Constructs for RNA expression

Abstract

Provided are synthetic constructs for providing an enhancer or variant thereof as a promoter to drive RNA expression, as well as nucleic acids, vectors, and cells encoding or comprising said constructs. Associated methods and pharmaceutical compositions for use in therapy are also provided herein.

Description

[0001] Constructs for RNA expression

[0002] Field of Invention

[0003] The invention relates to the field of novel constructs, nucleic acids, vectors, cells, composition and pharmaceutical compositions for providing an enhancer or variant thereof as a promoter to drive RNA expression. Associated methods for use in therapy are also provided herein.

[0004] Background

[0005] Constructs for driving RNA expression are widely used in modem molecular biology and gene therapy approaches worldwide. Crucial to such constructs is expression of a desired RNA sequence in the correct cell type(s). RNA expression is driven by two main types of DNA sequence elements in the genome; promoters and enhancers.

[0006] Promoters are found at the 5’ end of a gene that is to be transcribed into RNA and are capable of producing stable and properly processed mature RNA in a single direction in the genome. There are two classes of promoters; constitutive promoters (often found on housekeeping genes, also sometimes referred to as housekeeping promoters) which are active in every cell in the body, and cell-type specific promoters which are active in only a subset of cell types or under certain stimuli. Constructs require cell type specific promoters to drive RNA expression to avoid off-target RNA expression in the wrong cells.

[0007] However, cell type specific promoters have two problems. First of all, cell type specific promoters are rare and it is not possible to find a specific promoter for every cell type that exists. Secondly, cell type specific promoters often have overlapping expression in different cell types and thus are dependent on the addition of enhancers to give them added specificity. As cell type specific promoters are dependent on enhancers for their activity, an enhancer specific to the target cell type(s) has to be engineered into a construct in conjunction with a minimal promoter element at the 5’ of whichever RNA is to be expressed.

[0008] Because cell type specific promoters are rare, there is a limited known repertoire of enhancer and promoter combinations that work efficiently together and have the level of cell type specificity needed, thus limiting the cell types, stages of cellular differentiation and types of function that can be targeted by a construct. The selection of enhancer and promoter combinations available is vastly inadequate compared to the many different scenarios where one might want to achieve specific cell expression, developmental stage specific expression or context specific expression (e.g. in a tumour microenvironment).

[0009] In addition, because promoters naturally depend on enhancers, a minimal promoter element in a construct can interact with endogenous enhancers in the locale and produce leaky expression or mis-expression outside of the desired RNA sequence.

[0010] Finally, another major limitation is the size of the payload (the RNA sequence desired to be expressed) that can be packaged into a construct. A construct can be introduced into a cell through a viral vector, which delivers the payload within the construct to every cell and cell type it enters. However, the size of a viral vector is limited. For example, the maximum length of a construct that can be efficiently packaged into a Lentiviral Vector is 8.5kb, an Adenovirus Vector is 9kb and Adeno Associated Virus Vector is 4.7kb. These limits include the machinery required to drive RNA expression and the RNA sequence itself; therefore the size of the required machinery for driving correct RNA expression limits the size of the RNA sequence that can be expressed. Larger payloads of up to 35kb are possible but require the use of additional helper viruses during packaging and present safety problems due to the need for the complete removal of the helper virus from the desired virally packaged vector.

[0011] As explained above, scientists looked to combine enhancers with promoters as an alternative to promoters alone for driving RNA expression. Unlike promoters, enhancers are distributed unpredictably around the gene they control, and can be found in the regions of the genome between genes (intergenic) as well as within the noncoding intronic regions of genes (intragenic). Enhancers are regulatory DNA sequences that, when bound by transcription factors, activate or enhance the transcription from the promoter of an associated gene. There are estimated to be millions of cell type-specific enhancers in the human genome and enhancers are able to maintain their specificity when removed from their genomic context (natural environment). RNA transcripts produced from enhancers (eRNAs) are considered to be weak sources of transcription. eRNAs are short, are produced bidirectionally, and are unstable RNA transcripts that are rapidly degraded.

[0012] The observation was made that eRNA transcripts arising from intragenic enhancers could produce stable, spliced and polyadenylated transcripts. The art describes a strategy of using intragenic positioning of these enhancers to rely on the host’s native splicing signal and native polyadenylation signal in order to drive cell type specific stable RNA expression. The limitation of this configuration, due to its dependence on the host’s native splicing signal and native polyadenylation signal, is that it can only control the expression of the sequence of the gene the intragenic enhancer is embedded in and only those sequences between the position the enhancer is located and the host’s native polyadenylation site.

[0013] Accordingly, there is a need for providing an improved construct for driving RNA expression of any desired transcript in an increased range of cell types in an increased range of cell differentiation stages, whilst maintaining specific expression to only the desired target cell type or state, in constructs that can be easily delivered to the target cell type. In addition, there is a need for providing an improved construct for driving RNA expression that has a decreased size in the machinery required to drive RNA expression.

[0014] Brief Summary of the Disclosure

[0015] The invention provides a synthetic construct for providing an enhancer or variant thereof as a promoter to drive RNA expression, wherein the synthetic construct comprises:

[0016] (a) one or more transgenes for RNA expression;

[0017] (b) one or more stabiliser motifs; and

[0018] (c) one or more nuclear export signals.

[0019] Suitably, the enhancer is a native enhancer

[0020] Suitably, the native enhancer is in a native environment.

[0021] Suitably, the synthetic construct further comprises an enhancer.

[0022] Suitably, the enhancer is a native enhancer, a synthetic enhancer or an optimised native enhancer.

[0023] Suitably, the native enhancer is in a non-native environment. Suitably, the one or more stabiliser motifs is a synthetic stabiliser motif.

[0024] Suitably, the one or more stabiliser motifs is selected from a group consisting of polyadenylation (polyA) tail or Hairpin Stabilisation Sequence (HSS).

[0025] Suitably, the one or more stabiliser motifs is a polyadenylation (polyA) tail.

[0026] Suitably, the one or more stabiliser motifs is a Hairpin Stabilisation Sequence (HSS).

[0027] Suitably, the one or more nuclear export signals is a synthetic nuclear export signal Suitably, the one or more nuclear export signals is selected from a group consisting of an intron or a Cytoplasmic Accumulation Region motif E (CAR-E).

[0028] Suitably, the one or more nuclear export signals is an intron.

[0029] Suitably, the one or more nuclear export signals is a Cytoplasmic Accumulation Region motif E (CAR-E).

[0030] Suitably, the function of the one or more nuclear export signals and the function of the one or more stabiliser motifs are provided by Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE).

[0031] Suitably, the synthetic construct further comprises one or more flanking motifs.

[0032] Suitably, the synthetic construct comprises two or more stabiliser motifs, two or more nuclear export signals and two or more transgenes.

[0033] Suitably, the synthetic construct comprises two stabiliser motifs, two nuclear export signals and two transgenes.

[0034] The invention provides a nucleic acid encoding a synthetic construct disclosed herein.

[0035] The invention provides a vector comprising a synthetic construct or nucleic acid disclosed herein.

[0036] The invention provides a cell comprising a synthetic construct, nucleic acid or vector disclosed herein.

[0037] The invention provides a composition comprising a synthetic construct, nucleic acid, vector or cell disclosed herein.

[0038] The invention provides a pharmaceutical composition comprising a synthetic construct, nucleic acid, vector or cell disclosed herein.

[0039] The invention provides a composition, pharmaceutical composition, synthetic construct, nucleic acid, vector or cell disclosed herein for use in therapy.

[0040] The invention provides a composition, pharmaceutical composition, synthetic construct, nucleic acid, vector or cell according to any preceding claim for use in gene therapy. Brief Description of the Figures

[0041] Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which:

[0042] Figure 1Ato C: (A) Schematic of a basic TRANCER construct using a synthetic intron and synthetic polyA site. The “enhancer” box represents the positioning of the interchangeable enhancer sequence to direct differing patterns of expression. The 5’ and 3’HOM boxes represent the position of optional flanking homology arms to direct CRISPR mediated homologous integration into specific regions of the genome if required. (B) FACs analysis of the production of the mNeon green reporter gene that represents protein production from the TRANCER construct. Positive cells are labelled in dark grey (above the dashed line at 2x102), negative cells in light grey. The name of the enhancer element used for each construct is above each FACs plot. Scramble control is the sequence of the Nanog enhancer which has been complemented but not reversed to destroy the transcription factor binding sites without changing the overall base composition of the DNA segment. (C) A graph of the quantification of the FACs plots in Figure 1 B, with percentage of positive cells for dark grey on the Y axis.

[0043] Figure 2Ato B: (A)The left panel in Figure 2A shows the same FACs results from Figure 1 B for the Nanog enhancer, showing the heterogenous expression of the Nanog TRANCER. The positive cells from this population were sorted and replated for 48hrs to create a new population (arrow labelled +mNG) and FACs analysis shows that this positive population returns the same heterogenous population mixture of expressing and non-expressing cells. Similarly, the negative expression population was replated for 48hrs (arrow labelled -mNG) and FACs analysis revealed a reversion to the original heterogenous population of expressing and nonexpressing cells. (B) Single cell analysis of mES cells (cells on the X axis, genes on the Y axis) showing the relative expression of the genes indicated. Underscoring the heterogenous results with the Nanog TRANCER element, Nanog endogenous gene expression (outlined box) is also highly heterogeneous at the single cell level. The colour of each bar indicates different expression levels with a darker colour indicating lower expression. This shows that Nanog gene expression naturally fluctuates within a population of mES cells similar to TRANCER expression driven by the Nanog enhancer.

[0044] Figure 3A to B: (A) A schematic of TRANCER constructs (described in Figure 1A) where (i) represents a standard TRANCER (described in Figure 1A) and (ii) represents a TRANCER where the 225bp polyA addition site is replaced with a 78bp Hairpin Stabilisation Sequence (HSS) from the MALAT1 non-coding gene. In (iii) the control for these experiments is shown where both the PolyA and HSS are removed so the construct contains no stabiliser motif. These experiments were performed for the Pou5f1 enhancer and using Scramble (reversed, non-complementary Nanog sequence) (B) FACs analysis quantification for neon green levels produced from these TRANCER constructs (see Figure 1 B and Figure 1 C) in addition to control constructs (Scramble PolyA and Scramble MSS), which are constructs where the PolyA or HSS site is replaced with the complemented sequence of the PolyA or HSS sequence, respectively.

[0045] Figure 4Ato C: (A) A construct containing an HSS but with the intron removed no longer produces detectable mNeon green expression. Thus, indicating that a method of engagement with the TREX pathway for RNA export from the nucleus is essential for overall TRANCER function. Experiments performed using the Pou5f1 enhancer. (B) Using the Tmprss13 enhancer, 3 to 5 copies of the 10bp Cytoplasmic Accumulation Region (CAR) motif can replace the intron sequence and regain mNeon green expression in a copy number dependant manner. (C) The effect of the inclusion of the Woodchuck Posttranscriptional Response Element (WPRE) into a standard TRANCER construct (A standard TRANCER without the WPRE is the first construct in the schematic on the right hand side. The enhancer sequence used in this construct and the subsequent constructs is the Tmprss13 enhancer. The second construct down is this standard construction with WPRE sequence added). With the WPRE present, mNeon green expression is maintained in the absence of PolyA site (third construct down in schematic) and in the absence of both PolyA and an intron (fourth construct down). The quantification of mNeon green positive cells is shown in the left hand panel with the output of each construct annotated by the pattern shown in both the graph bars and beside the construct schematics. The independent scrambled enhancer control constructs (scramble) abolish mNeon green expression. Figure 5Ato E: (A) The schematic of a bidirectional TRANCER construct designed to drive the expression of two reporter proteins simultaneously. Homology arms for genome integration (5’ and 3’ HOM), synthetic introns (intron), puromycin resistance (PuroR) and, enhancer position (using Nanog enhancer) and mNeon green reporter (mNG) are displayed as in Figure 1A. The new mCherry reporter is also displayed (mCherry). The design of this construct allows for the simultaneous expression of the mNeon green reporter and the mCherry reporter using the bidirectional transcription of the centrally placed enhancer sequence. (B) FACs analysis of a bidirectional construct driven by the heterogeneous Nanog enhancer (Figure 1 B) with mNeon Green expression on the Y Axis and mCherry expression on the X axis, showing coexpression of both reporter genes in the expressing cells. (C) A separate analysis of the mNeon Green reporter channel and mCherry channel showing that both reporters have the same heterogenous pattern typical of the Nanog TRANCER (See Figure 1 B and Figure 2A). (D-E) Fixing a mutated GFP using a bidirectional TRANCER construct. (D) Schematic of the bidirectional TRANCER construct, which simultaneously expresses an adenine base editor (ABE) and a single guide RNA (sgRNA) in a tissue-specific manner. (E) Diagram of a genomically integrated EF1a promoter driving constitutive expression of a GFP gene carrying an early stop codon (TAG), which disrupts translation. This mutation can be corrected to a tryptophan codon (TGG) via ABE-mediated base editing, thereby restoring GFP expression. Flow cytometry plots showing GFP expression levels across different transfection conditions.

[0046] Figure 6: A quantification of the relative numbers of positive cells for the mES specific Nanog enhancer (triangle) and scrambled control sequence (circle) in three different cell types. High level expression is only present in mouse ES cells (mESC) where the Nanog enhancer is active. Fibroblast cells (T3T) and Human embryonic kidney cells (HEK293T) have an inactive Nanog enhancer, and the Nanog TRANCER also displays non-significant expression levels.

[0047] Figure 7Ato E: (A) ATAC-seq data in differentiated mouse erythroid cells and mouse ES cells showing that open chromatin activity for the R2 enhancer (in bold, with genomic position highlighted with a grey bar) is restricted to Erythroid cells. (B) Top: The schematic of a TRANCER construct containing erythroid R2 element as specific the transcriptional source and replacing the mNeon Green reporter gene with the coding sequence of human CD19. Bottom: Stages in the generation of erythroid cells from mES cells. Experimental steps (left to right), from formation of embryoid body organoids, to immunopurification of red blood cell precursors from the complex mixture of Ectoderm, Mesoderm and Endoderm lineages produced in this system. (C) FACs analysis of total organoid tissue derived from the embryoid body protocol. Human CD19 can only be detected in organoid derived cells which have been edited with the erythroid specific TRANCER. (D) Quantitation of the FACs analysis of WT and TRANCER containing organoids from Figure 7C. (E) LIMAP visualizations derived from multiome data illustrating that the R2-tCD19 TRANCER construct is specifically expressed in erythroid cells, as indicated by its colocalization with Hba-a1 expression. The overlap between TRANCER activity and erythroid marker expression demonstrates the high tissue specificity of the construct, supporting the targeted and selective nature of TRANCER technology, (i) LIMAP projection showing cell type annotations based on transcriptom ic profiles, (ii) LIMAP of the same dataset, where increasingly darker colours represent higher counts of the inserted R2-tCD19 TRANCER construct, (iii) Expression of the erythroid marker gene Hba-a1, highlighting its spatial overlap with cells expressing Cd19.

[0048] Figure 8Ato C: (A) A comparison of active chromatin marks between stimulated (stim) and unstimulated (naive) Jurkat human T-cell model (top panel) and primary human T-Cells (bottom panel) identify a stimulated T-cell enhancer. The epigenetic marks include open chromatin based on ATAC-seq (ATAC) and histone H3 lysine 27 acetyl ChlP-seq (H3K27ac). Gene annotation is shown on the top and an example of a stimulation dependent enhancer is highlighted by a light grey bar. (B) A schematic of the strategy of integrating TRANCER machinery into the genome adjacent to a pre-existing enhancer to use the endogenous enhancer as a transcriptional source, rather than supply the enhancer as a synthesised exogenous DNA sequence. (C) FACs analysis shows activation dependant increase in Reporter Gene (mNeon green) expression upon stimulation of the Jurkat cells.

[0049] Figure 9: General summary figure of the TRANCER technology. In the genome, enhancers provide tissue specificity while promoters establish the transcriptional start site of the gene. Enhancers and promoters need to communicate through enhancer-promoter contacts. However, identifying these interactions is not trivial and can affect expression levels of synthetic constructs. The TRANCER technology bypasses the need for a promoter by stabilising the short-lived eRNA expression into productive transcription through the addition of sequences capable of interacting with the TREX complex (leading to export) and 3’ stabilisation. The table in Figure 9 exemplifies the different elements of the TRANCER technology and their distinct applications of stability and export. The Table also highlights the Figures of this application where the different elements of the TRANCER technology and their distinct applications of stability and export are demonstrated. The TRANCER technology can be applied either exogenously via stable integrations (Figures 1-5,7) or transiently (Figure 6); additionally, the natural enhancer can be used (Figure 8).

[0050] Figure 10: Lentiviral delivery of TRANCER constructs. (A) Diagram of the lenti-TRANCER being used (B) Using top-performing synthetic enhancers. This panel demonstrates that TRANCER constructs can be effectively delivered using lentiviral vectors in K562 cells. Both integrated and episomal versions show measurable activity, confirming that lentiviral systems are a viable platform for TRANCER delivery and functional expression. Top enhancer from the synthetic enhancer publication were made into a lentiviral construct and tested in K562 cells1. Two lentiviral versions were tested, integrated and episomal, and activity was measured using flow cytometry. N=3, error bars show SEM. (C) Using Nanog (natural) and Scramble (negative control) using the integrated lentiviral vector tested in mESC cells.

[0051] Figure 11 : This figure shows that the synthetic enhancer designed for K562 cells drives gene expression specifically in this cell type. In contrast, the HepG2- specific enhancer does not induce expression in K562 cells, confirming its lack of activity outside its intended context. As expected, the scrambled control fails to drive expression. Together, these results demonstrate the cell-type specificity of synthetic enhancers and their ability to function as a TRANCER. Enhancers were selected for their MinGap value with 1 being the highest value. The scrambled control was used as a negative control. Plot was normalised over scrambled control. Differences among treatment groups were assessed using a one-way ANOVA. Tukey’s post-hoc test was performed to identify specific group differences, and adjusted P-values were used to determine statistical significance between group means. Significance thresholds were set at P < 0.05, with results reported as not significant (ns) or with the appropriate significance level. Statistical analyses were conducted using GraphPad Prism.

[0052] Figure 12. Stable CD19 expression over time in K562 transfected cells. K562 cells were transduced with TRANCER containing either a Synthetic or HS2 promoter driving CD19 expression. Following antibiotic selection for two weeks and complete removal, CD19 surface expression was monitored weekly by flow cytometry, and the percentage of CD19-APC-positive cells was quantified. Both constructs maintained stable expression over three weeks post-selection, with the Synthetic promoter showing consistently higher CD19 expression levels compared to HS2. Data are presented as mean percentage of CD19-positive gated cells over time.

[0053] Figure 13. T-cell mediated killing of K562 target cells transfected with a CD19 expressing TRANCER construct driven by either a synthetic or a HS2 enhancer. A) Schematic depiction of the TRANCER lentiviral constructs used containing either a synthetic or a HS2 enhancer. Lentiviral vectors were transfected into K562 and selected in puromycin for 2 weeks. CD19 antibodies were used to confirm CD19 expression. (B) Percentage of live / dead K562 cells following co-culture with T-cell expressing either Anti-hCD19- |3Gal (control, black) or Anti-hCD19-CD3 (bispecific engager, grey). Target cells were incubated with effector T-cells for 72 h. Bars represent the mean ± SD of biological replicates. Anti-hCD19-CD3 treatment significantly increased target cell killing compared to Anti-hCD19-[3Gal in both conditions, demonstrating effective T-cell activation and cytotoxicity.

[0054] Various aspects of the invention are described in further detail below.

[0055] Definitions

[0056] As used herein, “synthetic construct” is interchangeable with construct or TRANCER (Transcriptionally Autonomous Enhancers) or TRANCER construct. In the present invention, a synthetic construct comprises one or more transgenes, one or more stabiliser motifs and one or more nuclear export signals.

[0057] As used herein, the terms "enhancer" or “enhancer sequence” or “enhancer element” mean an autonomous piece of DNAthat is bound by transcription factors and can initiate transcription in a context dependent manner. An enhancer element may be a “native” enhancer that is a sequence with the desired activity that is found in nature in the genome of any species. The enhancer element may also be a “synthetic” enhancer which is an artificial combination of sequences such as Transcription Factor binding sites learned through the investigation of Transcription Factor binding sites used in native enhancers to cause the synthetic enhancer to have the desired activity. The synthetic enhancer may be designed using prior knowledge from the field derived from the study of native enhancers or from machine learning based approaches trained on the sequences of native enhancers. The enhancer may also be an “optimised” native enhancer which is a sequence naturally found in nature which has had its sequence altered based on prior knowledge from the field derived from the study of native enhancers or from machine learning based approaches trained on the sequences of native enhancers. The alteration of the optimised enhancer may alter the amount of transcription of the original native enhancer or alter the context in which it is transcribed. An enhancer may be used so as to enhance the expression efficiency of a gene of interest. A plurality of enhancers or a single enhancer may be used.

[0058] As used herein, “native” refers to a naturally occurring element. For example, a “native enhancer” refers to an enhancer found naturally within an organism. A native enhancer can be found in a native environment or a non-native environment. A native enhancer in a native environment is a naturally occurring enhancer in their natural genomic context. A native enhancer in a non-native environment is a naturally occurring enhancer in an environment the enhancer is not naturally found in. Examples of native enhancers include Nanog, Pou5f1 and Sox2, a-globin R2 (also referred to as R2 enhancer), Tmprss13, B3gnt7, Stimulated T-cell activation ATAC site.

[0059] As used herein, the term “native intergenic enhancer” refers to a naturally occurring enhancer found in the regions of the genome between genes.

[0060] As used herein, the term “native intragenic enhancer” refers to a naturally occurring enhancer found within the noncoding intronic regions of genes.

[0061] As used herein, the term "variant" is interchangeable with “modified” or “modification”, and refers to a substance, such as a polypeptide, polynucleotide, or the like, which differs partially from the original substance. Examples of such a variant include a substitution variant, an addition variant, a deletion variant, a truncated variant, and an allelic variant.

[0062] As used herein, the term “functional variant" refers to a variant which retains a biological activity (in particular, promoter activity) which the sequence of standard is responsible for.

[0063] As used herein, the term “promoter” or “promoter sequence" refers a DNA region that determines the initiation site of transcription of a gene and directly regulates the frequency of transcription, and is a base sequence to which RNA polymerase usually binds to initiate transcription.

[0064] As used herein, the term “drive RNA expression” refers to expression of a gene, transgene, a polynucleotide, a polypeptide, or the like, and indicates that the gene or the like are transcribed and translated into polypeptides. In one aspect of the expression, genes may be transcribed into mRNA.

[0065] As used herein, the term “transgene” is interchangeable with “gene of interest" or “a sequence of interest” and refers to a DNA sequence that is transcribed into RNA.

[0066] As used herein, the term “payload” refers to a transgene within a synthetic construct.

[0067] As used herein, the term “stabiliser motifs” refers to a structure in single-stranded DNA or RNA. A stabiliser motif prevents a transcript from being degraded by the exosome. Examples of stabiliser motifs include poly(A) tails and HSS. In one embodiment, the stabiliser motif is a tertiary hairpin structure in single-stranded DNA or RNA. In one embodiment, a stabiliser motif is modified from their natural genomic context. In one embodiment, a synthetic stabiliser motif is a modified endogenous stabiliser motif. In one embodiment, a stabiliser motif is a synthetic stabiliser motif.

[0068] As used herein, the term “nuclear export signal” refers to a single stranded DNA or RNA sequence that signals to a nuclear transport pathway that a transcript is to be exported from the nucleus to the cytoplasm. Examples of nuclear export signals include introns and CAR-E motifs. In one embodiment, a nuclear export signal is modified from their natural genomic context. In one embodiment, a nuclear export signal is a modified endogenous nuclear export signal. In one embodiment, a nuclear export signal is a synthetic nuclear export signal. As used herein, "intron" refers to a gene region provided as an intervening sequence that is present in DNA and included in a primary transcript, but not included in the final functional mature RNA, and removed by splicing.

[0069] As used herein, “flanking motifs” is interchangeable with “flanking amino acids” and “flanking sequences” and refers to residues adjacent to a specific sequence of interest.

[0070] As used herein “nucleic acid sequence”, “polynucleotide”, “nucleic acid” and “nucleic acid molecule” are used interchangeably to refer to an oligonucleotide sequence or polynucleotide sequence. The nucleotide sequence may be of genomic, synthetic or recombinant origin, and may be double-stranded or single-stranded (representing the sense or antisense strand). The term "nucleotide sequence" includes genomic DNA, cDNA, synthetic DNA, and RNA (e.g. mRNA) and analogs of the DNA or RNA generated, e.g., by the use of nucleotide analogs.

[0071] As used herein, the term “vector” refers to a nucleic acid sequence capable of transporting another nucleic acid sequence to which it has been operably linked. The vector can be capable of autonomous replication or it can integrate into a host DNA. The vector may include restriction enzyme sites for insertion of recombinant DNA and may include one or more selectable markers or suicide genes. The vector can be a nucleic acid sequence in the form of a plasmid, a bacteriophage or a cosmid.

[0072] As used herein, the term "cell" is interchangeable with “host cell” or “modified cell” and includes any cell into which the synthetic construct, nucleic acid or vector described herein may be introduced. Once a synthetic construct, nucleic acid, vector has been introduced into the cell, it may be referred to as a “modified cell” herein. Once the synthetic construct, nucleic acid, vector, is introduced into the host cell, the resultant modified cell should be capable of expressing the one or more transgene for RNA expression.

[0073] As used herein, a pharmaceutical composition may comprise a synthetic construct, nucleic acid or vector described herein along with a pharmaceutically acceptable excipient, adjuvant, diluent and / or carrier.

[0074] As used herein, "pharmaceutically acceptable" refers to a material that is not biologically or otherwise undesirable, i.e. , the material may be administered to an individual along with the selected synthetic construct, nucleic acid, or vector without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained. Pharmaceutically acceptable excipients are well known in the art. A suitable excipient is therefore easily identifiable by one of ordinary skill in the art. By way of example, suitable pharmaceutically acceptable excipients include water, saline, aqueous dextrose, glycerol, ethanol, and the like.

[0075] Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of them mean “including but not limited to”, and they are not intended to (and do not) exclude other moieties, additives, components, integers or steps.

[0076] Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

[0077] Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.

[0078] Terms such as "a", "an" and "the" are not intended to refer to only a singular entity, but include the general class of which a specific example may be used for illustration. The terminology herein is used to describe specific embodiments of the invention, but their usage does not delimit the invention, except as outlined in the claims.

[0079] Unless otherwise indicated, nucleic acid molecules are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively. It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described, as these may vary, depending upon the context they are used by those of skill in the art.

[0080] Various aspects of the invention are described in further detail below.

[0081] Detailed Description The present invention provides a synthetic construct for providing an enhancer or variant thereof as a promoter to drive RNA expression, wherein the synthetic construct comprises:

[0082] (a) one or more transgenes for RNA expression;

[0083] (b) one or more stabiliser motifs; and

[0084] (c) one or more nuclear export signals.

[0085] The inventors surprisingly discovered that by adding a stabiliser motif and a nuclear export signal, an enhancer can functionally act as an alternative to a promoter and drive productive levels of RNA expression. By integrating mechanisms that will protect a transcript from exosome degradation and that will cause a transcript to be recognised by a nuclear transport pathway, the inventors surprisingly discovered that enhancers, including native, synthetic and optimised native enhancers, are able to drive RNA expression and protein production. The stabiliser motif is able to protect the enhancer transcript from exosome degradation whilst the nuclear export signal allows the transcript to be recognised by the nuclear transport pathway and be transported into the cytoplasm. The combination of the one or more stabiliser motifs and the one or more nuclear export signals mean that enhancers are capable of producing high levels of stable, long, and properly processed mature RNA.

[0086] The claimed synthetic construct is therefore able to drive expression in any cell type, context or developmental stage desired. Furthermore, the claimed invention provides highly specific activity that is predictable and entirely dependent on the target enhancer. Finally, the claimed synthetic construct is more compact in size, allowing for a larger payload.

[0087] In one embodiment, the native enhancer is in a native environment. A synthetic construct according to the present invention may be inserted within a genome next to a native enhancer. The synthetic construct is able to utilise the native enhancer as a transcriptional source to drive RNA expression of the one or more transgenes. Advantageously, this removes the need for the inclusion of an enhancer sequence in the synthetic construct, providing a smaller synthetic construct and allowing for a larger payload. Also, advantageously, the synthetic construct can be used with any native enhancer in their native genomic context to drive cell specific expression of any desired transgene.

[0088] A synthetic construct according to the invention can be inserted into the genome using targeted integration. “Targeted integration” and “targeted insertion”, refers to a process involving insertion of one or more of the synthetic constructs according to the invention at pre-selected sites in the genome, with or without deletion of an endogenous sequence at the insertion site. Various methods and compositions for targeted integration are known in the art. These methods often involve the use of engineered cleavage systems to induce a double strand break (DSB) or a nick in a target DNA sequence such that repair of the break by an error-prone process such as non-homologous end joining (NHEJ) or repair using a repair template (homology directed repair or HDR) can result in the insertion of a synthetic construct according to the present invention. Cleavage can occur through the use of specific nucleases such as engineered zinc finger nucleases (ZFN), transcription-activator like effector nucleases (TALENs) or CRISPR / Cas systems with an engineered crRNA / tracr RNA (“single guide RNA”) to guide specific cleavage.

[0089] In one embodiment, the synthetic construct further comprises an enhancer and the synthetic construct can be supplied as an exogenous construct. In one embodiment, the synthetic construct comprises an enhancer, one or more transgenes for RNA expression, one or more stabilisation motifs and one or more nuclear export signals. The enhancer is included within the synthetic construct and used to drive RNA expression. In one embodiment, the enhancer is a native enhancer, a synthetic enhancer or an optimised native enhancer. In one embodiment, the enhancer is a native enhancer. In one embodiment, the enhancer is a synthetic enhancer. In one embodiment, the enhancer is an optimised native enhancer. In one embodiment, the native enhancer is in a non-native environment. Advantageously, any enhancers can be inserted into the synthetic construct, and the synthetic construct can be used to drive RNA expression at any location within the genome, regardless of whether the enhancer is a native enhancer and / or regardless of whether the native enhancer is in a native or non-native environment.

[0090] In one embodiment, the native enhancer is an intergenic native enhancer.

[0091] In one embodiment, the native enhancer is an intragenic native enhancer. In one embodiment, the one or more stabiliser motifs is a synthetic stabiliser motif.

[0092] In one embodiment, the one or more stabiliser motifs is selected from a group consisting of polyA tail or Hairpin Stabilisation Sequence (HSS).

[0093] In a preferred embodiment, the one or more stabiliser motif is a polyA tail. In a preferred embodiment, the one or more stabiliser motif is a synthetic polyA tail. The transcription of a polyadenylation (polyA) signal at the end of the 3’ UTR guides the enzymic addition of a polyA tail, which is known to protect transcripts from degradation and stabilises them. The inventors surprisingly discovered that the addition of a polyA signal could increase the levels of the enhancer derived transcripts in the cell by preventing enhancer transcript degradation.

[0094] In a preferred embodiment, the one or more stabiliser motifs is a Hairpin Stabilisation Sequence (HSS). In a preferred embodiment, the one or more stabiliser motifs is a synthetic Hairpin Stabilisation Sequence (HSS). The highly compact Hairpin Stabilisation Sequence (HSS) is employed by highly stable but non-coding transcripts such as tRNAs and the non-coding transcript MALAT1. Advantageously, HSS is able to protect transcripts from degradation, and is very small in size (78bp) providing a smaller synthetic construct and allowing for a larger payload.

[0095] In a preferred embodiment, the one or more nuclear export signal is a synthetic nuclear export signal.

[0096] In a preferred embodiment, the one or more nuclear export signal is selected from a group consisting of an intron or a Cytoplasmic Accumulation Region motif E (CAR-E). RNA transcripts need to be efficiently transported through the nuclear pore and into the cytoplasm. This process is known to be dependent on the recognition of splicing events by adaptors for the TREX complex. Splicing takes place in the nucleus. Splicing involves removing introns (intervening sequences), while exons (coding sequences) are joined together. Splicing consists of two sequential branching and ligation steps; both these reactions are trans-esterification reactions classified as nucleophilic substitutions. Conserved consensus cis-acting sequences within the pre-mRNA allow the identification of introns. These sequences include the 5' donor site, or 5' splice site (5'SS), which features a conserved Gil dinucleotide within a larger, less conserved region. Additionally, the 3' acceptor site, or 3' splice site (3'SS) contains an AG dinucleotide situated and a branchpoint sequence that forms a lariat structure 3. These elements are recognised by the spliceosome and are required for its assembly2The stepwise assembly of the spliceosome can be classified into 3 phases: assembly and activation, execution of the splicing reactions and disassembly of the spliceosome.

[0097] Coupled with RNA processing, the mature mRNA is exported from the nucleus through the nuclear pore complex (NPC) into the cytoplasm. RNA export through the NPC includes three major steps: mRNA docking at the nuclear basket, trafficking though the central channel and releasing from the cytoplasmic filaments into the cytoplasm. The transport / export (TREX) complex is the main mediator coupling transcription and processing to export. Therefore, the addition of a nuclear export signal such as an intron would cause the enhancer transcripts, stabilised by the addition of stabiliser motifs, to be also exported to cytoplasm and translated.

[0098] In one embodiment, the one or more nuclear export signal is an intron. In one embodiment, the one or more nuclear export signal is a synthetic intron.

[0099] In one embodiment, the one or more nuclear export signal is a Cytoplasmic Accumulation Region motif E (CAR-E). In one embodiment, the one or more nuclear export signal is a synthetic Cytoplasmic Accumulation Region motif E (CAR-E). Advantageously, CAR-E is still able to interact with TREX export machinery and export transcription to the cytoplasm and is very small in size providing a smaller synthetic construct and allowing for a larger payload.

[0100] In one embodiment, the invention provides a synthetic construct for providing a enhancer or variant thereof as a promoter to drive RNA expression, wherein the synthetic construct comprises one or more transgenes for RNA expression and WPRE. WPRE has dual functionality and can direct both stabilisation of the RNA and its subsequent export to the cytoplasm for translation. WPRE is thus able to function as a stabiliser motif and a nuclear export signal within the claimed synthetic construct.

[0101] The WPRE element is a DNA element derived from the genome of the Woodchuck Hepatitis virus, which when transcribed into RNA adopts a series of tertiary hairpin structures. Structural prediction of WPRE RNA structure has identified 3 hairpin structures within its sequence that likely direct these dual functions.

[0102] In one embodiment, the invention provides a synthetic construct for providing a enhancer or variant thereof as a promoter to drive RNA expression, wherein the synthetic construct comprises one or more transgenes for RNA expression and one or more WPRE hairpin structures.

[0103] Advantageously, WPRE is relatively small in size as it is a single element, therefore providing a smaller synthetic construct and allowing for a larger payload.

[0104] In one embodiment, the synthetic construct further comprises one or more flanking motifs. Advantageously, flanking motifs facilitate homology directed repair, allowing the construct to be inserted into a specific genomic location.

[0105] In one embodiment, the invention provides a synthetic construct for providing a enhancer or variant thereof as a promoter to drive RNA expression, wherein the synthetic construct comprises:

[0106] (a) two or more transgenes for RNA expression;

[0107] (b) two or more stabiliser motifs; and

[0108] (c) two or more nuclear export signals.

[0109] In one embodiment, the invention provides a synthetic construct for providing a enhancer or variant thereof as a promoter to drive RNA expression, wherein the synthetic construct comprises:

[0110] (a) two transgenes for RNA expression;

[0111] (b) two stabiliser motifs; and

[0112] (c) two nuclear export signals.

[0113] The natural bidirectional nature of enhancer transcription means that a claimed synthetic construct comprising two or more stabiliser motifs, two or more nuclear export signals and two or more transgenes is able to express two or more transcripts simultaneously. Advantageously, the claimed synthetic construct can express two or more transcripts simultaneously and synchronously in the same cell. For example, the claimed synthetic construct is able to drive expression of a CRISPR guide RNA as well as the Cas9 protein itself.

[0114] In one embodiment, a nucleic acid encodes a synthetic construct according to the invention.

[0115] In one embodiment, a vector comprises a synthetic construct or nucleic acid according to the invention. In one embodiment, a cell comprises a synthetic construct, nucleic acid or vector according to the invention. The cell is typically a eukaryotic cell, and particularly a human cell. Suitably, the cell may be a human immune cell, for example a T cell, NK cell, or an innate lymphoid cell (ILC). Suitably, the cell is a T cell.

[0116] In one embodiment, a composition comprises a synthetic construct, nucleic acid, vector or cell according to the invention.

[0117] In one embodiment, a pharmaceutical composition comprises a synthetic construct, nucleic acid, vector or cell according to the invention.

[0118] In one embodiment, a composition, pharmaceutical composition, synthetic construct, nucleic acid, vector or cell according to the invention is for use in therapy.

[0119] In one embodiment, a composition, pharmaceutical composition, synthetic construct, nucleic acid, vector or cell according to the invention is for use in gene therapy.

[0120] In one embodiment, a synthetic construct according to the invention may be used to express a specific gene in any target cell type or developmental stage desired, and then the biological outcomes can be monitored. For example, a gene could be expressed only in a subset of T-Cells, such as invariant natural killer cells (iNKT- cells), to determine if the gene induces a novel biological activity. In another example, a synthetic construct according to the invention may be used to express a gene in the same manner as a damaged gene in gene replacement therapy.

[0121] In one embodiment, a synthetic construct according to the invention may be used to express a fluorescent marker or a novel receptor in a subset of cells (e.g. Regulatory T-cells or Tregs) which can be easily isolated using flow cytometry.

[0122] In one embodiment, a synthetic construct according to the invention may be used to specifically target stem cells to induce stem cell expansion either in vitro or in vivo that turns off once the cells differentiate. In a different application of the same concept, a synthetic construct according to the invention may be used to provide T- cell subset specific expression to prevent T-cell exhaustion in immunotherapy.

[0123] In one embodiment, a synthetic construct according to the invention may be used for cell type specific CRISPR / Cas9 expression. Advantageously, the synthetic construct allows editing only in the target cells of interest. The reader's attention is directed to all papers and documents which are filed concurrently with or previous to this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference.

[0124] All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and / or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive.

[0125] Each feature disclosed in this specification (including any accompanying claims, abstract and drawings), may be replaced by alternative features serving the same, equivalent, or similar purpose, unless expressly stated otherwise. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features.

[0126] The invention is not restricted to the details of any foregoing embodiments. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

[0127] Aspects of the invention are demonstrated by the following non-limiting examples.

[0128] Examples

[0129] EXAMPLE 1: The inclusion of a synthetic Polyadenylation site in combination with a synthetic intron is sufficient to convert enhancers eRNAs into productive protein coding transcripts.

[0130] We generated vectors containing a basic TRANCER construct comprising a native enhancer, synthetic intron and a synthetic PolyA site, 5’ and 3’ homology arms and a neon green fluorescent reporter gene mNG (Figure 1 A).

[0131] We show the FACs analysis of 6 TRANCER constructs containing as their transcriptional source 5 randomly selected enhancer sequences from mouse ES cells (named by their target gene if known or the most proximal gene if not). In the sixth construct we include a scrambled control sequence (SEQ ID NO:1) in place of an enhancer (the complementary sequence of the Nanog enhancer, termed “scrambled” or “Ser”) (Figure 1 B).

[0132] As shown from the FACs plots, all 5 enhancer sequences (SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6) drive high levels of neon green protein production, though the actual level varies between the enhancers (Figure 1 c). The known enhancer of the Nanog gene (SEQ ID NO: 2) consistently gave heterogenous levels of expression. This heterogeneity of expression persists even when the high expressing cells are sorted from the population and replated (Figure 2A). After being cultured for 48 hours, these homogenously expressing cells revert back to the same heterogenous pattern. Furthermore, if the non-expressing cells (- mNG) are sorted from the mixture they also revert in culture to the same ratio of expressing and non-expressing cells. This indicates that this behaviour is intrinsic to the enhancer sequence used rather than being a technical artifact. Consistent with published work3, analysis of Nanog expression using single cell RNA-seq of mES cells shows that the expression of the Nanog gene itself is naturally heterogenous in mES cells (Figure 2B). As the expression pattern of genes are dictated by their enhancers this suggests that this behaviour is intrinsic to the Nanog enhancer sequence. This result combined with the differing levels of expression of the 5 enhancers tested, highlights the claim that TRANCERs can provide a spectrum of cell type specific transcriptional control as rich and varied as genes found in nature.

[0133] EXAMPLE 2: Other methods of transcript stabilisation can be used to form TRANCERs further decreasing the size of TRANCER constructs.

[0134] We wished to test whether other mechanisms employed in the nucleus could be used to form TRANCER constructs. In particular, we wanted to test if the highly compact Hairpin Stabilisation Sequence (HSS) employed by some highly stable but non-coding transcripts such as tRNAs and the non-coding transcript MALAT1 could replace the large PolyA site (225bp). The main advantage of using this sequence is its highly compact size of 78bp. To that end, we replaced the polyadenylation sequence of a TRANCER construct containing the highly transcriptionally efficient enhancer of the Pou5f1 gene (SEQ ID NO: 5) with the HSS sequence of MALAT1 (SEQ ID NO: 8) in place of the PolyA site (see i and ii in Figure 3A). As a control, we also generated a construct lacking any stabilisation sequence, removing both PolyA and HSS sequences (see iii in Figure 3A). As can be seen in Figure 3B, the MALAT1 Hairpin stabilisation sequence (MSS) is capable of the generation of equivalent if not greater levels of neon green protein than constructs with polyA even though it is derived from non-coding transcripts. Importantly, the construct that contains neither a PolyA nor HSS or contains a scrambled (complement sequence) version of the MSS or PolyA sequence generates no expression, equivalent to the scrambled enhancer controls (Figure 3B). This emphasises the critical role of stabilisation sequences in the TRANCER constructs and that the differing classes of sequence that perform this function in the genome, whether they are from coding or non-coding transcripts can perform this function in TRANCER and likely also canonical promoter-based constructs.

[0135] EXAMPLE 3: Engagement with the TREX nuclear export pathway is critical for protein production from TRANCERS

[0136] We next tested the necessity and variety of means of engaging the synthetic stabiliser motifs with the TREX pathway for export from the nucleus. To establish the baseline necessity, we removed the synthetic intron sequence from the HSS containing construct with the Pou5F1 enhancer that generates high levels of expression in mES cells. As can be seen in Figure 4A, the removal of the intronic sequence completely abrogates neon green expression, so that engagement with TREX is critical for TRANCER function. The necessity for splicing has previously been determined for canonical promoter-based constructs for spliced genes, however introns are large sequences and will decrease the cargo capacity of the TRANCER.

[0137] We investigated other potential sequences capable of directing engagement with the TREX pathway. It has been shown that the ability of intronless genes to bypass the need for an intronic sequence is due to sequence motifs in their RNA which can be bound by components of the TREX pathway and enable the export of the transcript to the cytoplasm. Such motifs could be used in place of the large intron sequence in TRANCERs, with the benefit of a substantial decrease in the size of the required TRANCER constructs and increase in payload capacity. While several possible motifs have been suggested, the best characterised of these in the Cytoplasmic Accumulation Region motif E (CAR-E). This is a 10 base pair consensus motif derived from 3 intronless genes and it has been shown that it has the capacity to interact with Prp19 complex and U2Af2 proteins of the TREX export machinery.

[0138] To test whether the same motif could replace the need for an intron in TRANCERs we added a tandem array of 3-5 copies of the 10 base pair CAR-E sequence consensus (30-50bp) into TRANCER constructs adjacent to the PolyA motif and removed the intronic sequence. As can been in Figure 4B, the inclusion of the CAR- E sequence (SEQ ID NO: 9) was able to drive the expression of neon green protein in a copy number dependant fashion in the absence of an intronic sequence. This shows that RNA motifs found naturally in the sequence of intronless genes can bypass the need for large intron sequences in TRANCER constructs and likely also in canonical expression constructs. This currently means that the TRANCER machinery has been reduced to a compact 3’ UTR sequence formed from a combination of a multimer of a TREX engagement motif and a sequence stabilisation motif (PolyA or HSS, Figure 4B).

[0139] EXAMPLE 4: The Woodchuck Posttranscriptional Response Element (WPRE) represent a combined TREX engagement and RNA stabilisation motif in TRANCER constructs.

[0140] The WPRE element is a DNA element derived from the genome of the Woodchuck Hepatitis virus, which when transcribed into RNA adopts a series of tertiary hairpin structures. We added WPRE (SEQ ID NO: 10) to our standard TRANCER construct with both polyA and intron sequences to see if this would also boost the output of TRANCER constructs (Figure 4C). We also tested a standard TRANCER construct with WPRE alone, a standard TRANCER construct with a polyA sequence, and a standard TRANCER construct with WPRE and an intron.

[0141] It is extremely surprising that, while the inclusion of the WPRE did not seem to boost neon green production compared to the standard TRANCER, the removal of the polyA site or both the intronic sequence and polyA site had no impact on neon green production so long as the WPRE was present (Figure 4C). This surprising result shows that the WPRE element is multifunctional and can direct both stabilisation of the RNA and its subsequent export to the cytoplasm for translation of a TRANCER. While the WPRE maybe be larger than the current CAR-E + polyA and CAR-E + HSS versions, structural prediction of WPRE RNA structure has identified 3 hairpin structures within its sequence that likely direct these dual functions. Therefore, the inclusion of different combinations of these structures and the deletion of the intervening spaces and testing in TRANCER constructs opens up the possibility of developing more minimal versions of a WPRE that can perform both the roles of stabilisation and export promotion.

[0142] EXAMPLE 5: The natural bidirectional nature of enhancer transcription means that TRANCERS can express two transcripts simultaneously.

[0143] It is often highly desirable to express two transcripts simultaneously and synchronously in the same cell. The most obvious example would be the expression of a CRISPR guide RNA as well as the CAS9 protein itself. One of the defining features of enhancer is that it is highly bi-directional in its transcription. To test this potential, we generated a bi-directional TRANCER construct. Transcription from one side of the enhancer would produce transcripts and protein for the mNeon green fluorophore while the other side would produce transcripts and protein for the mCherry red fluorophore (Figure 5A). Due to the different wavelengths of their emissions, they can be detected separately in FACs analysis. As the transcriptional source we used the Nanog mES cell enhancer and performed the experiments in mES cells. We choose the Nanog enhancer as we have previously shown it to have a highly distinctive heterogenous activity pattern compared to other enhancers tested and we wanted to see if both transcripts from the enhancer showed the same distinctive pattern. In figure 5B in the left panel the combined analysis shows a very large population of cells that simultaneously express similar amounts of both neon green and mCherry. Highlighting the conservation of the biological specificity of the bidirectional TRANCER, figure 5B show substantively similar and distinctive patterns of heterogeneity in both neon green and mCherry signal.

[0144] There are two classes of base editors: cytosine base editors (CBEs) which convert a C to T substitutions and adenine base editors (ABEs) which convert A to G base pairs. ABEs are generally considered less toxic as typically they cause less DNA damage than CBEs. ABEs do not create double-strand breaks limiting the acquisition of unintended mutations and chromosomal rearrangements. Additionally, ABEs show better cell survival and often higher editing precision4Adenine base editors catalyse the deamination of adenine to inosine, which is recognized as guanine during DNA replication, thus generating A>G transitions and have decreased toxicity when expressed in cells.

[0145] The inventors engineered an mESC cell line edited at the Rosa26 locus where they integrated a GFP containing a stop codon (TAG) at the 5’ end of a GFP cDNAto prevent translation under a constitutive EF1a promoter5. Following the same experimental design as Katti A et al (2020), the inventors designed mutated GFP with a stop codon at the start of the reading frame that can be repaired with an ABE.

[0146] This is shown in Figure 5D and 5E. The TAG stop codon can be fixed by an ABE creating a TGG (Trp) codon.

[0147] The clonal population was transiently transfected with the bidirectional TRANCER showing GFP fixing capacity. Additional controls include:

[0148] Negative control: a non-transfected line (left) and a mutated guide sequence (right).

[0149] Positive control: canonical repair performed by the co-transfection of the ABE and guide plasmids (second of the left).

[0150] EXAMPLE 6: TRANCER constructs are highly cell type specific.

[0151] Enhancer sequences are driven by the binding and action of cell type specific combinations of transcription factors and their activity is highly restricted to specific cell types, lineages and types of stimulation. Therefore, as transcription is a hall mark of enhancer activity the transcription of TRANCERs will also be highly cell type specific. The Nanog gene is a very well characterised pluripotency factor and its enhancers are highly specific to embryonic stem cells. To test the cell type specificity of this TRANCER we transfect the standard polyA+intron TRANCER with the Nanog enhancer as transcriptional source into mouse ES (Embryonic stem) and T3T (fibroblast) cells and human HEK293T (embryonic kidney) cells and showed that the TRANCER was only active in the mES cells (Figure 6). To test this further we transfected mES cells with a polyA+intron TRANCER where the transcriptional source was now the well characterised R2 enhancer (SEQ ID NO: 7) of the red blood cell specific alpha globin genes which is an erythroid cell specific element (Figure 7 A). Our ultimate goal was to differentiate these cells into red blood cells via the “embryoid body” organoid system to test if this red blood cell based TRANCER would become activated (Figure 7B). However, the high haemoglobin content of these cells caused a technical problem with using fluorophores as a readout. To address this problem, we switched the reporter gene from mNeon green to a human cell surface receptor not present in the mouse genome (tCD19, SEQ ID NO: 11), and detected this with anti CD19 antibodies.

[0152] As judged by CD19 production the TRANCER was inactive in mES as expected and when differentiated via the embryoid body organoid system CD19+ cells could be detected in the TRANCER containing cells but not the wild type control (Figure 7C and Figure 7D). Only a relatively small proportion of cells were positive however, that was to be expected as embryoid bodies are composed of a complex mixture of Ectoderm, Mesoderm and Endoderm of which terminally differentiated red cells only represent a small fraction (Figure 7B). This experiment shows the ability of TRANCERs to target small subgroups very specifically within a complex cellular entity such as an organoid.

[0153] As well as looking at transcriptional output using qPCR, the inventors performed multiome on R2-integrated cells. Enrichment of CD19+ cells was performed though sorting, with 40% of the cells being gated to express this cell surface marker. The results are shown in Figure 7E.

[0154] EXAMPLE 7: TRANCERs can be used to provide context specific expression using engineered cells.

[0155] We propose that TRANCERs would be to reactivate T-cells or prevent exhaustion of CAR T-cells through the expression of key “payload proteins” specifically under conditions of T-cell activation.

[0156] To determine if TRANCERs could be used in a context of immune cell activation, we used Jurkat cells as an in vitro cell line model for T-cells and their activation. The immune activation of both native immune cells such as T-Cells and Jurkat cells causes the induction of activation specific enhancer elements across the genome, which direct the upregulation of the genes of activation specific processes (Figure 8A)._We chose to even further simplify the deployment of the TRANCERs by engineering the TRANCER payload (i.e. a protein payload with a TREX engagement / 3’ stabilisation sequence) adjacent to native enhancers in the genome removing the need to supply an exogenous synthetic enhancer sequence as the transcriptional driver (Figure 8B). The TRANCER payload expression is therefore under the control of the activation pattern of this endogenous enhancer. We chose two jurkat activation specific enhancer regions, on chromosomes 1 and 16 (SEQ ID NO: 12 and SEQ ID NO: 13, respectively), as examples of endogenous engineering of a TRANCER payload. With both these engineered cells lines, we were able to show that the endogenous enhancers were also capable of driving TRANCER expression and that this expression was dependant on stimulation of Jurkat cells. This data highlights the potential for the use of endogenous enhancers as transcriptional sources of TRANCERs, decreasing the payload further and their use for the programmed expression of protein and RNAs under exquisitely specific cell stimulations- e.g. when a T-cell becomes activated in the tumour microenvironment.

[0157] EXAMPLE 8: TRANCERs can be delivered via lentivirus with very low off-target background.

[0158] Derived from the human immunodeficiency virus (HIV), lentiviral vectors (LV) have been engineered and optimised to deliver genetic material into target cells with high efficiency and long-term stability. Their unique characteristics, including their ability to integrate into the host genome, transduce both dividing and non-dividing cells, and exhibit low immunogenicity, making them an invaluable asset in the field of gene therapy67.

[0159] In Figure 10, the inventors showcase that the TRANCER technology can be delivered using lentiviral vectors (LV). Two LV were tested, a stably integrating virus with stable long-term expression (integrated) and an integration-deficient variant (episomal) with transient expression that is lost during cell division89.

[0160] One of the limitations in the use of lentiviral vectors carrying therapeutic transgenes in gene therapy are the packaging constraints, as viral packaging efficiency dramatically decreases as the sized of the cargo increases. A viral cargo of less than 10 kb in length is optimal10. The advantage of using a TRANCER is that it reduces the amount of DNA required as compact enhancers can be used instead of the large promoter / enhancer combinations that are typically used.

[0161] A further advantage of delivering the TRANCER technology using lentiviral vectors (LV) is that there is very low off-target background produced. EXAMPLE 9: Synthetic enhancers work as TRANCERs.

[0162] Due to the finite number of naturally found enhancers, in recent years, with artificial intelligence emerging there has been an increasing pursuit for the creation of synthetic enhancers with the aim of precisely tuning gene expression for clinical and biotechnological applications11. Deep-learning models are being trained in the complex patterns from existing genomic datasets enabling the generation of novel sequences1’12“16.

[0163] In a recent Nature publication leveraging deep neural networks and reporter assays, the authors generated and tested thousands of enhancers using a construct containing a canonical minimal promoter sequence 2. The study was conducted in three human cell lines: K562 (chronic myelogenous leukaemia representing erythroid precursors), HepG2 (hepatocyte carcinoma cell line) and SK-N-SH (neuroblastoma cell line). The top enhancers from the publication were selected based on their MinGap value which takes into consideration both predicted and experimental expression values. A higher MinGap value indicates greater specificity. Here, as shown in Figure 11 , we selected the two highest MinGap enhancers for both K562 and HepG2 cells. Due to cell availability, the experiment was performed using K562 cells where, as expected, the HepG2 and scrambled (previously used) control displayed minimal expression compared to the predicted K562 enhancers. Additionally, there is a significance difference in the expression level of the two selected K562 enhancers as predicted by the model. Thus, the TRANCER constructs also mimic the predicted expression patterns of completely synthetic enhancers.

[0164] EXAMPLE 10: TRANCERs are stable in their expression and do not get silenced

[0165] The Human Silencing Hub (HUSH) is an epigenetic complex that protects the genome by silencing invasive genetic elements like retroviruses and transposons. HUSH recruits chromatin-modifying effectors to the target DNA, leading to assembly of repressive histone modifications, namely H3K9me319’20. HUSH is known to target newly integrated DNA through a mechanism that is largely unknown. However, it has been shown to only target active transcripts and it is guided, at least in part, by the presence of nascent RNA transcripts19. The HUSH protective layer is therefore an issue in the integration of synthetic constructs used for gene expression, gene therapy or synthetic biology, thus limiting stable and robust expression. The characteristic effects of HUSH in vitro are a loss of expression over time, generally leading to heterogenous expression where some cells may escape silencing and others do not, leading to reduced predictability and limiting applicability.

[0166] The TRANCER technology provides a new tool by which transgene constructs can be designed. To test whether these constructs would still be silenced by the HUSH complex, base (intron + polyA) TRANCER lentiviral vectors containing a CD19 cell surface marker were designed to be driven by either a synthetic or an HS2 enhancer. Lentiviral vectors were then transduced into K562 cells. Cells were selected in 1 pg / mL of puromycin for 2 weeks to confirm integration of the construct in all cells. Then, selection was removed, and cells were grown in complete media. With traditional constructs, in many cell lines repression can be detected within the first week after transduction19’21’22For TRANCER-integrated cell lines, a sample was taken for flow cytometry once a week. As seen in Figure 12 there is no repression being observed overtime in these cells. Hence, TRANCERS seem to mitigate HUSH- mediated silencing and achieve stable and long-term expression.

[0167] EXAMPLE 11 : TRANCERs can be used in immune-oncology applications

[0168] Bispecific antibodies, or bispecific T-cell engagers (BiTEs), are engineered antibody constructs that simultaneously bind an antigen on a tumour cell and a surface molecule on T cells, most commonly CD323 24. By bridging tumour and T cells, BiTEs promote the formation of an immunological synapse, triggering T-cell activation, proliferation, and cytotoxic activity through the release of perforins and granzymes, ultimately inducing apoptosis in the tumour cell. A key advantage of BiTEs is their ability to elicit T-cell-mediated killing even against tumour cells with low antigen expression, without requiring ex vivo T-cell manipulation23 24. However, their efficacy is limited by the potential loss of target antigen expression on malignant cells, which can lead to relapse25’26TRANCERs allow for the selective expression of any cargo in any cell or tissue with high control. Knowing that treatment failure of BiTEs is mainly due to antigen loss, the inventors sought to identify if the TRANCER technology could be used as an approach to mitigate relapse to these drugs or even as a tagging strategy for a tumour cell that does not express a particular cell surface marker.

[0169] Blinatumomab is a clinically approved BiTE used in the treatment of B-cell acute lymphoblastic leukemia (B-ALL). This bispecific antibody targets CD19 on B cells and tumour cells, while simultaneously binding CD3 on T cells27. Although highly effective in many patients, blinatumomab therapy is often challenged by relapse due to antigen loss, wherein malignant cells cease expression of CD19 in a substantial fraction of treated individuals. Moreover, its efficacy is restricted to tumours that retain CD19 expression27. To address these limitations, a TRANCER construct could be used to ectopically express CD19 in non-B-cell tumours or in malignant cells that have lost CD19, thereby restoring susceptibility to BiTE-mediated cytotoxicity.

[0170] Lentiviral vectors expressing tCD19 were designed containing the base TRANCER construct approach (splice site + polyA). The testing cell-type of choice were K562 cells, a chronic myelogenous leukaemia (CML) cell-line that does not normally express CD19. TRANCER expression was directed under a synthetic K562 enhancer, previously used in Figure 10.B or a HS2 enhancer known to be active in these cells. Following lentiviral transduction, cells were treated either with Blinatumomab or a [3-gal negative control showing selective killing in the dug condition (Figure 13) once again highlighting the specificity of the TRANCER technology. Additionally, these experiments confirm the ability of the TRANCER technology to be used in immune-oncology applications.

[0171] Materials and Methods

[0172] Cell culture of mouse embryonic stem cells

[0173] The ES-E14TG2a.IV mouse embryonic stem cell (mESC) line is used for all mESC work. This cell line is a subclone of the male HPRT-deficient male ES-E14TG2a cell line, originally derived from the 129 / Ola mouse strain (Nichols, Evans and Smith, 1990; Smith, 1991 ). Cells were expended and frozen in liquid nitrogen stocks. Adherent mESC E14 cells were grown in tissue culture plates coated with 0.1 % Gelatin in PBS and cultured in Glasgow’s Minimal Essential Medium supplemented with 10% foetal bovine serum (FBS), 1 mM sodium pyruvate, 1x minimum Essential Medium Non-Essential Amino Acids, 2 mM L-Glutamine, 1000 LI / mL LIF and 0.1 mM 2- Mercaptoethanol. Cells are grown under controlled conditions in an incubator set to 37°C and 5% CO2.

[0174] Other cell types used: HEK293T and T3T.

[0175] Additional to the use of mESC, other cell types are used to study cell and tissue specific expression under transient transfection with plasmid donors. These included Human embryonic kidney 293(T) cells (HEK293T) and fibroblast cell line that was isolated from a mouse NIH / Swiss embryo (NIH / T3T) cells.

[0176] HEK293T cells were grown Dulbecco's Modified Eagle Medium, high glucose supplemented with 10% foetal bovine serum (FBS), 1x minimum Essential Medium Non-Essential Amino Acids and 2 mM L- Glutamine. T3T cells were grown in Dulbecco's Modified Eagle Medium with the addition of 10% FBS.

[0177] Creation of donor plasmids

[0178] Initial TRANCER donor plasmid was cloned by the genome engineering facility (WIMM). Subsequent designs were generated using Gibson cloning approaches by replacing the enhancer (insert) and modifying the entry vector using relevant primer combinations. Initial TRANCER donor plasmid was cloned by the genome engineering facility (WIMM). Variations of the original construct (Figure 1 A) were performed using Gibson assembly. Primers were designed to amplify the different inserts, either different enhancer sequences (scramble (SEQ ID NO:1 ), Nanog (SEQ ID NO:2), Tmprss13 (SEQ ID NO:4), Pou5f1 (SEQ ID NO:5), Sox2 (SEQ ID NO:6)) or parts (polyA, intron, HSS, CAR-E, WPRE) with 20 bp overlaps matching the ends of the linearised entry plasmid which was also amplified using Q5 High-Fidelity DNA polymerase (NEB). Both vector and insert were purified using a PCR purification kit (Qiagen) and assembled using the NEBulder HiFi DNA assembly Master Mix (NEB) according to manufacturer’s protocol. Following assembly, the product was transformed into chemically competent E. coli DH5a cells and plated using ampicillin selection. The final construct was confirmed by Sanger sequencing.

[0179] Creating edited stable cell line

[0180] The introduction of the different TRANCER constructs into the neutral loci selected on chromosome X was achieved using CRISPR-Cas9 mediated HDR in mESCs. We used a previously tested sgRNAs which is clones into pX458 a plasmid encoding Cas9 from Streptococcus pyogenes and enhanced green fluorescent protein (eGFP) (Ran et al., 2013). For each TRANCER donor DNAwe used templates containing 500 bp homology arms to the ChrX artificial locus. sgRNA / Cas9 plasmid DNA and donor plasmid DNAwere prepared for transfection using QIAprep Spin Miniprep Kit (Qiagen) according to manufacturer’s instructions. Mouse ES cells (~4 x 105) were transfected using Lipofectamine LTX Reagent with PLUS Reagent (Invitrogen, ThermoFisher) which were performed in 6-well format by plating freshly trypsinised cells in 2 ml media supplemented with the transfection mix prepared following manufacturer’s instructions: 6 pl LTX reagent, a total of 2.5 pg purified plasmid DNA (1 :3 ratio of sgRNA / Cas9 plasmid DNA:donor plasmid DNA), 2.5 pl PLUS reagent, and 250 pl Opti-MEM. After 24 hours incubation at 37 °C, transfected cells were selected based on the presence of eGFP by FACS. eGFP+ cells were plated at low densities (~3,000 cells / 10 cm dish) in 0.6pg / mL puromycin media to allow for isolation of colonies originating from single cells. After seven days of growth, individual colonies were picked into 96-well plates which were then screened by PCR. Edit was confirmed using Sanger sequencing (Eurofins) and downstream analysis was performed on positive clones.

[0181] Creation of a mESC line with a targeted Rosa26 GFP non-sense mutation for ABE repair

[0182] To generate a reporter system for site-specific suppression of a premature stop codon, the inventors engineered a mouse embryonic stem cell (mESC) line with a GFP transgene containing a TAG nonsense mutation at the 5' end of the GFP open reading frame, following the design of Katti et al. (2020). In contrast to their use of PiggyBac transposition AdGO2, the inventors’ construct was stably integrated into the Rosa26 locus via CRISPR / Cas9-mediated homologous recombination (creating edited cell lines, materials and methods). The transgene was placed under the control of a constitutive EF1a promoter within a custom plasmid backbone.

[0183] Fluorescence-Activated Cell Sorting (FACS)

[0184] FACS was used to (1 ) identify and sort successfully transfected cells or (2) select cells for further expansion. FACS was performed with the assist of the MRC WIMM Flow Cytometry Facility on live cells re-suspended in FACS buffer, generated by adding 10% FBS to PBS using either a BD FACSAriaTM III Cell Sorter or a BD FACSAriaTM Fusion Cell Sorter.

[0185] Flow cytometry analysis

[0186] Attune NxT (ThermoFisher) machines were used to assess the expression patterns and brightness levels of TRANCER signal. The experiment was performed either on a 96-well plate format or individual Eppendorf tubes. A wild-type non-expressing cell was used for gating. Cellular debris and doublets were excluded based on the forward scatter (FSC) and side scatter (SSC) parameters. PRISM software was used to plot the results of multiple replicates in a bar chart.

[0187] Differentiation of mESCs into embryoid bodies (EBs)

[0188] EB cultures were generated as described by Francis et al., (2022). 48h prior to differentiation, mESCs were induced by passaging into IMDM based media supplemented with LIF17. To start the differentiation culture (dO), cells growing in IMDM were trypsinized and plated in differentiation media in 10 cm dishes at 3x103 cells for seven days with daily gentle shaking of the dishes to disrupt potential EB attachment to the bottom of the dish. EBs were harvested and disaggregated in 0.25% trypsin for 5 minutes at 37°C. Cells from disaggregated EBs were used for FACS sorting to detect mNG+ populations.

[0189] Single cell analysis (Smart-seq3)

[0190] Bioinformatics analysis was performed by loading the data and performing quality control metrics using Seurat. Then enrichment analysis was conducted using EnrichR. Highly variable genes were identified, clusters were identified using the Leiden algorithm, and batch correction was performed using Com Bat. Post- correction visualizations were generated using Seurat and Scanpy included key marker and gene set heatmaps.

[0191] Jurkat cell culture and stimulation

[0192] Jurkat E6.1 derived from Jurkat FHCRC was grown in Roswell Park Memorial Institute (RPMI) 1640 supplemented with 2 mM Glutamine and 10% FBS at 37°C, 5% CO2.

[0193] TRANCER donor construct was targeted to a native enhancer, edit was done using a CRISPR guide to each individual genomic loci. Puromycin selection was applied 48 hours post electroporation of the cells.

[0194] After two weeks of selection, Jurkat stimulation was performed following the same approach as in18in which 100 ng / ml phorbol 12-myristate 13-acetate and 5 pg / ml anti-CD3 were added in the media for 4 hours. After that time media is washed from the cells and flow cytometry analysis was performed.

[0195] Single-Cell Multiome (ATAC + Gene Expression) Sequencing in Embryoid Bodies (EBs) Derived from mESCs

[0196] Sample Preparation

[0197] Mouse embryonic stem cells (mESCs) with a stable integration of the R2-tCD19 construct were differentiated into embryoid bodies (EBs) as previously described. Nuclei were isolated following the 10x Genomics CG000365-Rev C protocol using the low-cell input workflow.

[0198] Library Preparation and Sequencing

[0199] ATAC and gene expression (GEX) sequencing libraries were generated by Maria Greco (MRC WIMM Single Cell Facility) following the 10x Genomics CG000338-Rev E protocol. Libraries were sequenced on a NovaSeq instrument (PE-50 and PE-150) at SourceBioscience (UK).

[0200] Bioinformatic Analysis

[0201] Preprocessing and Quality Control Read Alignment and Genome Customization

[0202] Raw sequencing reads were processed using Cell Ranger ARC (10x Genomics, version 2.0.2) with default settings. A custom reference genome was used, incorporating the R2-tCD19 locus as an additional chromosome.

[0203] RNA-seq Analysis

[0204] RNA-seq data processing was performed using muon (vO.1.7) in Python, with the following filtering and normalization steps:

[0205] • Removal of technical artifacts using CellBender (version 0.3.0).

[0206] • Merging of raw and CellBender-corrected data into an AnnData object.

[0207] • Filtering out cells with <100 expressed genes.

[0208] • Calculation of mitochondrial, ribosomal, and hemoglobin gene content.

[0209] • Removal of outliers based on the Median Absolute Deviation (MAD) method.

[0210] • Doublet removal using Scrublet.

[0211] Post-filtering, total count normalization was applied, followed by logarithmic transformation. Highly variable genes were selected, and Principal Component Analysis (PCA) was performed for dimensionality reduction. For visualization, nearest neighbors were calculated, and LIMAP embedding was used. Clustering was conducted with the Leiden algorithm at varying resolutions to identify subpopulations.

[0212] ATAC-seq Analysis

[0213] ATAC-seq data was analyzed using ArchR in R with the following steps:

[0214] • Conversion of aligned data into Arrow files.

[0215] • Doublet filtering.

[0216] • Latent Semantic Indexing (LSI) for iterative dimensionality reduction.

[0217] • Seurat clustering (resolution = 0.8) and UMAP embedding.

[0218] • Peak calling using MACS2 for cluster characterization.

[0219] Multiome Data Integration The ATAC-seq AnnData object was integrated with the RNA-seq AnnData object into a unified MuData object using the Muon package. This allowed for joint analysis of RNA and ATAC-seq data. The integrated dataset was further annotated using cellxgene transfer learning models.

[0220] R2-tCD19 Locus Analysis

[0221] Post-mapping, reads associated with the R2-tCD19 locus were extracted using pysam and added as a new feature in the AnnData object for downstream analysis. The processed data was visualized using LIMAP embedding.

[0222] Lentiviral Production

[0223] Lentiviral particles were generated using HEK293T cells fusing a second-generation transfection system. The packaging plasmid psPAX2 (Addgene #12260) was used for stable integration, or psPAX2-D64V (Addgene #63586) was used for integrasedeficient lentivirus production, both providing the Gag and Pol proteins. The envelope plasmid pMD2.G(Addgene #12259) was used to encode the VSV-G envelope protein. The transfer plasmid Lenti_MCP-LSD1_Hygro (Addgene #138457) was digested with Nhel and Kpnl, and the cargo was cloned using HiFi DNA Assembly (NEB).

[0224] Transfection and Virus Collection

[0225] HEK293T cells were seeded in DMEM supplemented with 10% FBS and 1x GlutaMAX™ (Gibco)and incubated at 37°C with 5% CO2. Once 70-80% confluency was reached, cells were transfected with the three plasmids using Lipofectamine 2000 (Invitrogen) following the manufacturer’s protocol. The supernatant containing lentiviral particles was collected 48 hours post-transfection, filtered through a 0.45 pm filter and stored at -80°C until use.

[0226] Lentiviral Transduction of K562 Cells

[0227] K562 cells were spinoculated by centrifugation at 800 x g for 60 minutes at 32°C in the presence of 4 pg / mL polybrene (Sigma-Aldrich) to enhance transduction efficiency. Cells were maintained in RPMI-1640 medium supplemented with 10% FBS andlx GlutaMAX™ and incubated at 37°C with 5% C02. Transduced cells were selected with 1 ug / ml puromycin for 72 hours prior to analysis.

[0228] Flow Cytometry Analysis mNG gene expression was assessed 72 hours post-infection using an Attune NxT Flow Cytometer (Thermo Fisher Scientific).

[0229] Cloning of the synthetic enhancer constructs

[0230] The enhancer sequence was ordered as a synthetic custom DNA gBIock from IDT containing flanking homology regions of 20 bp. The entry vector containing a splice site, mNeon Green, WPRE and polyA with a puromycin selection cassette was digested using Mlul and Xmal and NEB HiFi assembly was used to insert the synthetic enhancer sequences into the vector.. The construct was validated using Sanger sequencing (SourceBioscience).

[0231] Nucleofection into K562 cells Transfection was performed using SF Cell line 4D nucleofector X Kit L (Lonza Bioscience) according to manufacturer’s protocol.

[0232] Listing

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[0260] 1 . A synthetic construct for providing a native enhancer or variant thereof as a promoter to drive RNA expression, wherein the synthetic construct comprises:

[0261] (a) one or more transgenes for RNA expression;

[0262] (b) one or more stabiliser motifs; and

[0263] (c) one or more nuclear export signals.

[0264] 2. The synthetic construct according to embodiment 1 , wherein the native enhancer is in a native environment.

[0265] 3. The synthetic construct according to embodiment 1 , wherein the synthetic construct further comprises a native enhancer.

[0266] 4. The synthetic construct according to embodiment 1 or embodiment 3, wherein the native enhancer is in a non-native environment.

[0267] 5. The synthetic construct according to any preceding embodiment, wherein the native enhancer is an intragenic native enhancer.

[0268] 6. The synthetic construct according to any preceding embodiment, wherein the one or more stabiliser motifs is a synthetic stabiliser motif.

[0269] 7. The synthetic construct according to any preceding embodiment, wherein the one or more stabiliser motifs is selected from a group consisting of polyadenylation (polyA) tail or Hairpin Stabilisation Sequence (HSS).

[0270] 8. The synthetic construct according to embodiment 7, wherein the one or more stabiliser motif is a polyadenylation (polyA) tail.

[0271] 9. The synthetic construct according to embodiment 7, wherein the one or more stabiliser motif is Hairpin Stabilisation Sequence (HSS).

[0272] 10. The synthetic construct according to any preceding embodiment, wherein the one or more nuclear export signals is a synthetic nuclear export signal.

[0273] 11 . The synthetic construct according to any preceding embodiment, wherein the one or more nuclear export signal is selected from a group consisting of an intron or a Cytoplasmic Accumulation Region motif E (CAR-E).

[0274] 12. The synthetic construct according to embodiment 11 , wherein the one or more nuclear export signal is an intron.

[0275] 13. The synthetic construct according to embodiment 11 , wherein the one or more nuclear export signal is a Cytoplasmic Accumulation Region motif E (CAR-E). 14. The synthetic construct according to any preceding embodiment, wherein the function of the one or more nuclear export signals and the function of the one or more stabiliser motifs are provided by Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE).

[0276] 15. The synthetic construct according to any preceding embodiment, wherein the synthetic construct further comprises one or more flanking motifs.

[0277] 16. The synthetic construct according to any preceding embodiment, wherein the synthetic construct comprises two or more stabiliser motifs, two or more nuclear export signals and two or more transgenes.

[0278] 17. The synthetic construct according to embodiment 16, wherein the synthetic construct comprises two stabiliser motifs, two nuclear export signals and two transgenes.

[0279] 18. A nucleic acid encoding the synthetic construct according to any one of the preceding embodiments.

[0280] 19. A vector comprising the synthetic construct or nucleic acid according to any preceding embodiment.

[0281] 20. A cell comprising one or more synthetic construct, nucleic acid or vector according to any preceding embodiment.

[0282] 21 . A composition comprising a synthetic construct, nucleic acid, vector or cell according to any preceding embodiment.

[0283] 22. A pharmaceutical composition comprising a synthetic construct, nucleic acid, vector or cell according to any preceding embodiment.

[0284] 23. A pharmaceutical composition according to embodiment 22, for use as a medicament.

[0285] 24. A composition, pharmaceutical composition, synthetic construct, nucleic acid, vector or cell according to any preceding embodiment for use in therapy.

[0286] 25. A composition, pharmaceutical composition, synthetic construct, nucleic acid, vector or cell according to any preceding embodiment for use in gene therapy.

Claims

Claims1 . A synthetic construct for providing an enhancer or variant thereof as a promoter to drive RNA expression, wherein the synthetic construct comprises:(a) one or more transgenes for RNA expression;(b) one or more stabiliser motifs; and(c) one or more nuclear export signals.

2. The synthetic construct according to claim 1 , wherein the enhancer is a native enhancer.

3. The synthetic construct according to claim 2, wherein the native enhancer is in a native environment.

4. The synthetic construct according to claim 1 , wherein the synthetic construct further comprises an enhancer.

5. The synthetic construct according to claim 4, wherein the enhancer is a native enhancer, a synthetic enhancer or an optimised native enhancer.

6. The synthetic construct according to claim 5, wherein the native enhancer is in a non-native environment.

7. The synthetic construct according to any preceding claim, wherein the one or more stabiliser motifs is a synthetic stabiliser motif.

8. The synthetic construct according to any preceding claim, wherein the one or more stabiliser motifs is selected from a group consisting of polyadenylation (polyA) tail or Hairpin Stabilisation Sequence (HSS).

9. The synthetic construct according to claim 8, wherein the one or more stabiliser motif is a polyadenylation (polyA) tail.

10. The synthetic construct according to claim 8, wherein the one or more stabiliser motif is Hairpin Stabilisation Sequence (HSS).11 . The synthetic construct according to any preceding claim, wherein the one or more nuclear export signals is a synthetic nuclear export signal.

12. The synthetic construct according to any preceding claim, wherein the one or more nuclear export signal is selected from a group consisting of an intron or a Cytoplasmic Accumulation Region motif E (CAR-E).

13. The synthetic construct according to claim 12, wherein the one or more nuclear export signal is an intron.

14. The synthetic construct according to claim 12, wherein the one or more nuclear export signal is a Cytoplasmic Accumulation Region motif E (CAR-E).

15. The synthetic construct according to any preceding claim, wherein the function of the one or more nuclear export signals and the function of the one or more stabiliser motifs are provided by Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE).

16. The synthetic construct according to any preceding claim, wherein the synthetic construct further comprises one or more flanking motifs.

17. The synthetic construct according to any preceding claim, wherein the synthetic construct comprises two or more stabiliser motifs, two or more nuclear export signals and two or more transgenes.

18. The synthetic construct according to claim 17, wherein the synthetic construct comprises two stabiliser motifs, two nuclear export signals and two transgenes.

19. A nucleic acid encoding the synthetic construct according to any one of the preceding claims.

20. A vector comprising the synthetic construct or nucleic acid according to any preceding claim.21 . A cell comprising one or more synthetic construct, nucleic acid or vector according to any preceding claim.

22. A composition comprising a synthetic construct, nucleic acid, vector or cell according to any preceding claim.

23. A pharmaceutical composition comprising a synthetic construct, nucleic acid, vector or cell according to any preceding claim.

24. A pharmaceutical composition according to claim 23, for use as a medicament.

25. A composition, pharmaceutical composition, synthetic construct, nucleic acid, vector or cell according to any preceding claim for use in therapy.

26. A composition, pharmaceutical composition, synthetic construct, nucleic acid, vector or cell according to any preceding claim for use in gene therapy.

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