that produces ddCt values. These values ​​were further standardized to calibrator samples (GSC7) to obtain ddCt.

[0378] Production of entry and final vectors using the Scalable Modular Mammalian Assembly (EMMA) toolkit

[0379] Vectors were designed and produced according to Martella et al., 2017. In brief, for domestication of a new part, the BsaI and BsmBI sites were removed. The new part was PCR amplified using primers that generate fusion sites and overhangs of the BsaI site. This ensures simple and efficient cloning into the part entry vector. All part entry vectors contain a red fluorescent protein (RFP) that recombines with the part to be cloned, which increases the cloning efficiency because only white colonies can contain this part. To assemble the expression vector, all parts were mixed with the recipient vector in an equimolar ratio. To increase the cloning efficiency, the recipient vector contains the bacterial suicide cassette ccdB that recombines with a part of the expression vector. Therefore, bacteria that take up the unmodified receptor will not be able to form colonies.

[0380] Western blotting

[0381] Cells were scraped in PBS and centrifuged twice at 0.3 g to remove all liquid. The cell pellet was resuspended in 70 μl RIPA buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% NP-40, 0.5% deoxycholate, 0.1% SDS and protease inhibitors (Complete, Roche, 11697498001)) and incubated on ice for 5 min. The lysate was centrifuged at 4 ° C for 10 min at 13000 rpm (centrifuge 5415D, Eppendorf). The supernatant was collected in a new tube. According to the manufacturer's instructions, the protein extract was quantified using the Pierce BCA protein assay kit (Thermo Scientific, catalog number: 23225).

[0382] 5% of the total volume of 4x lithium dodecyl sulfate (LDS) buffer containing 50mM DTT was added to the cell extract, and the samples were denatured at 95°C for 10 min. The samples were loaded into a 4-12% polyacrylamide gel prepared by Dr. Carla Blin, with a spectral polychromatic wide range protein ladder (Thermo Scientific, catalog number: 26634) or a BioRad Precision Plus protein dual color standard (catalog number: 1610377). The protein bands were transferred to Immobilon PVDF membranes (Millipore, IPVH00010) by wet electroblotting or semi-dry blotting, using the Biorad Trans-blot turbo system according to the manufacturer's instructions. The membranes were blocked at room temperature for 1 hour with a 5% milk (TBS+0.1% Tween-20) solution in TBS-T, followed by primary antibody incubation in 5% milk in TBS-T, while shaking overnight (Table 4). The next morning, the membrane was washed 3 times in TBS-T at room temperature for 5 min each time and incubated with secondary antibody conjugated to horseradish peroxidase in 5% milk in TBS-T for 1 h at room temperature (Table 4). Again, the membrane was washed 3 times in TBS-T for 5 min each time, developed using homemade enhanced chemiluminescence (ECL) solution or Clarity ECL Western Blotting Substrate (Bio-Rad, catalog: 170-5061), and visualized using X-ray film or Bio-Rad ChemiDoc TM Imager imaging.

[0383] Table 4: List of antibodies used in Western blot experiments

[0384]

[0385] Nuclear extract

[0386] All buffers were prepared the day before, passed through 22 μm filters (except dialysis buffer) and placed overnight at 4°C. DTT and protease inhibitors were added before use. Cells were scraped on culture medium and collected on 50ml falcons, centrifuged at 4°C for 5min at 1350rcf. The precipitation was resuspended in 50ml ice-cold PBS, aliquots were taken for cell counting, and rotated at 4°C for 5min at 1350rcf again. The precipitation was then resuspended in ice-cold buffer A (10mM HEPES pH 7.9, 1.5mM MgCl2, 10mM KCl, 0.5mM DTT, protease inhibitors (Complete, Roche, 11697498001)) of 5ml / 40 million cells, and incubated on ice for 10min. The cell suspension was transferred to a glass Dounce homogenizer and homogenized 40 times on ice. The cell suspension was transferred to a falcon, and centrifuged at 4°C for 10min at 1350rcf. The supernatant (cytosolic extract) was discarded and the pellet was resuspended in 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 inhibitors (Complete, Roche, 11697498001)) at 100 μl / 10 million cells. The suspension was rotated at 4°C for 30 min and transferred to a dialysis membrane (SnakeSkin, Thermo Scientific, catalog 68100). Dialysis was performed 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 inhibitors (Complete, Roche, 11697498001)) at 4°C with rotation for 2 h. Replace dialysis buffer (500 ml) and continue dialysis overnight at 4 ° C. The extract is collected in a 1.5 ml tube and spun at maximum speed for 15 min at 4 ° C (centrifuge 5415D Eppendorf). The supernatant is collected in a new 1.5 ml tube and quantified (Pierce BCA protein assay kit, Thermo Scientific, catalog 23225). 100 μg aliquots are snap-frozen in liquid nitrogen and stored at -80 ° C.

[0387] PCR amplification and purification using biotinylated primers

[0388] Enhancers were amplified using PrimeStar Max (Takara) with 5' biotinylated primers (Table 5) according to the manufacturer's instructions.

[0389] Table 5: 5' biotinylated primers for transcription factor pull-down

[0390] Primer No. sequence SEQ ID NO. Forward primer 595 TGATCCGTCTCgCCCTactaggttatactggtgcatgc 81 Reverse primer 596 ACTAACGTCTCgGAGCacccaaactattggagcgag 82

[0391] PCR purification was performed using Agencourt AMPure XP magnetic beads (Beckman Coulter) according to the manufacturer's instructions. PCR product quantification was performed using Tapestation and reagents according to the manufacturer's instructions.

[0392] Precipitation of interacting proteins

[0393] Streptavidin magnetic beads (NEB, S1420S) were used according to the manufacturer's instructions. Briefly, 10 μl of beads were aliquoted into LoBind tubes and washed three times with binding buffer (20 mM Tris-HCl pH 7.5, 0.5 M NaCl, 1 mM EDTA) on a magnetic stand. 20 μl of biotinylated DNA (approximately 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 (total volume 50 μl) was added. The beads, DNA and nuclear extract were added to 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 inhibitors (Complete, Roche, 11697498001)) and 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 blotting, proteins were eluted in 20 μl loading buffer (lithium dodecyl sulfate (LDS) buffer containing 50 mM DTT) and boiled for 5 min to denature the proteins. For mass spectrometry, the beads were dried and sent to the instrument.

[0394] Zebrafish experiments

[0395] All embryos were obtained by natural spawning and collected in conditioned aquarium water containing 0.00001% methylene blue. During the experiment, embryos were treated with 200 μM N-phenylthiourea (PTU) (Sigma) from 6 hours post fertilization (hpf) to inhibit pigmentation (Karlsson, Von Hofsten and Olsson 2001).

[0396] Fertilized eggs were injected at the one-cell developmental stage. Eggs were preserved in 1.5% agarose (Sigma) molds for microinjection as described by Nusslein-Volhard and Dahm 2002. Microinjection needles were pulled from glass capillaries (Harvard Apparatus, USA) using a P-97 Flaming / Brown micropipette puller (Sutter Instruments, USA). Needle pulling parameters were as follows: heat: 550; pull force: 200; viscosity: 55; time: 150. Microinjections were performed on a PV820 pneumatic micropump (World Precision Instruments [WPI], USA) system. DNA constructs were created using the Tol2Kit system (Kawakami 2007, Kwan et al. 2007). Approximately 2 nL of plasmid DNA (30 ng / μL) containing Tol2 capped mRNA (20 ng / μL) was injected, supplemented with 0.2% w / v phenol red (Sigma) to facilitate visualization of the injection volume. After anesthesia with tricaine, embryos were live imaged on a wide-field fluorescence microscope at 24-48 hpf.

[0397] Adeno-associated virus (AAV) transduction assay

[0398] One day before transfection, HEK293 cells were seeded into 6-well plates so that the fusion rate could be 60-70% on the day of transfection. Before transfecting HEK293 cells with PEI, a medium change was performed because the medium will be conditioned with the virus over a two-day period. In the following days, HEK293 or GSC7 cells were seeded at low density (10-20%) in the same or smaller plate format (6 or 12-well plates). The AAV-conditioned medium was centrifuged at 1300 rpm for 4 min to remove contaminating cells, and the supernatant was transferred to the HEK293 cells seeded at low density. Cells were analyzed on a microscope or flow cytometer.

[0399] Cell seeding and transduction for killing assays

[0400] On the day of cell seeding, cells were isolated using the method described above. Cells were counted using a hemocytometer and seeded into 96-well (1,000 cells in 50 μL per well), 24-well (30,000 cells in 500 μL per well), or 6-well (60,000 cells in 2 mL per well) Corning plates and placed in a 37°C / 5% CO2 incubator. The next day, AAV virus stock was thawed at room temperature and the appropriate amount of virus stock was added to the required amount of culture medium to reach 5 × 10 5Without replacing the existing medium, add the medium containing the viral particles to the cells. Return the cells to 37°C / 5% CO2.

[0401] Drug treatment

[0402] Lyophilized GCV was diluted in DMSO to achieve a stock concentration of 100 mM. The GCV stock solution was aliquoted and stored at -20°C for no longer than 1 month. To prepare a working stock concentration, GCV was diluted 1:100 in an appropriate culture medium and 20 μL of this working stock solution was added to a well already containing 80 μL of culture medium (achieving another 1:5 dilution). The final concentration of GCV on the cells was 200 μM.

[0403] As a negative control, dilute DMSO 1:100 in the appropriate culture medium to obtain a working stock. Add 20 μL of the working stock to a well already containing 80 μL of culture medium. As a positive control, add 20 μL of DMSO to a well containing 80 μL of culture medium to achieve a final concentration of 20% DMSO.

[0404] Incucyte Live Cell Imaging

[0405] To track cell proliferation and morphological changes during treatment, cells were monitored using the Incucyte live cell imaging system. Whole-well imaging of Corning 96-well plates was performed every 4 hours. Basic confluence scoring analysis software (Incucyte) was used to assess confluence. Images at specific time points were extracted to verify cell confluence and morphology.

[0406] MTT assay

[0407] On the day of the assay, replace the medium with 0.3 mg / mL MTT solution (diluted in a medium appropriate for the cell line). Place the cells in an incubator at 37°C / 5% CO2 for 3 h. After incubation, remove the medium and add 70 μL DMSO to each well. Place each plate in the dark at 37°C for 20 min with occasional shaking. Before reading the plate, visually inspect each well to ensure that all (formazan) crystals have dissolved. Read the plate at an absorbance of 560 nm using a plate reader.

[0408] Data analysis

[0409] Most data analyses were performed using Microsoft Excel version 16.23 for Mac and GraphPad Prism 7. Error bars are shown as standard deviation of the mean. Some data analyses were performed using RStudio version 1.1.456 (RStudio Team 2015). To generate illustrations, biorender.com and Adobe Illustrator 22.0.1 were used. Open source programs such as bedtools (Quinlan and Hall, 2010), GREAT (Mclean et al., 2010), fastaspliot (Stothard, 2000), and MEME (Bailey et al., 2009) were also used for various analyses.

[0410] Example 1 - Identification of functional SOX2 enhancers

[0411] To first establish proof of concept whether synergistic activity could be achieved when individually active enhancers were combined into clustered multipartite arrays, we first identified a small set of candidate enhancers. These enhancers were selected based on proximal genes with known roles in neural stem cell (NSC) self-renewal (POU3F2, POU3F3, CHD7, ASCL1, SOX6, ETV1) and using available differential expression data for NSCs relative to primary human fibroblasts. Five SOX2 candidate auto-regulatory enhancers were also included. These enhancers (typically ~500-800 bp) were cloned into plasmid expression vectors carrying mNeonGreen and luciferase reporter gene cassettes and their individual enhancer activity was tested using two independent patient-derived glioblastoma stem cell (GSC) cell lines (G7 and G328). Figure 1 Six of these enhancers were functional, increasing NanoLuc expression by more than 10-fold compared to mCMV (254, 270, 282, 292, 312, and 316; range 10-300-fold). None of these were active in HEK293 cells (negative controls).

[0412] Table 6: Predicted target genes and distance from enhancer fragment to transcription start site (TSS)

[0413]

[0414] Example 2 - Assembly of four different enhancers results in a synergistic increase in transcriptional activity

[0415] To investigate whether combining multiple enhancers could increase activity, we first constructed concatemer versions (i.e., multiple copies of the same enhancer) of two enhancers (2x), four enhancers (4x), or eight enhancers (8x) (concatemers). We selected three different individual enhancers with different strengths: 270 (approximately 100-fold), 254 (approximately 20-fold), and 312 (approximately 10-fold). For 270, we noted only modest synergy compared to individual enhancer activity in both the 2-part and 4-part enhancers. The 4-part enhancer produced approximately 500-fold more activity ( 100-fold more activity) compared to 100-fold activity from a single enhancer. Figure 1 F). Surprisingly, we observed no additional gain by increasing the copy number to 8 partial concatemers. A similar trend was observed for the independent patient cell line GSC328. For all constructs, only background levels of luciferase activation were observed in HEK293 cells, comparable to the mCMV negative control.

[0416] We next tested whether mixing different enhancers from different genes would result in improved synergy ( Figure 1 K). Therefore, we combined the first four individual enhancers identified in G7 (270, 282, 292, and 316) into a new synthetic enhancer (termed T4). Indeed, we noticed that the expression of T4 was significantly increased by more than 5000-fold relative to mCMV using Nanoluc reporter assays (Nano-Glo Luciferase Assay and Nano-Glo Dual Luciferase Reporter Assay, both Promega) Figure 1 L). This was further verified by scoring mNGreen in G7 cells using flow cytometry ( Figure 1 I), which indicated that the level of T4 was approximately 50-60% of the full-length CMV observed in G7 cells. Thus, combining enhancers from different genes produces a synergistic effect and significantly increases enhancer activity in GSCs, but does not increase background expression in HEK293 cells. These cell type-specific synthetic enhancer clusters are subsequently referred to as "synthetic super enhancers" (SSEs).

[0417] Example 3 - Activity of the T4 synthetic super enhancer is reduced in GSCs after astrocyte differentiation

[0418] We next tested whether the SSE-T4 synthetic super-enhancer is specific to the GSC state and therefore disappears when they differentiate into astrocytes with SOX downregulation. G7 cells can be efficiently differentiated into astrocyte-like cells after exposure to fetal calf serum (FCS) for 10-15 days. Therefore, GSCs (G7) were stably transfected with the SSE-T4 expression cassette (using the PiggyBac transposase system) and mNGreen+ cells were sorted using fluorescence-activated cell sorting (FAC). The next day they were plated in 5% FCS and mNGreen expression was measured using flow cytometry every 5 days over a 15-day time course ( Figure 1 HJ). Based on live cell fluorescence imaging, the expression of mNGreen was significantly reduced at day 5. This was confirmed by flow cytometry, which showed that approximately 50% of cells lost mNGreen at day 5 and approximately 80% of cells lost mNGreen at day 15. These data indicate that SSE-T4 is highly active in GSCs but not in their differentiated astrocyte-like progeny, and therefore SSE is cell type specific.

[0419] Example 4 - Systematic Screening of SOX2 Enhancer Array Plasmid Library

[0420] The above data provide proof-of-principle that combining enhancers into a four-part array can increase transcriptional activity without compromising background expression in non-GSC cells. We believe that a functional screen of enhancer fragments (160 bp) can be performed using the full set of candidate genome-wide SOX2 enhancers in a high-throughput 384-well plate-based luciferase assay to systematically identify functional enhancers with optimal performance and small size when used in SSE. By performing this screen in patient-derived GSCs, we can overcome the limitations of existing serum-grown cancer cell lines that do not have the appropriate characterization and associated enhancers.

[0421] 160 bp was chosen because this provides sufficient size for nucleosome binding and may be an important feature of grammatical evolution within functional enhancers (Soufi et al., 2015). In addition, 160 bp is a convenient size for synthesizing pooled oligonucleotide libraries that incorporate 20 bp ends for subsequent construction of arrayed plasmid libraries. Compared to other pooled library screening strategies, such as STARR-seq (Muerdter et al., 2018, Arnold et al., 2013), we prefer arrayed plasmid libraries constructed from synthetic oligonucleotides because this ensures improved signal-to-noise ratios and direct validation of hits.

[0422] We therefore designed a set of oligonucleotides that could be synthesized as a library. We reanalyzed the dataset of Suvà et al., 2014 and defined a set of GSC-specific SOX2 binding peaks that overlapped with H3K27ac, thereby identifying 1710 different enhancers. These enhancers were approximately 400 bp on average (range: 115-3366 bp). We in silico partitioned these sequences into 160 bp elements that overlapped by 100 bp. 9523 different oligonucleotide sequences were then synthesized as a library, PCR amplified, and cloned into arrayed 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 (approximately 70%) of these plasmids contained the appropriate enhancers.

[0423] The SOX2 enhancer library was screened in a 384-well format using an optimized luciferase assay. 135 plasmids were identified as functional, with a >10-fold increase over mCMV. 52 of these 135 initial hits were then validated using an independent Nanoglo DLR assay in triplicate ( Figure 2 ). 16 of these 52 were found more than once. The hits were Sanger sequenced, confirmed to contain an enhancer, and mapped back to the human genome using the expected fragments from the original design set.

[0424] We next validated the top 17 fragments (with a >30-fold increase over mCMV) across multiple cell lines using independent flow cytometry assays ( Figure 2 ). These fragments were active in an independent patient-derived GSC cell line (GSC328), but not in non-neural cells tested (HEK293 cells and human primary fibroblasts, huFb170). We found that most of the top few hits were sequences that are evolutionarily conserved in vertebrates. The top 5 enhancers were each over 100-fold compared to the NanoLuc-based mCMV (range: 100-260-fold). These were validated by flow cytometry.

[0425] ID1101 is located in the intron of PRCP; ID2904 is located in the intron region of MYO18 gene, close to SEZ6L; ID0109 is located in the gene enrichment region between TPK1 and CNTNAP2; ID4328 is located in the intron region of zinc finger transcription factor ZNF438. ID0876 is located in the intron region of NWD2.

[0426] Table 7: Predicted target genes and distance from enhancer fragment to transcription start site (TSS)

[0427]

[0428] Thus, our functional screen led to the identification of a number of 160 bp enhancer fragments that had significantly increased activity compared to the full-length enhancers (described above), despite being 4-5 fold smaller in size. Example 5 - Generation of strong and selective synthetic super-enhancers using the newly identified SOX2 enhancer

[0429] We constructed synthetic super-enhancers by clustering the most individually active fragments from the screen into 4-part arrays to identify whether their strengths increased synergistically while retaining cell-type selectivity.

[0430] To create the strongest possible synthetic super-enhancers, we designed a set of four new 4-part arrays using sequences from the top 17 enhancers. These were assembled upstream of mCMV. The fragments were ranked according to their median fold change to mCMV. The 4 most active enhancers (ID1101, ID2904, ID0109, ID4328, indicated by '1') were clustered into 4-part arrays to form synthetic super-enhancers. Enhancers comprising the second group (indicated by '2') were clustered into synthetic super-enhancers, and so on. We hypothesized that the four strongest enhancers would show the highest expression levels, the next group would be the second highest, and so on. In addition, by testing the synthetic super-enhancers in different cell types (GSC7, GSC328, HEK293, and huFb170), we also investigated their cell type selectivity.

[0431] All four constructs tested (C1, C3, C5, and C7) induced significant and selective expression in GSC7 and GSC328, while only inducing background expression in HEK293. No expression was detected in huFb170. In GSC7 and GSC328, each of these synthetic super-enhancers (C1, C3, C5, and C7) showed higher fluorescence intensity and a higher percentage of mNGreen+ cells than SSE-T4 (the first generation SSE discussed above), which was the most active synthetic super-enhancer using the candidate enhancers. Notably, C1 and C7 induced higher expression levels than the positive control CMV, which itself is one of the strongest viral promoters. Therefore, we observed a clear synergistic effect of constructing a 4-part enhancer array, resulting in very strong and cell type-selective synthetic super-enhancers.

[0432] We further reduced the size of the synthetic super-enhancers by removing the adaptors and spacers (e.g., 974 bp to 640 bp for C1). These synthetic super-enhancers were named SSE 1-7 because they are 7 constructs and their design follows the same principles as C1, C3, C5, C7. All constructs showed similar activity levels. This was comparable to levels seen in intact CMV and in some cases higher than CMV (SSE1, 2, 3, 5 by Nanoglo DLR assay and SSE-1, 2, 3, 5, 7 by flow cytometry in GSC7. SSE-1, 2, 5, and 7 also showed higher intensities in GSC7 than in CMV. GSC328, HEK293, and huFb170 were also analyzed for mNGreen expression by flow cytometry. The trend was similar to that of GSC328. The mean fluorescence intensity (MFI) was similar to that of CMV, and the intensity of SSE-6 was significantly lower than that of CMV. In HEK293 cells, all synthetic super-enhancers induced transcription to levels similar to that of mCMV. mCMV showed 32.5% mNGreen+ cells compared to untransfected cells. SSE expression was slightly increased in HEK293 compared to mCMV. The activity of SSE1-7 in huFb170 was undetectable (more than 1%). In both reporter gene assays (Nanoglo In DLR assays and flow cytometry), a trend was observed for each construct (C1 vs. SSE-1; C3 vs. SSE-3, etc.) that the smaller synthetic super-enhancer had higher activity ( Figure 3 B, C, D). This was confirmed by live cell images ( Figure 3 E) Each of the synthetic super-enhancers tested, SSE-1, 3, 5, and 7, is similar in activity to CMV.

[0433] Example 6 - Synthetic super-enhancers are less active in differentiated GSCs

[0434] To investigate the activity of SSE-2-SSE-7 under differentiation conditions, GSC7 cells were stably transfected using the Piggybac transposase system and mNGreen+ cells were sorted. Similar numbers of viable cells were sorted for mCMV and untransfected controls. Cells were then placed in medium without growth factors but with 5% FCS to induce astrocyte differentiation.

[0435] During differentiation, mNGreen expression was measured at days 0, 1, 5, 10, and 15 ( Figure 4 E and G). We observed a decrease in signal over time, suggesting that synthetic super-enhancers are most highly expressed in GSCs and become less active upon differentiation.

[0436] Example 7 - Zebrafish embryos reveal highly selective region-specific expression in the forebrain

[0437] The zebrafish is well suited as an experimental model to study tissue specificity of enhancers. We identified tissues shared between all structures, such as ventral spinal cord expression, otic placodes, and midbrain / hindbrain and forebrain. Some segments had additional activity, such as foregut / hindgut. We typically observed low expression at 24 hpf that continued to increase until the latest measured time point at 48 hpf. Regarding controls, we observed widespread, non-specific expression of EGFP in CMV controls and no expression in mCMV using the same assay ( Figure 4 The mCMV control showed green fluorescent eyes, which may be due to spurious transcription from the lens eye promoter ( Figure 4 K).

[0438] Example 8 - SSE can be used in adeno-associated viruses to induce selective killing of GSCs

[0439] To test whether SSE is suitable for driving cytotoxic payloads and selectively killing GSCs, we transduced GSC7 and huFB170 cell lines with adeno-associated virus 2 (AAV2) containing the suicide gene thymidine kinase (TK) from herpes simplex virus (HSV) in a polycistronic mRNA linked to the reporter gene mCherry via p2A. These expression cassettes were either driven by the constitutively active CMV promoter (CMV_HSV-TK-v5_P2A_mCherry) as a positive control or by 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) into a cytotoxic product by phosphorylation. Phosphorylated GCV competes with dGTP for the polymerase substrate, thereby interfering with DNA synthesis, leading to premature termination and cytotoxicity.

[0440] Flow cytometry and western blotting confirmed that SSE-7 drives mCherry and HSV-TKv5 expression in GSC7 but not in fibroblasts (huFb170) ( Figure 5 ). Cell confluence was recorded throughout the cytotoxicity experiments. A decrease in cell proliferation and confluence was particularly observed in cells transduced with CMV or SSE-7 driven TKv5-P2A-mCherry and receiving 200 μM GCV ( Figure 6 A). No significant negative effects on cell confluence were observed in cells treated with virus or ganciclovir alone. MTT assay data indicate loss (or lack) of metabolic activity, which can be used as an approximate readout of cell viability in GSC7 cells treated with CMV or SSE-7 driven TKv5-P2A-mCherry and GCV ( Figure 6 B). There was no statistical difference between CMV and SSE-7 (13% vs. 12% viability) (p value = 0.7792). Ganciclovir alone had a small effect on GSC7 cell viability (89% viability). Live cell imaging confirmed these findings, showing lower cell density and morphological changes associated with cell stress, damage or death (rounded cells) after treatment ( Figure 6 C). In conclusion, both CMV and SSE-7 can drive the expression of the HSV-TK-v5-P2A-mCherry construct in GSC7 cells to sufficient levels to induce cell death in the presence of ganciclovir.

[0441] Based on live cell imaging data at the end of the experiment (day 17), the majority of huFB170 cells transduced with CMV-driven, but not SSE-7-driven, viruses were killed ( Figure 6 D). This was confirmed with MTT data demonstrating 6.3% viability in cells transduced with CMV driven virus relative to full viability in cells transduced with SSE-7 driven TKv5-P2A-mCherry ( Figure 6 E, n=1).

[0442] Example 9 - SSE shows activity in a range of GSCs

[0443] In the proposed gene therapy, SSE needs to be highly expressed in different patient-derived glioblastoma cell lines but 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). These cell lines were chosen for this experiment because they cover a range of genetic and transcriptional GBM subtypes and have been shown to induce tumors in the mouse brain. In summary, SSE7 is active in all cell lines, but to varying degrees: it is highly expressed in the positive control cell line GSC7; as well as in E17 and E28 ( Figure 7 C) is highly expressed and comparable to the full-length CMV ( Figure 7 C), all of which belong to the classical subtype, but have lower expression frequencies in E21, E31, and E34 ( Figure 7 C).

[0444] Example 10 - De novo motif analysis identified a SOX dimer motif enriched in the top 32 hits

[0445] We used the MEME tool, which can identify de novo motifs (Bailey et al., 2009). We used the first 32 hits as input. Unexpectedly, the significant motif that emerged was the partial palindromic SOX motif ( Figure 8A). SOX2 does not form homo- or heterodimers with other SOX factors; however, members of the SOXE group are well established as dimers (SOX8, SOX9, SOX10). Interestingly, SOX9 is a well-known regulator of NSCs and GSCs but has not been studied as intensively as SOX2 (Bulstrode et al., 2017, Huang et al., 2015, Mateo et al., 2015, DK Singh et al., 2017). This motif directly suggests that SOX2 and SOX9 may act on shared enhancers ( Figure 8 C).

[0446] We then investigated whether this dimer motif was a general feature of the oligonucleotide sequence pool compared to the validated unconfirmed hits and 10 random sequences of the initial oligonucleotide pool and mapped it to the top 52 hits (all sequences with a 10-fold change compared to mCMV). The motif appeared at high frequency in the top 32 hits and at lower frequencies in the remaining top 33-top 52 hits.

[0447] Example 11 - The SOX dimer motif is essential for the activity of ID2904 in GSCs and suggests cooperative action of SOX2 and SOX9 at key enhancers of GSC function

[0448] We next investigated whether the SOX dimer is essential for enhancer function. We generated a series of variants of the ID2904 motif. The synthesized variants had: 1) reduced spacing between half-sites (-2n); 2) increased spacing between motif half-sites; 3) and 4) deletion of one half-site; 5) replacement of the flanking sequence with random sequences; 6) inversion of the motif; 7) mutation of the core nucleotides; 8) substitution with a high affinity motif as reported by Jolma et al., 2013; 9) random sequences as negative controls; and 10) the motif was tandem four times. These different variants allowed us to functionally dissect the key sequences in the dimer motif ( Figure 8 ).

[0449] We observed that almost all variants of the SOX dimer motif in ID2904 showed almost complete loss of activity, confirming the importance of this dimer motif for enhancer function. One exception was an altered distance between the two half-sites, which resulted in increased activity. This is interesting because it is consistent with the concept that spacing can be “suboptimal” and that a 2 bp reduction may 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. The reduction in spacing is also consistent with Huang et al., 2015, who defined the optimal spacing for the motifs of SOXE factors.

[0450] Next, we aimed to confirm that SOX2 and SOX9 indeed bind to enhancers and SOX dimer motifs. To this end, we performed pull-down assays in which ID1101 (the first hit in the screen) and ID2901 were biotinylated and incubated with nuclear extracts. Western blotting and mass spectrometry were performed to identify key transcription factors that interact with the fragments ( Figure 8 ). For data analysis, ID1101 was normalized (in each cell line) to the mean of the random sequence 599. In both cell lines, the cutoff was set to two-fold enrichment above 599. 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.

[0451] In co-immunoprecipitation experiments, SOX2 was specifically pulled down by ID1101 in GSC7 and GSC328, demonstrating that SOX2 binds to enhancers ( Figure 8 ). This was confirmed in mass spectrometry, as SOX2 was enriched 23-fold in GSC328 and 4.4-fold in G7, confirming the binding of this transcription factor.

[0452] In addition, the above and Figure 8 The data shown in J indicate that the sequence context (syntax) surrounding the motif is functionally important, as substitutions 20 bp upstream and downstream of the wild-type (WT) sequence resulted in a complete loss of activity.

[0453] Example 12 - SOX dimer motif helps filter active enhancers in silico simulations

[0454] We hypothesized that the presence of the motif might increase our predictive power about which enhancers are active. To test this hypothesis, we mapped this motif to all full-length enhancers from the original SOX2 ChIP-seq dataset. 70 regions were selected for a primary screen based on their predicted target genes with known functions in GBM or neural stem cells and clustering within the genome, implying regulation of the same predicted target genes. They were tested in the Nanoglo DLR assay. These are referred to as “clustered SOX dimer motif enhancers” (CSEs). Thus, using the SOX dimer motif frequency and rational design, we increased the success rate of identifying positive enhancers by approximately 11-fold; from approximately 2% (52 / 2610) in the original screen to approximately 22.8% (16 / 70) in our rational selection. Positive hits were further validated by flow cytometry in GSC7, GSC328, HEK293, and huFb170 ( Fig. 9 ).

[0455] Additional screening was performed with 209 enhancers to further identify active enhancers. Enhancers were reduced to 160 bp based on their predicted motifs (called minCSEs - see Table 1). Based on data from the Human Protein Atlas (https: / / www.proteinatlas.org / ), 160 bp enhancer fragments from different target genes were mixed to cover pathways such as proliferation, stem cell properties or brain-enriched / brain-specific or glioma-enriched / glioma-specific predicted target genes to generate further SSEs for screening.

[0456] Table 8: Validated CSE hits with genomic coordinates, predicted target genes and their distance from enhancer fragment to TSS

[0457]

[0458] in conclusion

[0459] Mapping of transcription factor binding sites, chromatin accessibility or enhancer-associated histone marks is widely used to search for key transcription factors (TFs) and associated enhancers. However, a major limitation is that TF binding is rather promiscuous and functional enhancers represent only a small subset. We demonstrate here that combining an array of functional enhancer sequences can lead to the formation of synthetic super-enhancers. By exploiting the natural “grammar” of endogenous functional enhancer sequences, but combining these sequences in optimal sizes and clusters, we were able to obtain SSEs that synergistically increase transcriptional activation without sacrificing cell-type selectivity. Thus, our data support that individual enhancers can be combined to generate synthetic super-enhancers that retain selectivity but are very strong in driving transcription.

[0460] References

[0461] Arnold et al.2013 "Genome-wide quantitative enhancer activity maps identified by STARR-seq." Science 339(6123):1074-1077

[0462] Bailey et al. 2009. “MEME Suite: Tools for Motif Discovery and Searching.” Nucleic Acids Research 37(Suppl.2):202–8.

[0463] Boumahdi et al.2014。“SOX2 controls tumour initiation and cancer stem-cell functions in squamous-cell carcinoma.”Nature 511(7508):246-50.

[0464] Bulstrode et al.2017.“Elevated FOXG1 and SOX2 in GlioblastomaEnforces Neural Stem Cell Identity through Transcriptional Control of CellCycle and Epigenetic Regulators.”Genes&Development 31(8):757–73.

[0465] Corish and Tyler-Smith,1999.“Attenuation of Green Fluorescent ProteinHalf-Life in Mammalian Cells.”Protein Engineering 12(12):1035–40.

[0466] Farley et al.2015“Suboptimization of Developmental Enhancers.”Science(New York,N.Y.)350(6258):325–28.

[0467] Gangemi et al.2009“SOX2 Silencing in Glioblastoma Tumor-InitiatingCells Causes Stop of Proliferation and Loss of Tumorigenicity.”Stem Cells 27(1):40–48.

[0468] Gibson et al.2009“Enzymatic Assembly of DNA Molecules up to SeveralHundred Kilobases.”Nature Methods 6(5):343–45.

[0469] Guerra-Rebollo et al.2019“Targeting of Replicating CD133 and OCT4 / SOX2 Expressing GliomaStem Cells Selects a Cell Population That ReinitiatesTumors upon Release of TherapeuticPressure.”Scientific Reports 9(1):9549。

[0470] Huang et al.2015“SOXE Transcription Factors Form Selective Dimers onNon-Compact DNAMotifs through Multifaceted Interactions between Dimerizationand High-Mobility GroupDomains.”Scientific Reports 5(January):1–12.

[0471] Jolma et al.2013“Multiplexed Massively Parallel SELEX forCharacterization of HumanTranscription Factor Binding Specificities.”GenomeResearch 20(6):861–73.

[0472] Karlsson,Von Hofsten,and Olsson,2001“Generating TransparentZebrafish:A Refined Method toImprove Detection of Gene Expression duringEmbryonic Development.”Marine Biotechnology3(6):522–27.

[0473] Kawakami,2007“Tol2:A Versatile Gene Transfer Vector in Vertebrates.”Genome Biology 8(Suppl.1):1–10.

[0474] Kwan et al.2007“The Tol2kit:A Multisite Gateway-Based ConstructionKit for Tol2 TransposonTransgenesis Constructs.”Developmental Dynamics 236(11):3088–99.

[0475] Lopez-Bertoni et al.2015“DNMT-Dependent Suppression of MicroRNARegulates the Inductionof GBM Tumor-Propagating Phenotype by Oct4 and Sox2.”Oncogene 34(30):3994–4004.Lujan et al.2012“Direct Conversion of MouseFibroblasts to Self-Renewing,Tripotent NeuralPrecursor Cells.”PNAS 109(7):2527–32.

[0476] MacLeod et al.2019“Genome-Wide CRISPR-Cas9 Screens Expose GeneticVulnerabilities andMechanisms of Temozolomide Sensitivity in GlioblastomaStem Cells.”Cell Reports 27(3):971-986.e9.

[0477] Martella et al.2017“EMMA:An Extensible Mammalian Modular AssemblyToolkit for the RapidDesign and Production of Diverse Expression Vectors.”ACSSynthetic Biology 6(7):1380–92.Mclean et al.2010“GREAT Improves FunctionalInterpretation of Cis-Regulatory Regions.”NatureBiotechnology 28(5):495–501.

[0478] Muerdter et al.2018“Resolving Systematic Errors in Widely UsedEnhancer Activity Assays inHuman Cells.”Nature Methods 15(2):141–49.

[0479] Nusslein-Volhard and Dahm,2002“Zebrafish:a practical approach”NewYork:Oxford UniversityPress:303 pages.

[0480] Pollard et al.2009“Glioma stem cell lines expanded in adherentculture have tumor-specificphenotypes and are suitable for chemical andgenetic screens”Cell Stem Cell 4(6):568-80.Quinlan and Hall,2010“BEDTools:AFlexible Suite of Utilities for Comparing GenomicFeatures.”Bioinformatics 26(6):841–42.

[0481] D.K.Singh et al.2017“Oncogenes Activate an Autonomous TranscriptionalRegulatory CircuitThat Drives Glioblastoma.”Cell Reports 18(4):961–76.

[0482] S.K.Singh et al.2004“Identification of Human Brain Tumour InitiatingCells.”Nature432(7015):396–401.

[0483] Soufi et al. 2015 “Pioneer Transcription Factors Target Partial DNA Motifs on Nucleosomes to Initiate Reprogramming.” Cell 161(3):555–68.

[0484] Stothard, 2000 “Internet On-Ramp.” BioTechniques 28(6):1102–4.

[0485] 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. Suvà et al. 2014 “Reconstructing and Reprogramming the Tumor-Propagating Potential of Glioblastoma Stem-like Cells.” Cell 157(3):580–94. Table 1 - Sequences of Enhancers (without Linkers)

[0486]

[0487]

[0488]

[0489]

[0490]

[0491]

[0492]

[0493]

[0494]

[0495]

[0496]

[0497]

[0498]

[0499]

[0500] Note: Fold changes refer to the fold change in expression of individual enhancers compared to mCMV.

[0501] Table 2 – Sequence of the complete SSE

[0502]

[0503]

[0504]

[0505]

[0506]

[0507]

[0508]

[0509]

[0510]

[0511]

Claims

1. A construct or functional nucleic acid for (i) treating or preventing recurrent / secondary cancer / tumor and / or (ii) enhancing, eliciting or inducing an anti-recurrent / secondary cancer / tumor immune response, wherein the construct comprises a gene or transgene encoding an adverse payload.

2. The construct or functional nucleic acid according to claim 1, wherein the recurrent / secondary cancer is associated with a primary cancer that has been treated with the construct according to claim 1.

3. The construct or functional nucleic acid according to claim 1 or 2, wherein the gene or transgene is for expression in a target primary cancer cell and the payload is detrimental to the primary cancer cell.

4. The construct or functional nucleic acid according to claim 3, wherein the target primary cancer cell is a primary glioblastoma cancer cell and the recurrent / secondary cancer is a recurrent / secondary glioblastoma.

5. The construct or functional nucleic acid according to any one of the preceding claims, wherein the unfavorable payload comprises or encodes a protein that stimulates an immune response resulting in the recruitment of cytotoxic immune cells.

6. The construct or functional nucleic acid according to any one of the preceding claims, wherein the gene or transgene is operably linked to a synthetic super enhancer, wherein the synthetic super enhancer comprises two or more enhancer sequences derived from different genomic loci.

7. The construct or functional nucleic acid according to claim 6, wherein each enhancer sequence comprises a binding site for a transcription factor; and optionally, 20 to 400 nucleotides located upstream and / or downstream of the transcription factor binding site.

8. The construct or functional nucleic acid according to claim 6 or 7, wherein the enhancer sequences originating from different genomic loci are activated by a common transcription factor or a group of cell type-associated transcription factors.

9. The construct or functional nucleic acid according to any one of claims 6 to 8, wherein the synthetic super enhancer comprises at least one SOX motif and / or SOX dimer motif.

10. The construct or functional nucleic acid 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 construct or functional nucleic acid according to any one of claims 6 to 10, wherein the synthetic super enhancer is activated by one or more transcription factors of the SOX family.

12. The construct or functional nucleic acid according to any one of claims 6 to 11, wherein each enhancer sequence comprises or consists of 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 construct or functional nucleic acid according to any one of claims 6 to 12, wherein the synthetic super enhancer comprises or consists of a sequence having at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 64 to 80.

14. The construct or functional nucleic acid according to any one of the preceding claims, wherein the unfavorable payload comprises or encodes or further comprises or further encodes a cytotoxic substance.

15. The construct or functional nucleic acid according to any one of the preceding claims, wherein the unfavorable payload comprises or encodes a suicide gene encoding a protein capable of converting an inactive prodrug into a cytotoxic drug.

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

17. The construct or functional nucleic acid according to any one of the preceding claims, wherein the unfavorable payload comprises or encodes an IL-12 gene.

18. The construct or functional nucleic acid of any one of the preceding claims, wherein the construct is administered to a subject having a primary cancer and having been treated with the construct for the primary cancer, wherein treatment of the primary cancer with the construct induces, triggers or enhances an anti-recurrent / secondary cancer / tumor immune response that prevents or reduces the risk of recurrent / secondary cancers caused by the primary cancer.

19. A construct or functional nucleic acid molecule for (i) treating or preventing recurrent / secondary cancer / tumor and / or (ii) enhancing, eliciting 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).

20. The construct or functional nucleic acid according to claim 19, wherein the recurrent / secondary cancer / tumor is recurrent / secondary glioblastoma.

21. The construct or functional nucleic acid according to any one of the preceding claims, wherein the construct or functional nucleic acid is packaged in an AAV vector.

22. The construct or functional nucleic acid according to any one of the preceding claims, wherein the construct or functional nucleic acid is packaged in an AAV1 vector.

Images