Controllable expression in mammalian cells

Abstract

This invention provides a nucleic acid construct comprising; (i) a first nucleotide sequence for expression, (ii) a first promoter operably linked to the first nucleotide sequence, said first promoter being inducible, (iii) a first insulating sequence located upstream of the first nucleotide sequence and first promoter, (iv) a second insulating sequence located downstream of the first nucleotide sequence and first promoter, (v) a second nucleotide sequence encoding a transcription regulator protein that induces expression from the first promoter; and (vi) a second promoter operably linked to the second nucleotide sequence. The second nucleotide sequence and second promoter may be located either (a) upstream of the first insulating sequence or (b) downstream of the second insulating sequence. Also provided are vectors and mammalian cells comprising the construct, methods of inducibly expressing a nucleotide sequence in the construct, and methods of producing a mammalian cell by inducing expression from the construct.

Description

[0001] Controllable Expression in Mammalian Cells Field This invention relates to nucleic acid constructs, vectors, and methods for the controllable expression of heterologous nucleotide sequences in mammalian cells, in particular the controllable expression of transcription factors for programming of cells into different cell lineages and cell types. Background Somatic cells derived from pluripotent stem cells have broad applications in regenerative medicine, disease modelling, drug discovery, and cell transplantation. Initial methods of generating somatic cells from pluripotent stem cells involved directed differentiation of the stem cells along pathways that mimic natural differentiation processes using multi-stage cell culture with different combinations of growth factors. However, directed differentiation is often time consuming, requires considerable cost in cytokines, and is challenging technically. Other approaches for generating somatic cells include the programming of either pluripotent stem cells or somatic cells into the desired cell types without mimicking natural differentiation processes. In particular, forward programming has been widely adopted to directly convert pluripotent stem cells (PSCs) into somatic cell types. This involves the expression of a specific set of transcription factors in the PSCs. The set of transcription factors determines the somatic cell type into which the PSCs are programmed. Initially, forward programming techniques involved the transduction of PSCs with lentiviral vectors encoding the transcription factors. This often resulted in the variegated expression or complete silencing of randomly inserted transcription factors and additional purification steps were needed in order to isolate a sub- population expressing the required transcription factors. Clinical grade lentiviral vectors are also costly, difficult to use at large scale and introduce batch-to-batch variability that presents quality issues for the manufacture of cells for therapeutic use. Controllable expression systems were subsequently developed to avoid the need for lentiviral vectors. In these systems, the expression of the transcription factors driving the cell programming is chemically controlled using an inducer compound, commonly tetracycline or a tetracycline analogue. The expression of coding sequences randomly inserted into the mammalian cell genome is often adversely affected by epigenetic silencing and promoter interference. To address these problems, a dual targeting approach2, 15is commonly adopted. In one example of this approach, a vector containing a coding sequence for a reverse tetracycline-controlled transactivator (rtTA) is targeted to a first genomic safe harbour (GSH) locus, typically the Rosa26 locus. A separate vector containing coding sequences for a set of programming transcription factors operatively linked to a third generation Tet-responsive promoter element known as the Tet operator is targeted to a second GSH, typically AAVS1. However, dual targeting has drawbacks for inducible mammalian expression systems. It is technically challenging to generate cell lines with insertions at two separate GSH loci. It requires either the simultaneous use of 6 different vectors or sequential use of 3 vectors followed by selection, cloning and genotyping then a further 3 vectors followed by more selection, cloning and genotyping. Furthermore, the use of two GSH loci for the controllable expression system reduces the possible range of genetic modifications by reducing the number of established GSH loci that are available for other expression constructs.

[0002] The present inventors have developed a system for inducible expression in mammalian cells that targets a single genetic locus. This system simplifies the production of recombinant cell lines, for example for programming into different somatic cell types, and is resistant to promoter interference and epigenetic gene silencing.

[0003] A first aspect of the invention provides a nucleic acid construct comprising;

[0004] (i) a first nucleotide sequence for expression,

[0005] (ii) a first promoter operably linked to the first nucleotide sequence, said first promoter being inducible,

[0006] (iii) a first insulating sequence located upstream of the first nucleotide sequence and first promoter,

[0007] (iv) a second insulating sequence located downstream of the first nucleotide sequence and first promoter, and

[0008] (v) a second nucleotide sequence encoding a transcription regulator protein that induces expression from the first promoter; and

[0009] (vi) a second promoter operably linked to the second nucleotide sequence, wherein the second nucleotide sequence and second promoter are located either (a) upstream of the first insulating sequence or (b) downstream of the second insulating sequence.

[0010] A second aspect of the invention provides a vector comprising a nucleic acid construct of the first aspect.

[0011] A third aspect of the invention provides a method of producing a mammalian cell for controlled expression of a nucleotide sequence comprising; inserting a nucleic acid construct of the first aspect into a genomic safe harbour (GSH) locus within the genome of a mammalian cell.

[0012] A fourth aspect of the invention provides a mammalian cell comprising nucleic acid construct of the first aspect at a GSH locus within the genome of the cell.

[0013] A fifth aspect of the invention provides a method of expressing a nucleotide sequence comprising; a) providing a mammalian cell comprising a nucleic acid construct at a genomic safe harbour locus within its genome, the nucleic acid comprising;

[0014] (i) a first nucleotide sequence for expression,

[0015] (ii) a first promoter operably linked to the first nucleotide sequence, said first promoter being inducible,

[0016] (iii) a first insulating sequence located upstream of the first nucleotide sequence and first promoter,

[0017] (iv) a second insulating sequence located downstream of the first nucleotide sequence and first promoter, and (v) a second nucleotide sequence encoding a transcription regulator protein that induces expression from the first promoter;

[0018] (vi) a second promoter operably linked to the second nucleotide sequence, wherein; the second nucleotide sequence and second promoter are located either upstream of the first insulating sequence or downstream of the second insulating sequence; and the second nucleotide sequence is expressed in the mammalian cell to produce the transcription regulator protein in the mammalian cell, and b) activating the transcriptional regulator protein in the mammalian cell to induce the expression from the first promoter, such that the first nucleotide sequence is expressed.

[0019] Suitable mammalian cells for use in methods of the fifth aspect include cells of the fourth aspect and cells produced by methods of the third aspect.

[0020] In some embodiments, the first nucleotide sequence may encode one or more proteins or transcription factors. Methods ofthe fifth aspect may be useful for example in producing the one or more encoded proteins or transcription factors.

[0021] A sixth aspect of the invention provides a method of producing a mammalian cell or programming a first mammalian cell into a second mammalian cell comprising; a) providing a first mammalian cell comprising a nucleic acid construct at a genomic safe harbour locus within its genome, the nucleic acid comprising;

[0022] (i) a first nucleotide sequence for expression,

[0023] (ii) a first promoter operably linked to the first nucleotide sequence, said first promoter being inducible,

[0024] (iii) a first insulating sequence located upstream of the first nucleotide sequence and first promoter,

[0025] (iv) a second insulating sequence located downstream of the first nucleotide sequence and first promoter,

[0026] (v) a second nucleotide sequence encoding an activatable transcription regulator protein that induces expression from the first promoter;

[0027] (vi) a second promoter operably linked to the second nucleotide sequence, wherein; the second nucleotide sequence and second promoter are located either upstream of the first insulating sequence or downstream of the second insulating sequence; and the second nucleotide sequence is expressed in the PSC to produce the transcription regulator protein, b) activating the transcriptional regulator protein to induce the expression from the first promoter, such that the first nucleotide sequence is expressed to produce the set of transcription factors in the PSC and said transcription factors program the first mammalian cell into the second mammalian cell. In some preferred embodiments of the sixth aspect, the first mammalian cell may be a pluripotent stem cell and the second mammalian cell may be a somatic cell. For example, the somatic cell may be a haematopoietic cell, preferably a megakaryocyte. A method of producing a haematopoietic cell or programming a pluripotent cell into a haematopoietic cell may comprise; a) providing a PSC comprising a nucleic acid construct at a genomic safe harbour site within its genome, the nucleic acid comprising;

[0028] (i) a first nucleotide sequence comprising coding sequences for the transcription factors GATA1 , TAL1 and FLI1 ,

[0029] (ii) a first promoter operably linked to the first nucleotide sequence, said first promoter being inducible,

[0030] (iii) a first insulating sequence located upstream of the first nucleotide sequence and first promoter,

[0031] (iv) a second insulating sequence located downstream of the first nucleotide sequence and first promoter,

[0032] (v) a second nucleotide sequence encoding an activatable transcription regulator protein that induces expression from the first promoter;

[0033] (vi) a second promoter operably linked to the second nucleotide sequence, wherein the second nucleotide sequence and second promoter are located upstream of the first insulating sequence or downstream of the second insulating sequence; and the second nucleotide sequence is expressed in the PSC to produce the activatable transcription regulator protein, b) activating the transcriptional regulator protein to induce the expression from the first promoter, such that the first nucleotide sequence is expressed to produce the transcription factors GATA1 , TAL1 and FLI1 in the pluripotent stem cell, and

[0034] (c) culturing the cell, such that the transcription factors program the PSC to a haematopoietic cell.

[0035] Suitable first mammalian cells for use in methods of the sixth aspect include mammalian cells of the fourth aspect and mammalian cells produced by a method of the third aspect.

[0036] In other embodiments of the sixth aspect, the first mammalian cell may be a somatic cell of a first cell type, such as a fibroblast or mesenchyme cell, and the second mammalian cell may be a somatic cell of a second cell type, for example a dendrocyte or a haematopoietic cell, such as an erythrocyte; or the first mammalian cell may be a PSC of a first cell type, and the second mammalian cell may be a PSC of a second cell type.

[0037] The transcription regulator protein encoded by the second nucleotide sequence of the first to the sixth aspects may induce expression from the first promoter in the presence of an inducer compound. The transcriptional regulator protein may be activated to induce expression of the first nucleotide sequence by contacting the transcriptional regulator protein with the inducer compound.

[0038] Preferred genomic safe harbour loci of the first to the sixth aspects include the Adeno- Associated Virus Integration Site 1 (AAVS1) locus or the Rosa 26 locus. Suitable nucleic acid constructs of the third to the sixth aspects include nucleic acid constructs of the first aspect.

[0039] Other aspects and embodiments of the invention are described in more detail below.

[0040] Brief Description of the Figures

[0041] Figure 1 shows the genomic insulation of the polycistronic cassette by sequential addition of HS4 INS fragments HS4. Adding HS4 insulators in opposite orientations flanking the TET-ON element and transgene expression cassette prevents silencing of the transgenes.

[0042] Figure 2 shows flow cytometry histograms showing percentage of GFP expression as the proxy for expression of the 3TFs for forward programming with (bottom) and without (top) protection by insulating sequences. In both lines the inducible forward programming protocol was followed using 0.25pg / ml doxycycline. These clones demonstrate that insertion of insulators prolongs gene expression from the integrated vector. Clones were produced using a one - step cloning strategy incorporating a specific polycistronic cassette for MK production

[0043] Figure 3 shows that at respective best doses of doxycycline, clones with insulators have increased yields of viable CD41 / 42+ fully differentiated megakaryocytes. Numbers next to columns indicate the fold yields of mature cells per starting hiPSC. For the new clones IRCIB10.1 12 n=3, error bars ±1 x SEM. For iRCI B10.1 H this represents a 4,400 fold improvement over the original clone without insulation of the expression cassette.

[0044] Figure 4 shows a schematic representation of a version of the Aim (All in one) megakaryocyte vector specifically designed to create megakaryocytes on demand from hPSCs containing the 3TF construct designed for the production of megakaryocytes. The CAG promoter driving rtTA (TET-ON 3G) expression required to complete the inducible construct was introduced 3’ to the second HS4 insulator sequence (INS2). This allowed sufficient separation of the CAG promoter and the TRE element which is also based on a minimal CMV like promoter and avoiding potential cis detrimental effects of having two promoters close together.

[0045] Figure 5 compares megakaryocyte production from two Aim clones with a dual targeted clone from the same hiPSC parent line. Figure 5A shows the number of viable MK per 1 .00E+05 starting hiPSC, determined by flow cytometry at Day 10 to 30 of differentiation showing mature CD42 / 41++ MKs. The bar graph compares a clone created using a dual targeting system using 0.125pg / ml doxycycline, the determined best dose for this clone, to two new clones from the Aim system at 0.0625 and 0.125pg / ml doxycycline. Figure 5B shows a bar graph of viable platelets per MK determined by flow cytometry using CD41 and CD42 gated on the platelet window as determined using donor blood and Calcein AM viable platelets. Red arrows indicate flow cytometry plots of the relevant samples. Figure 5C shows expression of von Willebrand factor VWF by mature MKs at D25 of differentiation, as determined by intracellular flow cytometry, gated on CD42 positive viable MKs. As in 5A and 5B, Aim clones are shown alongside the best dual targeted clone. In all cases, changing from the dual to single targeting system decreases the dox concentration required for induction. Figure 6 shows that genetic modification using the Aim system has no effect on the pluripotent potential of lines and GFP expression is robust. Figure 6A shows flow cytometry dot plots for the original parent line for RCIB-10 and genetically modified inducible hiPSC lines showing that constructs have no impact on the pluripotency of the undifferentiated cultures, they remain positive for the pluripotency markers TRA-1-60 and SSEA4. Figure 6B shows flow cytometry histograms showing overlayed % GFP expression as the proxy for expression of the 3TFs for forward programming for iQOLG clones using both the dual targeting system (at the best dose for the clone) and the GFP expression of a new Aim clone being assessed at different doxycycline concentrations. At D2 of programming, robust expression of the transgene is observed.

[0046] Figure 7 shows data from 2 iQolg Aim clones (Figure 7A), 2 lines from hESCs iMS10.8A (Figure 7B) and iMS4.4A (Figure 7C) and a Thrombocytopeania Absent Radius (TAR) patient line alongside the sibling control line (Figure 7D). The Aim vector performs well using either control donor derived hiPSCs, hESCs or patient derived hiPSCs.

[0047] Figure 8 shows an AIM construct in which the reverse tetracycline-controlled transactivator (rtTA) constitutively expressed through a CAG promoter was inserted 3’ to the INS1 downstream of the 5’HAR arm. This was not the final configuration of the Aim vector and demonstrates cis interference between the promoter and operator elements controlling transcription of the key TFs. Clones generated using this version showed no GFP signal. The arrangement in the final construct (figure 4) is key to the efficient function of the system.

[0048] Figure 9 shows qPCR results showing the rtTA signal generated by the AIM construct of Figure 8 compared to the dual system with insulators. When the rTTA sequences were moved outside 3’ to INS2 sequences this was resolved giving the final construct demonstrating the importance of the arrangement of sequences for optimal function.

[0049] Detailed Description

[0050] This invention relates to inducible mammalian systems that allow the controlled expression of heterologous nucleic acid using an inducible promoter. The inducible promoter is regulated by a transcription regulator protein that is responsive to an inducer compound. The transcription regulator protein may stimulate expression from the inducible promoter in the absence, or more preferably in the presence, of the inducer compound in the culture medium, causing the heterologous nucleic acid to be expressed in mammalian cells.

[0051] The genetic components of the inducible mammalian expression systems described herein are located at a single genomic locus in the mammalian cells. This avoids the need to engineer two separate genetic loci and increases the ease and efficiency of producing new mammalian cell lines comprising the system. Despite being located at a single genomic locus in the mammalian cells, the inducible mammalian systems described herein are resistant to promoter interference and epigenetic silencing. The inducible mammalian systems may be useful for example in producing proteins of interest or for expressing sets of transcription factors, for example for the forward programming of pluripotent stem cells (PSCs) into somatic cells. This is exemplified herein by the forward programming of PSCs into megakaryocytes, but any somatic cell type may be generated using the appropriate set of transcription factors. A nucleic acid construct for use in an inducible expression system as described herein may be a heterologous or recombinant nucleic acid and may be partially or wholly synthetic. The construct may comprise; (a) a first nucleotide sequence operably linked to a first promoter; (b) first and second insulating sequences and (c) a second nucleotide sequence, operably linked to a second promoter. The second nucleotide sequence and second promoter are separated from the first nucleotide sequence and first promoter by the first or the second insulating sequence i.e. one of the first and second insulating sequences is located between elements (a) and (c). For example, element (c) may be located upstream of the first insulating sequence or downstream of the second insulating sequence.

[0052] The first nucleotide sequence may comprise one or more coding sequences i.e. it may be an expression cassette. For example, the first nucleotide sequence may comprise a single coding sequence for a transcription factor or protein or a polycistronic cassette comprising coding sequences for multiple transcription factors or proteins. The expression cassette may contain multiple coding sequences, for example 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more coding sequences. Suitable coding sequences include any nucleotide sequence that is capable of being transcribed into RNA by the first promoter. The one or more coding sequences in the first nucleotide sequence may be controllably expressed in the mammalian cells after the introduction of the nucleic acid construct into the genomic safe harbour locus of the mammalian cells.

[0053] A coding sequence within the first nucleotide sequence may be expressed to produce a gene product, such as a protein or non-coding RNA molecule, in the mammalian cell. A coding sequence for a non-coding RNA may consist of a nucleotide sequence that is transcribed in the mammalian cell from DNA into RNA but is not translated. Suitable non-coding RNA molecules may include for example shRNA, miRNA or long non-coding RNA (IncRNA). In some preferred embodiments, a coding sequence may encode a transcription factor or other protein. The coding sequence may be transcribed and translated in the mammalian cell following expression of the first nucleotide sequence in order to generate the encoded protein.

[0054] A coding sequence within the first nucleotide sequence may encode any protein for which increased expression or overexpression is desired. Suitable proteins include transcription factors, therapeutic proteins, such as clotting factors, such as Factor VIII, enzymes, toxins, hormones, antibody molecules, cytokines, secretory proteins, and receptors, for example chimeric antigen receptors (CARs) and T cell receptors (TCRs). Other suitable proteins include antigenic proteins, such as viral, bacterial and parasite protein antigens, and tumour antigens. Viral protein antigens may include coronavirus proteins, such as coronavirus Spike (S) protein (e.g. SARS-CoV-2 S protein). Tumour antigens may include tumour-specific and tumour- associated antigens. Other suitable proteins include research proteins, for example gene editing proteins, such as Cas9, immunological proteins, such as non-polymorphic HLA class I variants HLA-E and HLA-G, suicide proteins, such as caspase 9, and fluorescent proteins, such as GFP.

[0055] In some preferred embodiments, the first nucleotide sequence may comprise coding sequences for a set of transcription factors (TFs). A set of TFs may program a mammalian cell of a first type into a mammalian cell of a second type. For example, the first nucleotide sequence may comprise coding sequences for a set of TFs that program a pluripotent stem cell (PSC) into a haematopoietic cell. In other embodiments, the first nucleotide sequence may comprise coding sequences for a set of transcription factors (TFs) that program (i) a somatic cell into a PSC (ii) a first somatic cell into a second somatic cell or (iii) a first PSC into a second PSC.

[0056] Transcription factors are DNA binding proteins which regulate the expression of genes in cells. Preferably, the transcription factors encoded by the one or more coding sequences in the first nucleotide sequence are human transcription factors. The identities of the transcription factors in the set of transcription factors encoded by the one or more coding sequences depends on the somatic cell into which a PSC or other somatic cell is to be programmed. In some embodiments, the somatic cell may be a haematopoietic cell, such as a megakaryocyte or erythroid cell and the set of transcription factors may comprise or consist of GATA1 , TAL1 and FLI1. The transcription factors GATA1 , TAL1 and FLI1 may for example program PSCs into bipotent megakaryocytic-erythroid progenitors (MEPs) and then into either megakaryocytes or erythroid cells. The amino acid sequences of GATA1 , FLI1 and TAL1 are readily available on public databases. For example, the reference amino acid sequence of human GATA1 (GATA binding protein 1 ; also known as ERYF1 : Gene ID 2623) has the NCBI database entry NP_002040.1 Gl: 4503925; the reference amino acid sequence of human FLI1 (Friend leukaemia virus integration 1 , also known as EWSR1 , SIC-1 or ERGB; Gene ID 2313) has the NCBI database entry NP_002008.2 Gl: 7110593 and the reference amino acid sequence of human TAL1 (T cell acute lymphocytic leukemia protein 1 ; Gene No: 6886) has the NCBI database entry NP 003180.1 Gl: 4507363). The one or more coding sequences in the first nucleotide sequence may encode GATA1 , FLI1 and TAL1 with the reference amino sequences or with variants of these reference amino sequences.

[0057] In other embodiments, the somatic cell may be a hepatocyte and the set of transcription factors may comprise or consist of HNF6, HNF1 A, FOXA3, RORc, and optionally ERa; the somatic cell may be a macrophage and the set of transcription factors may comprise or consist of PU.1 , and m-CSFR, and optionally RARp, GATA6, and mCSF; the somatic cell may be a neuronal cell and the set of transcription factors may comprise or consist of Neurogenin 2 (NEUROG2, Ngn-2); or the somatic cell may be an oligodendrocyte and the set of transcription factors may comprise or consist of SOX10 and OLIG2.ln other embodiments, the somatic cell may be a myocyte and the set of transcription factors may comprise or consist of MYOD1 .

[0058] The expression cassette of the first nucleotide sequence may further comprise a coding sequence for a marker. This may be example allow the identification or selection of cells expressing the coding sequences in the first nucleotide sequence. Suitable labels include luminescent or fluorescent markers, such as luciferase, green fluorescent protein (GFP), mCherry, mRuby, LSS Orange, dTomato, or Turquoise 2.

[0059] The expression cassette of the first nucleotide sequence may be polycistronic. i.e. it may comprise multiple coding sequences that are expressed in a single RNA transcript. The coding sequences within the single RNA transcript may be translated as separate proteins in the mammalian cells or the transcript may be translated as a single protein that is subsequently processed to generate separate proteins. This allows the stoichiometric expression of each coding sequence in the cassette. In some embodiments, the coding sequences in a polycistronic expression cassette may be separated by sequences encoding self-cleaving oligopeptides, preferably 2A oligopeptides. A self-cleaving oligopeptide coding sequence may be located between the coding sequences in the expression cassette of the first nucleotide sequence. The self-cleaving oligopeptide causes cleavage of the nascent peptide chain during translation and separates the polypeptides encoded by the coding sequences in the polycistronic expression cassette. Suitable 2A peptides may include T2A, P2A, E2A and F2A peptides (Poddar et al (2018) supra; Kim et al (2011) PLoS ONE 6, e18556.) and may comprise the amino acid sequence of any one of SEQ ID NOs: 2, 4, 6, or 8 or a variant thereof. A self-cleaving peptide coding sequence may comprise the nucleotide sequence of any one of SEQ ID NOs: 1 , 3, 5, or 7 or a variant thereof.

[0060] A variant of a reference amino acid sequence or reference nucleotide sequence set out herein may comprise an amino acid sequence or a nucleotide sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% sequence identity to the reference sequence. Particular amino acid sequence variants may differ from the reference sequence by insertion, addition, substitution, or deletion of 1 amino acid, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more than 10 amino acids. Particular nucleotide sequence variants may differ from the reference sequence by insertion, addition, substitution, or deletion of 1 nucleotide, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more than 10 nucleotides.

[0061] Sequence similarity and identity are commonly defined with reference to the algorithm GAP (Wisconsin Package, Accelerys, San Diego USA). GAP uses the Needleman and Wunsch algorithm to align two complete sequences that maximizes the number of matches and minimizes the number of gaps. Generally, default parameters are used, with a gap creation penalty = 12 and gap extension penalty = 4. Use of GAP may be preferred but other algorithms may be used, e.g. BLAST (which uses the method of Altschul et al. (1990) J. Mol. Biol. 215: 405-410), FASTA (which uses the method of Pearson and Lipman (1988) PNAS USA 85: 2444-2448), or the Smith-Waterman algorithm (Smith and Waterman (1981) J. Mol Biol. 147'. 195- 197), or the TBLASTN program, of Altschul et al. (1990) supra, generally employing default parameters. In particular, the psi-Blast algorithm (Nucl. Acids Res. (1997) 25 3389-3402) may be used. Computerized implementations of these algorithms (GAP, BESTFIT, PASTA, and FASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wl) are available and publicly available computer software may be used such as ClustalOmega (Sbding, J. 2005, Bioinformatics 21 , 951 - 960), T-coffee (Notredame et al. 2000, J. Mol. Biol. (2000) 302, 205-217), Kalign (Lassmann and Sonnhammer 2005, BMC Bioinformatics, 6(298)), Genomequest™ software (Gene-IT, Worcester MA USA) and MAFFT (Katoh and Standley 2013, Molecular Biology and Evolution, 30(4) 772-780 software. When using such software, the default parameters, e.g. for gap penalty and extension penalty, are preferably used. A preferred example of algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., Nuc. Acids Res. 25:3389-3402 (1977) and Altschul etal., J. Mol. Biol. 215:403-410 (1990), respectively. Sequence comparison may be made over the full-length of the relevant sequence described herein.

[0062] An amino acid residue in a reference amino acid sequence may be altered or mutated by insertion, deletion or substitution, preferably substitution for a different amino acid residue, to produce a variant of the reference amino acid sequence. A nucleotide in a reference nucleotide sequence may be altered or mutated by insertion, deletion, or substitution, preferably substitution for a different nucleotide, to produce a variant of the reference nucleotide sequence.

[0063] The first nucleotide sequence is operably linked to the first promoter. A promoter that is operably linked to a nucleotide sequence is capable of affecting the expression of that nucleotide sequence when the appropriate transcription complex is present. The promoter need not be contiguous with the nucleotide sequence, so long as it functions to direct the expression of the operably linked nucleotide sequence. For example, intervening sequences that are transcribed but not translated may be present. A promoter and an operably linked nucleotide sequence may have any spacing or orientation which allows for initiation of transcription of the nucleotide sequence upon recognition of the promoter by a transcription complex. The first promoter effects the expression, in particular the transcription, of the first nucleotide sequence in the nucleic acid construct.

[0064] The first promoter may be an inducible promoter. An inducible promoter is only active under specific circumstances to cause the expression of an operably linked nucleotide sequence. In the absence of the specific conditions, the inducible promoter is inactive and does not cause expression of the operably linked nucleotide sequence. For example, an inducible promoter may be active in the presence of an analyte, cofactor or regulatory protein and inactive in the absence of the analyte, co-factor or regulatory protein. The first promoter may be active in the presence of an activated transcription regulator protein and inactive in the absence of an activated transcription regulator protein. The transcription regulator protein may be activated by the absence or more preferably the presence of an inducer compound. For example, the transcription regulator protein may induce expression from the first promoter in the absence of the inducer compound but not in the presence of the inducer compound; or the transcription regulator protein may induce expression from the first promoter in the presence of the inducer compound but not in the absence of the inducer compound.

[0065] In the nucleic acid construct described herein, the first promoter is induced to express the first nucleotide sequence by the transcription regulator protein that is encoded by the second nucleotide sequence. The first promoter may comprise sequence elements which are suitable for binding or interacting with the transcriptional regulator protein (often called a transcription factor-binding sites or response elements). The binding of the transcriptional regulator protein to the first promoter may be controlled by an inducer compound which may for example allow or block the binding of the transcriptional regulator protein with the first promoter.

[0066] A transcriptional regulator protein is a DNA binding factor that binds to a response element located in the first promoter. The binding of the transcriptional regulator protein to the response element represses or more preferably stimulates expression of the nucleotide sequence that is operably linked to the first promoter.

[0067] The transcriptional regulator protein may be activatable. An activatable transcriptional regulator protein may be active or activated under a first set of conditions to bind to the response element of the first promoter and stimulate expression of the first nucleotide sequence and inactive under a second set of conditions, so that it does not bind to the response element of the first promoter and the first nucleotide sequence is not expressed. For example, the activatable transcriptional regulator protein may be active in the presence of an inducer compound, such that it binds to the response element of the first promoter and stimulates expression. The activatable transcriptional regulator protein may be inactive in the absence of the inducer compound, such that it does not bind to the response element of the first promoter and does not stimulate expression.

[0068] Preferably, the transcriptional regulator protein is activated by the presence of the inducer compound to allow it to bind to the first promoter (ON systems). In some embodiments, the presence of the inducer compound may activate the transcriptional regulator protein. For example, the inducer compound may be introduced to the mammalian cells by addition to the medium in which the cells are cultured. Following the introduction of the inducer compound into the cells, it binds to the transcriptional regulator protein encoded by the second nucleotide sequence allowing binding of the transcriptional regulator protein to the response element of the first promoter and thereby stimulating expression. The transcriptional regulator protein thus binds to the first promoter to promote transcription and expresses the first nucleotide sequence in the presence of the inducer compound but does not bind to the first promoter or promote transcription in the absence of the inducer compound.

[0069] Alternatively, the transcriptional regulator protein may be inactivated by the presence of the inducer compound to prevent it from binding to the first promoter (OFF systems). The inducer compound may bind the transcriptional regulator protein encoded by the second nucleotide sequence and prevent it from binding to the first promoter, thereby preventing expression. In this format, the inducer compound may be withdrawn from the mammalian cells by removing it from the medium in which the cells are cultured. The removal of the inducer compound from the mammalian cell allows the transcriptional regulator protein encoded by the second nucleotide sequence to bind the first promoter sequence to stimulate expression.

[0070] The inducer compound can be any suitable substance that binds to or interacts with the transcriptional regulator protein and causes it to bind or not bind to the first promoter. Suitable substances include tetracycline, doxycycline, ponasterone A and mifepristone (RU486). Thus, the second nucleotide sequence encoding the transcriptional regulator protein within the nucleic acid construct provides the control mechanism for the expression of the first nucleotide sequence which is operably linked to the first promoter within the nucleic acid construct.

[0071] In some preferred embodiments, the inducible mammalian system may be a tetracycline-based system. For example, the response element of the first promoter may be the TRE tetracycline operator (tetO) and the transcriptional regulator protein may be the tetracycline repressor (tetR) or a derivative thereof. The inducer compound may be tetracycline or a tetracycline analogue, such as doxycycline, minocycline or tigecycline.

[0072] Preferred systems include “Tet-ON” systems, in which the presence of tetracycline (TET) or a tetracycline analogue activates the transcriptional regulator protein. The transcriptional regulator protein encoded by the second nucleotide sequence only binds to the first promoter in the presence of TET or TET analogue and induces expression of the first nucleotide sequence. The transcriptional regulator protein may be a reverse Tet repressor (rTetR), which has a “reversed” phenotype that relies on the presence of TET or a TET analogue for induction rather than repression of expression. Suitable transcriptional regulator proteins include a fusion protein comprising rTetR and VP16 (known as reverse tetracycline-controlled transactivator; rtTA)) or derivatives of rtTA, such as Tet-On Advanced transactivator (rtTA2s-M2) or Tet-On 3G (also known as rtTA-V10). Suitable transcriptional regulator proteins may comprise the amino acid sequence of SEQ ID NO: 14 or a variant thereof. A coding sequence for a transcriptional regulator protein may comprise the nucleotide sequence of SEQ ID NO: 13 or a variant thereof.

[0073] Alternatively, the system may be a “Tet-OFF” system in which the absence of TET or TET analogue activates the transcriptional regulator protein. Expression from the first promoter is reduced or blocked in the presence of TET or TET analogue. In the presence of TET or TET analogue, the TET or TET analogue binds to the transcriptional regulator protein. This precludes binding of the transcriptional regulator protein to the tetO sequences in the first promoter, resulting in reduced or blocked gene expression. In the absence of TET or TET analogue, the transcriptional regulator protein binds to the tetO sequences and promotes expression. Suitable transcriptional regulator proteins for use in such a system include tetracycline-controlled transactivator (tTA), which is a fusion protein comprising tetR and the C-terminal domain of HSV VP16 (virion protein 16). For example, a suitable transcriptional regulator protein may comprise the amino acid sequence of SEQ ID NO: 21 or a variant thereof. A coding sequence for a transcriptional regulator protein may comprise the nucleotide sequence of SEQ ID NO: 20 or a variant thereof. The sequences of other suitable transcriptional regulator proteins are well-known in the art.

[0074] The first promoter promotes expression of the first nucleotide sequence when the response element of the first promoter is bound by the transcriptional regulator protein (i.e. the activity of the first promoter is induced by the transcriptional regulator protein). In tetracycline-based systems, the response element of the first promoter may be a tetracycline response element (TRE). A TRE is a nucleotide sequence comprising multiple repeats of the 19-nucleotide tetracycline operator (tetO) sequence. Suitable TRE may comprise the nucleotide sequence of SEQ ID NO: 9 or a variant thereof. The TetO repeats may be separated by spacer sequences and linked to a minimal promoter, such as a minimal Cytomegalovirus (CMV) promoter, human phosphoglycerate kinase 1 (hPGK1) promoter, human elongation factor 1a promoter (EF1A), or viral simian virus 40 (SV40). A suitable minimal CMV promoter may comprise the nucleotide sequence of SEQ ID NO: 10 or a variant thereof.

[0075] In other embodiments, the inducible mammalian system may be a GAL-4-based system. The inducer compound may be the steroid RU486 (mifepristone). Suitable transcriptional regulator proteins for use with RU486 include fusion proteins comprising yeast GAL4 DNA binding domain protein, a ligand binding domain (LBD) from the human progesterone receptor, and a transactivator domain D from the human NF-kB protein. The first promoter and, optionally the second promoter, may comprise GAL4 upstream activating sequences (UAS). The introduction of the RU486 to the cell may activate the transcriptional regulator protein encoded by the second nucleotide sequence. The active transcriptional regulator protein promotes expression of the first nucleotide sequence. In some embodiments, it may also promote expression of the second nucleotide sequence operably linked to the second promoter. Suitable transcriptional regulator proteins and promoters are well known in the art (see for example Wang, Y. et al (1994) Proc. Natl. Acad. Sci. USA 91 , 8180-8184) and available from commercial suppliers (Thermofisher Scientific; Gene Switch ™).

[0076] In other embodiments, the inducible mammalian system may be an ecdysone receptor (EcR)-based system.

[0077] The inducer compound may be ponasterone A (ponA). Suitable transcriptional regulator proteins for use in such a system include fusion proteins comprising Drosophila ecdysone receptor (EcR), the DNA-binding domain of the glucocorticoid receptor (GR), and the transcriptional activation domain of HSV VP16. The first promoter may comprise an ecdysone- responsive element (EcRE) or derivative thereof, such as a E / GRE recognition sequence which comprises inverted half-site recognition elements for the retinoid-X-receptor (RXR) and GR binding domains. The EcR based transcriptional regulator protein blocks transcription from the first promoter. The introduction of PonA to the cell removes the repressive effect of the transcriptional regulator protein and activates expression of the first nucleotide sequence. Suitable transcriptional regulator proteins and promoters are well known in the art and available from commercial suppliers (e.g. Agilent Technologies USA).

[0078] The second promoter expresses the second nucleotide sequence that encodes the transcriptional regulator protein. Preferably, the second promoter is a constitutive promoter i.e. it directs expression of the second nucleotide sequence under standard culture conditions. For example, the transcriptional regulator protein may be constitutively expressed in a mammalian cell. Constitutive promoters may for example confer high levels of expression of the second nucleotide sequence when used in the systems described herein. Suitable second promoters are well known in the art and include constitutive promoters, such as the human p-actin promoter (ACTB), cytomegalovirus (CMV) promoter, elongation factor-la, (EFla) promoter, phosphoglycerate kinase 1 (PGK1) promoter, CAG promoter, and ubiquitinC (UbC) promoter.

[0079] In some preferred embodiments, the second promoter is a CAG promoter. The CAG promoter is a strong synthetic promoter frequently used to drive high levels of gene expression and may comprise the following sequences: the cytomegalovirus (CMV) early enhancer element (SEQ ID NO: 10), the promoter, the first exon and the first intron of chicken beta-actin gene (SEQ ID NO: 11), and the splice acceptor of the rabbit beta-globin gene (SEQ ID NO: 12). For example, a second promoter may comprise the nucleotide sequence of bases 8626 to 10294 of SEQ ID NO: 19 or a variant thereof.

[0080] The first promoter and the first nucleotide sequence are flanked by first and second insulating sequences in the nucleic acid construct. Following insertion into the genomic safe harbour, the first and second insulating sequences isolate the first promoter and the first nucleotide sequence from the genomic DNA of the mammalian cell. One of the first and second insulating sequences also isolates the first promoter and the first nucleotide sequence from the second promoter and second coding sequence.

[0081] The first insulating sequence may be upstream (i.e. 5‘) of the first promoter and the first nucleotide sequence and the second insulating sequence may be downstream (i.e. 3’) of the first promoter and the first nucleotide sequence. The first promoter and the first nucleotide sequence may be separated from the second nucleotide sequence and second promoter by one of the first and second insulating sequences i.e. one of the first and second insulating sequences may be located in the construct between the first promoter and nucleotide sequence and the second promoter and coding sequence. For example, the second nucleotide sequence and second promoter may be located in the nucleic acid construct upstream of the first insulating sequence or downstream of the second insulating sequence.

[0082] The insulating sequences are genomic insulators that form boundaries between genomic regions with different transcriptional activity, such as transcriptionally active or inactive regions. For example, a genomic insulator may protect a gene from adjacent inactive condensed chromatin, heterochromatin or adjacent enhancers that might otherwise cause inappropriate expression.

[0083] Suitable insulating sequences are known in the art and include the p-globin chicken hypersensitivity site 4 (HS4). For example, the first insulating sequence may comprise the nucleotide sequence of SEQ ID NO; 15 or a variant thereof. The second insulating sequence may comprise the nucleotide sequence of SEQ ID NO; 16 or a variant thereof.

[0084] The first and second insulating sequences may be orientated in opposite directions. For example, the first insulating sequence may be in a 5' to 3’ orientation in the nucleic acid construct and the second insulating sequence may be in a 3’ to 5’ orientation in the nucleic acid construct.

[0085] The nucleic acid construct may further comprise additional elements, such as a selectable marker. A selectable marker may allow the selection of mammalian cells in which the nucleic acid construct has successfully inserted into the target locus within the genome. Suitable markers are well-known in the art and include resistance genes to antibiotics, such as puromycin or geneticin (G418).

[0086] In some embodiments, the nucleic acid construct may allow reversible insertion into the GSH locus of the mammalian cell. For example, the nucleic acid construct may be removed or replaced after the controlled expression of the one or more coding sequences of the first nucleic acid in the mammalian cell. The nucleic acid construct may for example further comprise cleavable sequences. A cleavable sequence is a site or motif that is specifically cleaved by a factor with endonuclease activity, such as a restriction endonuclease, nuclease, recombinase, or ribozyme.

[0087] At least one cleavable sequence may be present in the nucleic acid construct, but preferably two or more are present. These cleavable sequences may be at any suitable point in the nucleic acid construct, such that a selected portion of the nucleic acid construct, or the entire nucleic acid construct, can be selectively removed from the GSH. This allows the removal and / or replacement of the nucleic acid construct or a portion thereof from the GSH after expression of the first nucleotide sequence. The cleavable sites may thus flank the part or all of the construct to be removed. The method may result in removal of the nucleic acid construct and / or portion. In some embodiments, the portion of the insertion flanked by the cleavable sites may include the promoter operably linked to the genetic sequence. Suitable cleavable sequences include the loxP site for Ore recombinase and the rox site for Dre recombinase.

[0088] The nucleic acid construct may further comprise elements to allow the insertion of the construct into a site within a target genomic locus, such as a genomic safe harbour locus., The construct comprises homology arms that mediate the targeting of the construct to a target locus by homologous recombination. Homology arms comprise a 5’ homology arm at the 5' end of the construct and a 3' homology arm at the 3’ end of the construct. The 5’ and 3’ homology arms may comprise a sequence of 30 to 1000 nucleotides that is complementary to the nucleotide sequence of the target genomic locus upstream or downstream respectively of the site of insertion within the locus. Suitable homology arms for the targeted insertion of nucleic acid constructs into genomic safe harbour loci, such as AAVS1 , are available in the art and may be generated using conventional techniques. For example, suitable homology arms for targeting the AAVS1 locus may comprise the nucleotide sequence of SEQ ID NO: 17 and SEQ ID NO: 18 or variants thereof.

[0089] The nucleic acid construct described above may be contained in a vector for the transduction of the mammalian cells.

[0090] A vector is a nucleic acid molecule which is used as a vehicle to artificially carry genetic material into a cell. The vector is generally a nucleic acid sequence that consists of an insert (such as a nucleic acid construct described herein) and a larger sequence that serves as the "backbone" of the vector. The vector may comprise the nucleic acid construct described herein and may be in any suitable format, including plasmids, minicircle, or linear DNA. In some embodiments, a vector may further comprise multi cloning restriction enzyme site, self-cleaving 2A oligopeptides, poly A sequences and ribosomal binding sites. A vector may further comprise sequences, such as origins of replication, promoter regions, and selectable markers, which allow for its selection, expression and replication in bacterial hosts such as E. coli. For further details see, for example, Molecular Cloning: a Laboratory Manual: 3rd edition, Russell et al., 2001 , Cold Spring Harbor Laboratory Press. Many known techniques and protocols for manipulation of nucleic acid, for example in preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, are described in detail in Current Protocols in Molecular Biology, Ausubel et al. eds. John Wiley & Sons, 1992.

[0091] The nucleic acid constructs and vectors described herein may be used to generate cells and cell lines that controllably express the first nucleotide sequence by means of the inducer compound. A method of producing a mammalian cell for controllable expression of a nucleotide sequence may comprise; inserting a nucleic acid construct described above into a genomic safe harbour (GSH) locus within the genome of the mammalian cell.

[0092] The nucleic acid construct may be inserted into a site within a GSH locus on one chromosome, or on both chromosomes of the mammalian cell. Insertion within both chromosomes may be preferred, since it may allow an increase in the level of transcription from the inserted first nucleotide sequence, thus achieving particularly high levels of expression of the coding sequences in the first nucleotide sequence.

[0093] A vector comprising the nucleic acid construct may be introduced into a mammalian cell in combination with one or more targeting vectors that facilitate insertion into the GSH locus. The introduction may employ any suitable available technique. Suitable techniques may depend on the vector and cell type and may include calcium phosphate transfection, DEAE-Dextran, electroporation or Nucleofection ©Technology (Lonza), liposome-mediated transfection and transduction using retrovirus or other virus, e.g. vaccinia.

[0094] The one or more targeting vectors may then facilitate the insertion of the nucleic acid construct into the GSH locus. Suitable techniques and targeting vectors for insertion of the nucleic acid construct into the GSH locus are well known the art. In some embodiments, a double-strand DNA break (DSB) may be generated at a site in the GSH locus. Cellular DNA repair mechanisms, such as homologous recombination (HR) dependent DNA DSB repair may then be exploited to repair the DSB and to insert the nucleic acid construct into GSH locus. The one or more targeting vectors may express suitable factors to generate a site-specific DNA DSB within a GSH of a mammalian cell.

[0095] Suitable techniques for generating a site-specific DNA DSB in a GSH include clustered regularly interspaced short palindromic repeats / CRISPR associated protein (CRISPR / Cas9), zinc finger nucleases (ZFNs), and transcription activator-like effector nucleases (TALENs) (see for example (Gaj, T, et al Trends Biotechnol, 31 :397-405, July 2013). The one or more targeting vectors may comprise sequences encoding ZFNs, TALENs or CRISPR / Cas9 that are capable of generating the site-specific DNA DSB in the GSH.

[0096] In some embodiments, CRISPR / Cas9 may be employed, The Cas9 protein maybe introduced into a cell by direct delivery of a ribonucleoprotein (RNP) consisting of the Cas9 protein in complex with a targeting guide RNA (gRNA). Cas9 RNPs are capable of cleaving genomic targets with similar efficiency to vector / plasmid- based expression of Cas9 / gRNA and are readily available in the art.

[0097] In some preferred embodiments, Zinc finger nucleases (ZFNs) may be employed. ZFNs are artificial enzymes which are generated by fusion of a zinc-finger DNA-binding domain to the nuclease domain of the restriction enzyme Fokl. The latter has a non-specific cleavage domain which must dimerise in order to cleave DNA. Two ZFN monomers may be required to allow dimerization of the Fokl domains and to cleave the DNA. The DNA binding domain may be designed to target any genomic sequence of interest and is a tandem array of Cys2His2 zinc fingers, each of which recognises three contiguous nucleotides in the target sequence. The two binding sites are separated by 5-7bp to allow optimal dimerisation of the Fokl domains. The enzyme thus is able to cleave DNA at a specific site, and target specificity is increased by ensuring that two proximal DNA-binding events must occur to achieve a double- strand break. In some embodiments, two targeting vectors, each encoding a different ZFN, may be employed, such that the expressed ZFNs dimerise and generate a DNA DSB.

[0098] A nucleic acid construct may comprise homology arms that are complementary to the sequence of the GSH locus at the site of the DNA DSB. When the DNA DSB is generated, it is repaired by the homologous recombination (HR) dependent DNA DSB repair pathway within the cell. During this repair, the nucleic acid construct is precisely inserted into the site.

[0099] A genomic safe harbour (GSH) is a locus within the genome of a mammalian cell into which a gene may be inserted without any deleterious effects on the cell or on the inserted gene. Insertion of a gene into a site within a GSH may avoid read-through expression of the gene from neighbouring genes and the disruption of critical endogenous gene expression in the mammalian cell. Suitable genomic safe harbour loci are well known in the art and include the citrate lyase beta like gene locus (CLYBL) gene locus in intron 2 (Cerbibi et al, 2015, PLOS One, DOI : 10.1371 ) , the Rosa 26 locus within the open reading frame (ORF) of the THUMPD3 long non-coding RNA (reverse strand) on chromosome 3 (3p25.3), the C-C chemokine receptor type 5 (CCR5) locus, and the Adeno- Associated Virus Integration Site 1 (AAVS1) locus in the first intron of PPP1 R12C (Hockemeyer 2009 Nature Biotech 27 851-857;Qian 2014 Stem Cells 32(5) 1230-1238; Carlson- Stevermer 2016 Stem Cell Reports 6(1) 109-120; Kotini 2015 Nat.Biotechnol 33 645-655). In some preferred embodiments, the genomic safe harbour locus is the AAVS1 locus. The nucleic acid construct may be conveniently targeted to the AAVS1 locus using zinc finger nucleases (ZFNs). Suitable ZFNs for targeting sites within the AAVS1 locus are well known in the art and include for example pZFN- AAVS1_ELD (Addgene; #159297) and pZFN-AAVS1_KKR (Addgene; #159298)). A method may comprise transfecting a mammalian cell with a vector comprising the nucleic acid construct and one or more ZFN vectors targeting the AAVS1 locus. The ZFNs expressed by the targeting vectors generate a DNA DSB at site within the AAVS1 locus. When the DNA DSB is repaired by the cell, the nucleic acid construct is inserted into the site.

[0100] Also disclosed herein is a mammalian cell that comprises a nucleic acid construct described above, at a genomic safe harbour locus, preferably the AAVS1 locus, within the genome of the cell. Suitable mammalian cells for use as described herein may be human cells or non-human mammalian cells. Preferably, the cells are isolated cells and the methods described herein are performed in vitro. Suitable mammalian cells include pluripotent stem cells (PSCs) and somatic or mature cells of any differentiated or partially differentiated cell type that contains an intact GSH locus.

[0101] In some preferred embodiments, the mammalian cells may be PSCs. PSCs may include embryonic stem cells, induced PSCs or naive stem cells.

[0102] PSCs are unspecialized, undifferentiated cells that are capable of replicating or self-renewing themselves and developing into specialized cells of all three primary germ layers i.e. ectoderm, mesoderm and endoderm but are not able to develop into all embryonic and extra-embryonic tissues, including trophectoderm (i.e. not totipotent). PSCs include embryonic stem (ES) cells and non-embryonic stem cells, including foetal and adult somatic stem cells and stem cells derived from non-pluripotent cells, for example induced pluripotent (iPS) cells which are derived from non-pluripotent cells. PSCs may express one or more of the following pluripotency associated markers: Oct4 (Pou5f1), Sox2, Alkaline Phosphatase, SSEA-3, Nanog, SSEA-4 and Tra-1-60. Preferably, pluripotent stem cells express Oct4 (Octamer-binding transcription factor 4 encoded by the Pou5f1 POU class 5 homeoboxl gene).

[0103] In some embodiments, the PSCs may be ES cells, for example human ES cells or non-human ES cells. Suitable ES cells may be obtained from a cultured hES cell line, such as Edi2, H9 or hSF-6. Further examples of suitable human embryonic stem cells are described in (Thomson JA et al Science 282: 1145- 1147 (1998); Reubinoff et al. Nat Biotechnol 18:399-404 (2000); Cowan, C.A. et al. N. Engl. J. Med. 350, 1353-1356(2004), Gage, F.H., et al. Ann. Rev. Neurosci. 18 159-192 (1995); and Gotlieb (2002) Annu. Rev. Neurosci 25 381-407); Carpenter et al. Stem Cells. 5(1): 79-88 (2003); see also: the NIH stem cell registry which is accessible online. Potentially clinical grade hESCs are described in Klimanskaya, I. et al. Lancet 365, 1636-1641 (2005); and Ludwig, T.E. et al. Nat. Biotechnol. 24, 185-187 (2006).

[0104] In other embodiments, the pluripotent stem cells may be iPS cells, for example human iPS cells. IPS cells are pluripotent cells which are derived from non-pluripotent ancestor cells, for example somatic cells, such as fibroblasts. Ancestor cells are typically reprogrammed into iPS cells through the introduction of reprogramming factors Oct4, Sox2, Klf4 and c-Myc into the cell. Other suitable reprogramming factors and combinations of reprogramming factors for inducing pluripotency are known in the art. (see, for example, Yu et al Science 318 2007 1917-1920, Tesar, P. J. et al. Nature 448, 196-199 (2007); Nichols, J. & Smith, A. Cell Stem Cell 4, 487-492(2009); Ying, Q. L. et al. Nature 453, 519-523 (2008), Hanna J, et al Proc Natl Acad Sci U S A. 2010 May 18;107(20):9222-7; Han DW, et al Nat Cell Biol. 2011 Jan;13(1):66-71 ; Silva J et al Cell. 2009 Aug 21 ; 138(4)722-37). Reprogramming factors and techniques for the production of iPS cells are well-known in the art and include introducing reprogramming factors by plasmid or viral transfection, direct protein delivery or direct delivery of nucleic acid, such as mRNA. (Yamanaka et al Nature (2007); 448:313-7; Yamanaka 6 (2007) Jun 7;1 (1):39-49. Kim et al. Nature. (2008) Jul 31 ;454(7204):646-50;

[0105] Takahashi Cell. (2007) Nov 30;131(5):861 -72. Park et al Nature. (2008) Jan 10; 451 (7175): 141 -6; Kimet al Cell Stem Cell. (2009) Jun 5;4(6):472-6; Vallier, L., et al. (2009) Stem Cells 27, 2655-66.).

[0106] In some preferred embodiments, the mammalian cell may be a pluripotent stem cell, and the first nucleotide sequence of the nucleic acid construct may comprise coding sequences for a set of transcription factors that are capable of forward programming the pluripotent stem cell into a somatic cell type. This may be useful for example for the efficient production of large populations of pure somatic cells in vitro. Use of the inducible expression system described herein for forward programming for example may remove the need for lentiviral vector transduction of vectors encoding the set of transcription factors.

[0107] In other embodiments, the mammalian cell may be a somatic cell, and the first nucleotide sequence of the nucleic acid construct may comprise coding sequences for a set of transcription factors that are capable of forward programming the somatic cell into a different somatic cell type. This may be useful for example for the efficient production of large populations of pure somatic cells in vitro. In other embodiments, the mammalian cell may be a somatic cell, and the first nucleotide sequence of the nucleic acid construct may comprise coding sequences for a set of transcription factors that are capable of forward programming the somatic cell into a pluripotent stem cell. This may be useful for example for the efficient production of large populations of pure pluripotent stem cells in vitro. In other embodiments, the mammalian cell may be a pluripotent stem cell, and the first nucleotide sequence of the nucleic acid construct may comprise coding sequences for a set of transcription factors that are capable of forward programming the somatic cell into a pluripotent stem cell of a different type. This may be useful for example for the efficient production of large populations of pure pluripotent stem cells in vitro.

[0108] A method of producing a somatic cell or programming a pluripotent stem cell into a somatic cell may comprise; a) providing a pluripotent stem cell comprising a nucleic acid construct described above at a genomic safe harbour site within its genome, wherein the first nucleotide sequence comprises coding sequences for a set of transcription factors that program a pluripotent stem cell (PSC) into the somatic cell and the second nucleotide sequence is expressed in the PSC to produce a transcriptional regulator protein, b) activating the transcriptional regulator protein to induce the expression from the first promoter, such that the first nucleotide sequence is expressed to produce the set of transcription factors in the PSC, wherein said transcription factors program the PSC into the somatic cell.

[0109] The transcriptional regulator protein may be activated by the presence of an inducer compound. Binding of the transcriptional regulator protein to the inducer compound allows binding to the first promoter as described herein and induces expression of the first nucleotide sequence. In some preferred embodiments, culturing the PSCs in the presence of the inducer compound activates the transcriptional regulator protein. In other embodiments, culturing the PSCs in the absence of the inducer compound activates the transcriptional regulator protein.

[0110] Expression of the set of transcription factors encoded by the coding sequences of the first nucleotide sequence within the PSC following induction of the first promoter by the transcriptional regulator protein promotes forward programming of the PSC and the development of a somatic cell phenotype.

[0111] The cell may be cultured under suitable conditions and for a sufficient period of time, following introduction of the transcription factors, to allow the cell to display a somatic cell phenotype. After programming, the somatic cell may be maintained in culture, expanded, stored, for example frozen using conventional techniques, or used in therapeutic or other applications as described herein.

[0112] Forward programming is the direct imposition of a more differentiated phenotype on a pluripotent stem cell or other precursor cell which bypasses normal differentiation pathway; i.e. the cell does not pass through the intermediate stages of differentiation taken during the in vivo development of that cell type. For example, a PSC which is forward programmed into a megakaryocyte does not differentiate through the mesoderm progenitor, haemogenic endothelium progenitor and hematopoietic progenitor stages that would occur in- vivo or a directed differentiation setting before displaying a megakaryocyte phenotype. In other words, during step (b), the cells said population may not progressively display the phenotype of each intermediate stage of differentiation. Differentiation occurs rapidly as if a compressed version of normal events which has the advantage of channelling more cells towards one pathway.

[0113] The somatic cell may be of any differentiated or partially differentiated cell type, depending on the transcription factors encoded by the first nucleotide sequence. For example, the somatic cell may be a nerve cell, myocyte, osteocyte, chondrocyte, epithelial cell, secretory cell, or blood cell, such as a megakaryocyte.

[0114] PSCs may be forward programmed in a chemically defined medium (CDM). A CDM is a nutritive solution for culturing cells which contains only specified components, preferably components of known chemical structure. A CDM is devoid of components which are not fully defined, for example serum or proteins isolated therefrom, such as Foetal Bovine Serum (FBS), Bovine Serum Albumin (BSA), and feeder or other cells. In some embodiments, a CDM may be humanised and may be devoid of components from non-human animals. Proteins in the CDM may be recombinant human proteins Suitable CDMs are well known in the art and described in more detail below.

[0115] Media and ingredients thereof may be obtained from commercial sources (e.g. Gibco, Roche, Sigma, Europa bioproducts, Cellgenix, Life Sciences, Promocell, CellGenix, Stem Cell technologies). In a humanised CDM, for example BSA may be replaced in CDM by Polyvinyl alcohol (PVA), human serum albumin, Plasmanate™ (human albumin, alpha-globulin and beta globulin: Talecris Biotherapeutics NC USA) or Buminate™ (human albumin: Baxter Healthcare), all of which are available from commercial sources. Suitable CDMs may be based on DMEM / F12 supplemented with 20% knockout serum replacement (KSR), 1 mM L-GIn, 100pM non-essential amino acids, 100pM 2-mercaptoethanol; Essential 6TM; Essential 8TM (Gibco); mTeSR™ (Ludwig et al Nat Biotech 2006 24 185); Stemfit 03 / 04 (Amsbio), N12 medium and Johansson and Wiles CDM (Johansson and Wiles (1995) Mol Cell Biol 15, 141 -151). These basal media may be supplemented with suitable cytokines or a a concentrated supplement for pluripotent stem cell culture.

[0116] During or after forward programming, the cells may be cultured in a medium adapted for the somatic cell type. For example, haematopoetic cells may be cultured in a suitable haematopoetic medium, such as AMK (Evans AL et al. Blood Adv. 2021 ;5(7):1977-1990), CellGenix® GMP SCGM (CellGenix) or StemSpan™ (Stem Cell Technologies).

[0117] In some embodiments, the somatic cell may be a myocyte and the transcription factors may be myogenic transcription factors. For example, the set of transcription factors may comprise or consist of MYOD1 . The PSCs may be cultured in the presence of retinoic acid (RA) following activation of the transcriptional regulator protein during the programming, such that the PSCs are forward programmed into myocytes. In other embodiments, the somatic cell may be a cardiomyocyte and the transcription factors may be cardiomyogenic transcription factors. For example, the set of transcription factors may comprise or consist of Gata4, Mef2c, Baf60c and Tbx5. In other embodiments, the somatic cell may be a bone cell, such as an osteocyte, and the transcription factors may be osteogenic transcription factors. For example, the set of transcription factors may comprise or consist of L-Myc (RXOL) Runx2, Osterix. In other embodiments, the somatic cell may be a chondrocyte, and the transcription factors may be chondrogenic transcription factors. For example, the set of transcription factors may comprise or consist of c-Myc Klf4, and SOX9. In other embodiments, the somatic cell may be a brown adipocyte and the transcription factors may be adipogenic transcription factors. For example, the set of transcription factors may comprise or consist of C / EBP-p and c- Myc. In other embodiments, the somatic cell may be an oligodendrocyte and the set of transcription factors may be oligodendrogenic transcription factors. For example, the set of transcription factors may comprise or consist of SOX-10, OLIG2, NKX2.2, and NKX6.2. In other embodiments, the somatic cell may be an astrocyte and the set of transcription factors may be astrocytic transcription factors. For example, the set of transcription factors may comprise or consist of NFIA, NFIB, and SOX9. In other embodiments, the somatic cell may be a neuron and the set of transcription factors may be neuronal transcription factors. For example, the set of transcription factors may comprise or consist of Ascii , neurogenin, and NeuroD or Pax6, Neurog2, Ascii , Dlx2, and NeuroD. In other embodiments, the somatic cell may be a haematopoetic cell and the set of transcription factors may be haematopoetic transcription factors. For example, the set of transcription factors may comprise or consist of GATA1 , TAL1 and FLI1 . In some embodiments, the set of transcription factors may consist of GATA1 , FLI1 and TAL1 i.e. the only transcription factors in introduced into the PSCs are GATA1 , FLI1 and TAL1. In other embodiments, the set of transcription factors may consist of GATA1 , FLI1 and TAL1 , with optionally, one, two, three or more, additional transcription factors. For example, additional transcription factors may include one or more of IKZF1 , HOXA5, RUNX1 , ZFPM2, ZFPM1 and GATA2. In other embodiments, the starting cell maybe a PSC or fibroblast and transcription factors may include MEF2A and MEF2D involved in terminal macrophage differentiation (Biochem J. 2010 Aude-Garcia et al.) or SPI1 / PU.1 (Blood 2010 Zakrzewska et al.), CEBPA (Pios One 2019 Repele et al) and MITF (Molecular Cancer 2020 Ballot! et al). In other embodiments, the somatic cell may be a hepatocyte and the set of transcription factors may be hepatocyte transcription factors. For example, the set of transcription factors may comprise or consist of HNF6, HNF1A, FOXA3, RORc, and optionally Era. In other embodiments, the somatic cell may be a macrophage and the set of transcription factors may be macrophage transcription factors. For example, the set of transcription factors may comprise or consist of PU.1 , and m-CSFR, and optionally RARp, GATA6, and mCSF. In other embodiments, the somatic cell may be a neuronal cell and the set of transcription factors be neuronal transcription factors. For example, the set of transcription factors may may comprise or consist of Neurogenin 2 (NEUROG2, Ngn-2), In other embodiments, the somatic cell may be an oligodendrocyte and the set of transcription factors may be oligodendrocyte transcription factors. For example, the set of transcription factors may comprise or consist of SOX10 and OLIG2.ln other embodiments, the somatic cell may be a myocyte and the set of transcription factors may be myocyte transcription factors. For example, the set of transcription factors may comprise or consist of MYOD1 .

[0118] In some preferred embodiments, the somatic cell may be a haematopoietic cell, preferably a megakaryocyte or a megakaryocyte progenitor cell and the set of transcription factors may be transcription factors for megakaryopoiesis. Megakaryocytes are non-proliferative bone marrow cells which are responsible for the production of platelets. Megakaryocytes may have the phenotype CD34+ / -, CD61 +, CD41 a+, CD42a+, CD42b+, GPVI+ or CD61+, CD41 a+, CD42a+, CD42b+, GPVI+, CD235a+ / -. Mature megakaryocytes are large (20-100um) polyploid cells (4-128N) which eventually produce platelets through pro-platelet formation. Megakaryocyte progenitors may include megakaryoblasts, pro-megakaryocytes and megakaryocytes (Chang et al Journal of Thrombosis and Haemostasis, 5 (Suppl. 1): 318-327).

[0119] For example, a method of producing a megakaryocyte or programming a pluripotent cell into a megakaryocyte may comprise; a) providing a pluripotent cell comprising a nucleic acid construct at a genomic safe harbour site within its genome, the nucleic acid comprising;

[0120] (i) a first nucleotide sequence comprising coding sequences for the transcription factors GATA1 , TAL1 and FLI1 ,

[0121] (ii) a first promoter operably linked to the first nucleotide sequence, said first promoter being inducible,

[0122] (iii) a first insulating sequence located upstream of the first nucleotide sequence and first promoter,

[0123] (iv) a second insulating sequence located downstream of the first nucleotide sequence and first promoter, and

[0124] (v) a second nucleotide sequence encoding an activatable transcription regulator protein that induces expression from the promoter;

[0125] (vi) a second promoter operably linked to the second nucleotide sequence, wherein; said second promoter and second nucleotide sequence are separated from the first nucleic acid and first promoter by the first or second insulating sequence; and the second nucleotide sequence is expressed in the pluripotent cell to produce the activatable transcription regulator protein, b) activating the transcriptional regulator protein to induce the expression from the first promoter, such that the first nucleotide sequence is expressed to produce the transcription factors GATA1 , TAL1 and FLI1 in the pluripotent cell, and

[0126] (c) culturing the cell, such that the transcription factors program the pluripotent cell into a megakaryocyte.

[0127] The cell may be cultured under suitable conditions and for a sufficient period of time, following introduction of the transcription factors, to allow one or more cells in the population to display a megakaryocyte phenotype, for example the stable expression of CD61 , CD42 and CD41 a; and / or the stable expression of CD61 , CD235a, CD42 and CD41 a in said cells. After programming, the megakaryocytes may be maintained in culture, expanded, stored, for example frozen using conventional techniques, or used in therapeutic or other applications as described herein. For example, the megakaryocytes may be expanded and / or matured by culturing in a medium supplemented with SCF and TPO. Suitable media include AMK.

[0128] Other aspects and embodiments of the invention provide the aspects and embodiments described above with the term “comprising” replaced by the term “consisting of” and the aspects and embodiments described above with the term “comprising” replaced by the term ’’consisting essentially of’.

[0129] The term “downstream” as used herein refers to the 5' to 3’ direction in a nucleic acid described herein and the term “upstream” as used herein refers to the 3’ to 5’ direction in a nucleic acid described herein.

[0130] Reference to a nucleotide sequence as set out herein encompasses a DNA molecule with the specified sequence, and encompasses a RNA molecule with the specified sequence in which U is substituted for T, unless context requires otherwise.

[0131] It is to be understood that the application discloses all combinations of any of the above aspects and embodiments described above with each other, unless the context demands otherwise. Similarly, the application discloses all combinations of the preferred and / or optional features either singly or together with any of the other aspects, unless the context demands otherwise.

[0132] Modifications of the above embodiments, further embodiments and modifications thereof will be apparent to the skilled person on reading this disclosure, and as such, these are within the scope of the present invention.

[0133] All documents and sequence database entries mentioned in this specification are incorporated herein by reference in their entirety for all purposes.

[0134] “and / or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example, “A and / or B” is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein.

[0135] Materials & Methods Generation of inducible targeting constructs

[0136] The All- in one Megakaryocyte (Aim) system exemplified below uses a TET-ON 3G, third generation reverse tetracycline-controlled transactivator (rtTA) constitutively expressed through a CAG promoter, a fusion of multiple TET operons and a minimal Cytomegalovirus (CMV) promoter. The construct also contains coding sequences for three megakaryopoiesis forward programming transcription factors plus a green fluorescent protein (GFP) as a polycistronic cassette conditionally expressed under the control of a tetracycline response element (TRE) regulated by the rtTA and responsive to doxycycline (dox). The GFP and three transcription factors are separated by different self-cleaving 2A oligopeptides, leading to the production of four individual proteins from one mRNA via ribosomal skipping. Dual insulating sequences (INS1 , INS2) based on the beta globin chicken hypersensitivity site 4 (HS4) are incorporated flanking 5' and 3' to the polycistronic cassette. To mitigate any interference between the CAG promoter driving rtTA expression and the TRE driving the polycistronic cassette, the CAG promoter was placed outside the second insulating sequence (INS2) 3’ to the polycistronic cassette (Figure 4).

[0137] The construct is then targeted to the Adeno Associated Virus Integration Site 1 (AAVS1) using Zinc finger nucleases (ZFNs). These targeting plasmids, pZFN-AAVS1_ELD (Addgene; #159297) and pZFN- AAVS1_KKR (Addgene; #159298)), contain codon optimised sequences for mammalian expression (Hockemeyer Nat Biotechnol. 2009). The combination of these two plasmids induces a specific double strand break between exons 1 and 2 of PPR1 R12C on chromosome 19 (AAVS1 locus).

[0138] Modifications to the polycistronic cassette designed for forward programming were performed using NEBuilder HiFi DNA assembly protocol. The positions of insertion of the genomic insulators used for the Aim vector are shown in Figure 1 , and the subsequent conversion to an all-in one system in Figure 4.

[0139] Addition of B-globin chicken hypersensitivity site 4 (HS4) sequences (Figurel)

[0140] Insertion of dual insulating sequences flanking 5’ and 3’ to the polycistronic cassette based on the p globin chicken hypersensitivity site 4 (HS4) sequence, designed originally for use in Xenopus, improves the GFP expression (Figure 2) and therefore transgene expression. Using sequences obtained from Addgene plasmid #74102 a 551 bp fragment was cloned seamlessly into the original pUC_AAVS1_eGFP_PC3 with the aid of specific primers. The first sequence was cloned 5’ to 3’ in orientation followed by sequential insertion of a 560bp fragment in the 3’ to 5' orientation (INS2). Note the sequences are in the same orientation in the chicken genome and a two-step method of cloning is necessary to avoid recombination during cloning since the flanking sequences are very similar and inverting the sequence exacerbates this tendency. Since the insulators do have directional dependent consequences, sequential cloning minimizes this risk.

[0141] This approach led to c / s-regulatory improvements in expression, minimizing endogenous regulatory silencing and allowing significant improvements in the number of viable megakaryocytes obtained (Figure 3).

[0142] Introduction of the reverse tetracycline-controlled transactivator (rtTA) to the AAVS1 targeting construct Building on the success of INS addition, a constitutively expressed rtTA was cloned using HiFi assembly as three fragments 3’ to INS2, the first linking the CAG promoter to INS2 plus a synthetic intron and the rtTA element. The resulting 14372 bp construct (Figure 4) allowed much simplified creation of inducible hiPSCs with the potential use for conversion to production of any cell type using appropriate transcription factors in replace of the 3TF used to create megakaryocytes. The creation of inducible lines using 3 vectors instead of the 6 vectors used in the dual approach was found to massively increase the ease and efficiency of edited line production, retaining the ability to form mature MKs with high CD42 / 41 levels (Figures 3,5)

[0143] Human pluripotent cell culture hPSC lines derived from dermal fibroblasts using the four Yamanaka's factors (OCT4, SOX2, KLF4, MYC) introduced by a variety of means (integrative retroviral vectors, episomal plasmid vectors and non-integrative Sendai vectors) and, hESCs were used in these experiments. hESCs and hPSCs were obtained from the UK Regenerative Medicine Platform (UKRMP), the Human Induced Pluripotent Stem Cell Initiative (HipSCi), Cell and Gene Therapy Catapult (Catapult). hPSC were maintained on a recombinant vitronectin (VTN-N) human substrate at 0.5pg / cm2(Thermo Fisher Scientific) or laminin-521 at 5pg / ml (BioLamina) in E8 media (Thermo Fisher Scientific) and passaged using EDTA / PBS or TrypLE Select (Thermo Fisher Scientific).

[0144] Generation of inducible hPSC for MK-FoP

[0145] Prior to genetic modification of each hPSC a puromycin survival curve was performed. Parental lines were seeded as single cells, following TrypLE Select (Thermo Fisher Scientific) dissociation, at 5.00+04 cells per well of a 12-well plate in E8 media plus rock inhibitor Y-27632 (Sigma) 10pM overnight at 37°C and 5% CO2. Colonies are allowed to grow for 48hrs changing the media daily then Puromycin (Sigma) 1 mg / ml stock in dH20 was added at 2, 1 , 0.25, 0.125, 0.0625, 0.03125 pg / ml final concentration to different wells. After monitoring daily for a week, the results are recorded generally cultures are killed by between 0.5 and 0.125 pg / ml puromycin.

[0146] To create inducible lines low passage number parental lines were dissociated to single cells using TrypLE Select, counted using a haemocytometer and 5.00+05 centrifuged gently at 1 15g for 3 minutes at room temperature. Vectors may be introduced using the Amaxa® 4D-Nucleofector® using manufacterer’s protocols. However, AIM clones described here were created using a very simple transfection technique using Lipofectamine Stem Transfection reagent (LSTR) (Gibco). Laminin LN-521 at 5pg / ml (BioLamina), diluted in DPBS with calcium and magnesium (ThermoFisher Scientific), plates are coated overnight at 4°C then the coating mix aspirated and E8 media plus rock inhibitor Y-27632 (Sigma) 10pM added to receive cells. Then cells were dissociated to single cells using TrypLE Select (Gibco), counted using a haemocytometer, and 2x 105transferred to a fresh tube and centrifuged for 5 mins 300g, while lipid mix is incubating. For lipid mix, the ratio of Cells: DNA : LSTR used was 2 x 105cells: 0.25pmol total vectors: 0.25pl LSTR. For example, Tube 1 containing 10OpI media + 0.25pmol DNA (x 3 plasmids.0.08333pmol per vector) was mixed with Tube 2 containing 10OpI media +2.5pl LSTR. The mixture was incubated at RT for 10 mins. As much of the supernatant as possible was aspirated from the centrifuged cells and the pellet gently resuspended. The DNA: LSTR mix was added to the cell pellet, mixed and plated into prepared plates at 1-2 x 105cell per well.

[0147] Transfected cells were incubated overnight at 37°C and 5% CO2 then fresh media without Y-27632 added. Selection with puromycin and puromycin selection was started 48hrs after transfection using line determined concentrations. Selection continued for 7 days or until all the cells on the non-transfected control well were dead. Colonies started to appear 7-10 days later and are picked into 12 well plates mechanically using a 200p pipette tip, colonies were picked relatively small to ensure maintenance of pluripotency. Colonies were expanded and transferred back to VTN coated plates by passage 3. Frozen vials and genomic DNA (gDNA) was prepared for genotyping.

[0148] Genotyping of inducible clones

[0149] Genomic DNA was extracted using the Purelink® Genomic DNA Mini Preparation kit (Invitrogen, Life Technologies) and eluted in 50p I dH20, the concentration determined using a Qubit® 2.0 Fluorometer (Invitrogen, Life Technologies) and gDNA diluted to 10ng / pl in aliquots for genotyping and 20ng used for each reaction. All genotyping PCRs were performed using Q5®Hot Start High-Fidelity DNA polymerase (NEB). In brief, PCR for integration at the AAVS1 GSH used 63°C for 32 cycles with 1 ,5-minute extension giving 1692bp fragment in wildtype cells and no band when integration is homozygous. Integration at the 3’ and 5’ AAVS1 sites used 55°C with 1 ,5minute extension for 35 cycles and 55°C for 1 minutes extension for 35 cycles respectively using specific primers. Key lines may then undergo whole genome sequencing as well as karyotyping analysis.

[0150] Inducible forward programming (iMK_FoP)

[0151] Following the same principles as viral forward programming including an initial mesoderm induction phase, except differentiation is initiated from inducible cassette on the addition of doxycycline. hPSCs are seeded for differentiation on day-1 onto vitronectin coated plates in E8 + Y27326 10pg / ml as single cells @ 5.00E+04 x 12 well plates (TrypLE split) and on day 0 mesoderm media (AE6 + FGF2 20ng / ml (4ul / ml) + BMP4 10ng / ml (1 ul / ml) + Dox (at the concentration determined in trial experiments). Day 1 media is supplemented with CHIR99021 3pM (Cayman Chemical Company). Mesoderm media is refreshed on day 2 plus doxycycline, and on D3 media replaced with MK media plus TPO 20ng / ml (Biotechne) and SCF 25ng / ml (ThermoFisher Scientific) plus doxycycline. Media is refreshed every 2 day and on day 10 cells harvested producing a single-cell suspension with TrypLE and seeded in fresh MK media, TPO 20ng / ml (Biotechne) and SCF 25ng / ml (ThermoFisher Scientific) plus doxycycline. Partial media changes are then performed every 2 days until D15-25 or longer when cells are mature, If cell culture becomes dense (>1x 10! cells / ml) then wells maybe split. MK media maybe AMK or suitable commercial media such as those described above.

[0152] Flow cytometry analysis

[0153] Flow cytometry was carried out using a Gallios flow cytometer (Beckman Coulter). Single cell suspensions were generated from hPSCs using TrypLE Select (Thermo Fisher Scientific) dissociation followed by a wash step in basal media at 300g for 5 minutes and then stained for 207RT in PBS 0.2%BSA 5mM EDTA using combinations of FITC, PE and APC conjugated antibodies. For GFP expression (Figure 2, 6B) cells were harvested for flow without staining apart from DAPI as a vital dye. For mature MK cultures in suspension (Figure 3, 5A, 7ABCD) the culture was gently mixed by pipetting and an aliquot stained. For inducible lines CD235aPeCy7, CD42aAPC and CD41 APCH7 antibodies were used. Background fluorescence was set against matched isotype control antibodies and a compensation matrix defined using single-color stained cells. Flow count fluorospheres (Beckman Coulter) and 4’-6’-diamidino-2-phenylindole (DAPI)1 pg / ml were used to determine viable cell count in samples. For pluripotency checks undifferentiated cells were stained using SSEA PE and Tra-1-60 FITC antibodies (Figure 6A).

[0154] Intracellular flow cytometry staining Intracellular detection for von Willebrand (VWF) (Figure5C) was performed using the Fix and Perm cell Permeabilization Kit (Thermo Fisher Scientific). Mature MK suspension cultures were first stained with the extracellular antigens CD42a APC, CD41 APC H7 and CD9 BV510 for 15 minutes, then fixed using reagent A for a further 15 minutes, washed used in PBS 0.2%BSA 5mM EDTA and permeabilized with reagent B plus the intracellular stain VWF for 20 minutes followed by a further wash. Samples were run on Gallios flow cytometer (Beckman Coulter).

[0155] Gene expression analysis by RT-qPCR

[0156] Total RNA was prepared using the Nucleospin® RNA plus kit (Macherey-Nagel) including a DNA removal step and 500ng RNA reverse transcribed using Superscript®IV reverse transcriptase and random hexamers (Thermo Fisher Scientific). Two-step qPCR was performed with SYBR green chemistry on the ABI 7500HT (Applied Biosystems) by the standard curve method using HMBS endogenous control gene and rTTA specific primers (Figure 9).

[0157] In the exemplified Aim system, presence of the insulating sequences ensured robust GFP and transgene expression while at the same time allowing sufficient physical separation of the CAG and TRE sequences resulting in efficient programming to MKs. Data from the first two clones tested using flow cytometry of surface marker expression CD42 and CD41 showed a pure MK population (Figure 5A) releasing viable CD42 / 41 positive platelets (Figure 5B). The MKs produced also expressed von Willebrand factor (VWF) a marker of a mature adult phenotype (Figure 5C). For some lines, such as iRCI B10.1 (Figures 3, 5), addition of insulators allowed the cell-line to become a useable tool in the study of MKs, when previous techniques had proved unsuccessful.

[0158] Using the Aim system for lines already known to be capable of robust MK production provided inducible lines which could be further manipulated using for example the Rosa26 GSH. In a detailed comparison of the new system alongside the dual system, with and without insulators, it was clear that the system was robust, while showing the expected variation of any system giving independent clones. Detailed comparison of the dual system with and without INS alongside 3 independent indicated that the system performed well, producing viable MKs which released platelets with comparable levels of intracellular VWF indicating mature MKs capable of endoreduplication.

[0159] Undifferentiated lines retained pluripotency when cultured in the same conditions as the original line (Figure 6A) and on differentiation, gave robust GFP signals (Figure 6B) and remained capable of forming all three germ layers given the correct cues. Expression levels of maturity markers and foetal markers showed comparable levels to those described. At reduced doxycycline doses, the addition of insulating sequences had a marked positive effect on yield with GFP levels (a proxy for transgene expression) maintained for longer in insulated compared to uninsulated clones. Insulating sequences have potential to reduce silencing of iPSC transgenes for the derivation of any cell type by these methods The Aim system would not be expected to make a massive change to the FoP pathway and the resulting MKs contained the test construct with the original 3TFs. The much-simplified system further opens this field of research in terms of accessibility and the potential to investigate megakaryocyte biology and platelet formation. Finally, as for mature MKs derived using the dual system, it is possible to freeze thaw these cells. The Aim system has now been tested in 5 hiPSC and 2 hESC lines with success. The most suitable doxycycline dose was empirically determined for each new clone. The Aim platform was robust as illustrated in Figure 7 showing data from 2 iQolg Aim clones (Figure 7A), 2 lines from hESCs iMS10.8A (Figure 7B) and iMS4.4A (Figure 7C) and a Thrombocytopaenia Absent Radius (TAR) patient line alongside the sibling control line (Figure 7D).

[0160] During the design of the Aim construct, other configurations of the promotors in relation to the insulator sequences were tested. Figure 8 shows a configuration (Aimv3) with the reverse tetracycline-controlled transactivator (rtTA) constitutively expressed through a CAG promoter inserted 3’ to the INS1 downstream of the 5'HAR arm. Clones were generated using this configuration, but no GFP signal was seen and the rTTA signal by qPCR compared to the dual system with insulators was reduced indicating cis interference between the rTTA sequences and the multiple TET operons (Figure 9). However, when the rTTA sequences were moved outside 3’ to INS2 sequences, this was resolved giving the final construct (Figure 4; Aim v7).

[0161] Simplification of the editing process also reduced the complexity of genotyping the new lines and all those shown herein are homologous knockins at the AAVS1 locus. Applied to the hiPSCs derived from patients with platelet disorders such as Gray Platelet Syndrome (GPS) or Thrombocytopaenia Absent Radius (TAR) this has already meant the difference between being able to use these frequently difficult lines to program and failing to produce enough platelets to give meaningful data. The above point is also relevant to any disease situation where hiPSC have proved difficult to program compared to control lines when using lentiviral systems.

[0162] Advances made in the programming of megakaryocytes towards the transfer to the clinic2 10are retained in the Aim clones. However, a key advantage of the Aim system is that by using only one of the two most described and tenable safe harbours of the human genome i.e. AAVS1 this leaves the other, the Rosa 26 locus, free to accommodate other constructs to enhance research into the understanding of MK biology and platelet disorders and in particular to address terminal differentiation and ploidy levels seen in hiPSC derived MKs. Additionally, use of ZFNs rather than CRISPR potentially simplifies the translational process. The Aim construct can be used as the basis to create AiDs or All in one Differentiation constructs by simply replacing our 3 megakaryocyte biased transcription factors with any other TFs of interest thus making it a universal inducible system.

[0163] References

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[0165] 2. Evans AL, Dalby A, Foster HR, et al. Blood Adv. 2021 ;5(7):1977-1990. 3. Doronina VA, Wu C, de Felipe P, Sachs MS, Ryan MD, Brown JD.. Molecular and

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[0167] 4. Yu Y, Lowy MM, Elble RC.. Metab Eng Commun.20'\6;3-.64-67.

[0168] 5. Bestor TH. Gene silencing as a threat to the success of gene therapy. The Journal of clinical investigation. 2000;105(4):409-411 . 6. Gossen M, Bujard H. T Proc Natl Acad Sci U S A. 1992;89(12):5547-5551.

[0169] 7. Wang C, Szaro BG. Dev B / o / .2015;398(1):11-23.

[0170] 8. Shearwin KE, Callen BP, Egan JB. Trends Genet. 2005;21 (6):339-345.

[0171] 9. Hasegawa K, Nakatsuji N. FEBS Lett. 2002;520(1-3):47-52.

[0172] 10. Lawrence M, Evans A, Moreau T, et al. European GMP. NPJ Regen Med. 2021 ;6(1):27. 11. Shin S, Kim SH, Shin SW, et al.ACS Synth Biol. 2020;9(6): 1263-1269.

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[0174] 13. Bibikova M, Golic M, Golic KG, Carroll D. Genetics. 2002;161 (3):1169-1175.

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[0176] 15. Pawlowski et al (2017) Stem Cell Reports 8 803-812

[0177] uences

[0178] GAGGGCAGAGGAAGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCCGGCCCT

[0179] SEQ ID NO: 1 coding sequence for self-cleaving 2A peptide from thosea asigna virus 2A capsid protein (T2A) 1261-1314 54bp

[0180] EGRGSLLTCGDVEENPGP

[0181] SEQ ID NO: 2 amino acid sequence for self-cleaving 2A peptide from thosea asigna virus 2A capsid protein (T2A)

[0182] GGAAGCGGACAGTGTACTAATTATGCTCTCTTGAAATTGGCTGGAGATGTTGAGAGCAACCCTGGACCT

[0183] SEQ ID NO: 3 coding sequence for self-cleaving 2A peptide from equine rhinitis A virus (bases 3870-3938 of

[0184] SEQ ID NO: 19) 69bp (E2A) 1261-1314 54bp

[0185] EADSVLIMLS *NWLEMLRATLDL

[0186] SEQ ID NO: 4 amino acid sequence for self-cleaving 2A peptide from equine rhinitis A virus (E2A)

[0187] GGAAGCGGAGCTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACCT

[0188] SEQ ID NO: 5 coding sequence for self-cleaving 2A peptide from porcine teschovirus-1 2A (P2A) 5199-5264 66bp

[0189] EAELLTSAC* SRLETWRRTLDL

[0190] SEQ ID NO: 6 amino acid sequence for self-cleaving 2A peptide from porcine teschovirus-1 2A (P2A)

[0191] GGAAGCGGAGAGGGCAGAGGAAGTCTGCTAACATGCGGTGACGTCGAGGAGAATCCTGGACCT

[0192] SEQ ID NO: 7 coding sequence for self-cleaving 2A peptide from thosea asigna virus 2A (bases 6300-6362 of SEQ ID NO: 19) 63bp (T2A)

[0193] EAERAEEVC*HAVTSRRILDL

[0194] SEQ ID NO: 8 amino acid sequence for self-cleaving 2A peptide from thosea asigna virus 2A

[0195] GAG T T T AC T C C C TAT C AG T GAT AGAGAAC G T AT GAAGAG T T T AC T C C C TAT C AG T GAT AGAGAAC G T AT G C AGAC T T T AC TCCCTATCAGTGATAGAGAACGTATAAGGAGTTTACTCCCTATCAGTGATAGAGAACGTATGACCAGTTTACTCCCTATC AGTGATAGAGAACGTATCTACAGTTTACTCCCTATCAGTGATAGAGAACGTATATCCAGTTTACTCCCTATCAGTGATAG AGAACGTATGTCGAGGTAGGCGTGTACGGTGGGCGCCTATAAAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGA GCAATTCCACAACACTTTTGTCTTATACTT

[0196] SEQ ID NO: 9 nucleotide sequence for Tet-responsive promoter (TRE3GV promoter) (bases 2743-3092 of

[0197] SEQ ID NO: 19) 350bp

[0198] CGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATG

[0199] TTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTA

[0200] CATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTA CATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATG SEQ ID NO: 10 human cytomegalovirus immediate early enhancer (bases 8626-8929 of SEQ ID NO: 19) 304bp

[0201] TCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTT

[0202] TAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGG

[0203] CGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGC

[0204] GGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCG

[0205] SEQ ID NO: 11 chicken p-actin promoter (bases 8931-9208 of SEQ ID NO: 19) 278bp

[0206] GGGAGTCGCTGCGACGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACC

[0207] GCGTTACTCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCGCTTGGTTTAATGACGGCTTGT

[0208] TTCTTTTCTGTGGCTGCGTGAAAGCCTTGAGGGGCTCCGGGAGGGCCCTTTGTGCGGGGGGAGCGGCTCGGGGGGTGCGT

[0209] GCGTGTGTGTGTGCGTGGGGAGCGCCGCGTGCGGCTCCGCGCTGCCCGGCGGCTGTGAGCGCTGCGGGCGCGGCGCGGGG

[0210] CTTTGTGCGCTCCGCAGTGTGCGCGAGGGGAGCGCGGCCGGGGGCGGTGCCCCGCGGTGCGGGGGGGGCTGCGAGGGGAA

[0211] CAAAGGCTGCGTGCGGGGTGTGTGCGTGGGGGGGTGAGCAGGGGGTGTGGGCGCGTCGGTCGGGCTGCAACCCCCCCTGC

[0212] ACCCCCCTCCCCGAGTTGCTGAGCACGGCCCGGCTTCGGGTGCGGGGCTCCGTACGGGGCGTGGCGCGGGGCTCGCCGTG

[0213] CCGGGCGGGGGGTGGCGGCAGGTGGGGGTGCCGGGCGGGGCGGGGCCGCCTCGGGCCGGGGAGGGCTCGGGGGAGGGGCG

[0214] CGGCGGCCCCCGGAGCGCCGGCGGCTGTCGAGGCGCGGCGAGCCGCAGCCATTGCCTTTTATGGTAATCGTGCGAGAGGG

[0215] CGCAGGGACTTCCTTTGTCCCAAATCTGTGCGGAGCCGAAATCTGGGAGGCGCCGCCGCACCCCCTCTAGCGGGCGCGGG

[0216] GCGAAGCGGTGCGGCGCCGGCAGGAAGGAAATGGGCGGGGAGGGCCTTCGTGCGTCGCCGCGCCGCCGTCCCCTTCTCCC

[0217] TCTCCAGCCTCGGGGCTGTCCGCGGGGGGACGGCTGCCTTCGGGGGGGACGGGGCAGGGCGGGGTTCGGCTTCTGGCGTG

[0218] TGACCGGCGGCTCTAGAGCCTCTGCTAACCATGTTCATGCCTTCTTCTTTTTCCTACAGCTCCTGGGCAACGTGCTGGTT

[0219] ATTGTGCTGTCTCATCATTTTGGCAAAGAATTAATTCGGATCCACC

[0220] SEQ ID NO: 12 synthetic intron (bases 9209-10294 of SEQ ID NO: 19) 1086bp

[0221] ATGTCTAGACTGGACAAGAGCAAAGTCATAAACTCTGCTCTGGAATTACTCAATGGAGTCGGTATCGAAGGCCTGACGAC

[0222] AAGGAAACTCGCTCAAAAGCTGGGAGTTGAGCAGCCTACCCTGTACTGGCACGTGAAGAACAAGCGGGCCCTGCTCGATG

[0223] CCCTGCCAATCGAGATGCTGGACAGGCATCATACCCACTCCTGCCCCCTGGAAGGCGAGTCATGGCAAGACTTTCTGCGG

[0224] AACAACGCCAAGTCATACCGCTGTGCTCTCCTCTCACATCGCGACGGGGCTAAAGTGCATCTCGGCACCCGCCCAACAGA

[0225] GAAACAGTACGAAACCCTGGAAAATCAGCTCGCGTTCCTGTGTCAGCAAGGCTTCTCCCTGGAGAACGCACTGTACGCTC

[0226] TGTCCGCCGTGGGCCACTTTACACTGGGCTGCGTATTGGAGGAACAGGAGCATCAAGTAGCAAAAGAGGAAAGAGAGACA

[0227] CCTACCACCGATTCTATGCCCCCACTTCTGAAACAAGCAATTGAGCTGTTCGACCGGCAGGGAGCCGAACCTGCCTTCCT

[0228] TTTCGGCCTGGAACTAATCATATGTGGCCTGGAGAAACAGCTAAAGTGCGAAAGCGGCGGGCCGACCGACGCCCTTGACG

[0229] ATTTTGACTTAGACATGCTCCCAGCCGATGCCCTTGACGACTTTGACCTTGATATGCTGCCTGCTGACGCTCTTGACGAT TTTGACCTTGACATGCTCCCCGGGTAA

[0230] SEQ ID NO: 13 rtTA coding sequence (bases 10295-11041 of SEQ ID NO: 19) 747bp

[0231] MSRLDKSKVINSALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHVKNKRALLDALPIEMLDRHHTHSCPLEGESWQDFLR

[0232] NNAKSYRCALLSHRDGAKVHLGTRPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERET

[0233] PTTDSMPPLLKQAIELFDRQGAEPAFLFGLELI I CGLEKQLKCESGGPTDALDDFDLDMLPADALDDFDLDMLPADALDD FDLDMLPG*

[0234] SEQ ID NO: 14 rtTA amino acid sequence (bases 10295-11041 of SEQ ID NO: 19) 249AA GTAGACAGACTCATATCCGCGGGAGCTCACGGGGACAGCCCCCCCCCCAAAGCCCCCAGGGCTGTAATTGCGTTCCTCTCCCGCTAGGGGGCGGCAGCGA

[0235] GCCGCCCAGGGCTCCGCTCCGGCCCGACGCTCCCCCCGCATCCCCGAGCCGGAGCCCGCAGCGTGCGGGGACAGCCCGGCACGGGGAAGGTGGCACGGGA

[0236] TCGCTTTCCTCTGAACGCTTCTCGCTGCTCTTTGAGCCTGCAGACACCTGGGGGGATACGGGGAAAAAGCTTTATCTAGATCCGCGGGAGCTCACGGGGA

[0237] CAGCCCCCCCCCAAAGCCCCCAGGGCTGTAATTGCGTTCCTCTCCCGCTAGGGGGCAGCAGCGAGCCGCCCGGGGCTCCGCTCCGGCCCGACGCTCCCCC

[0238] CGCATCCCCGAGCCGGAGCCCGCAGCGTGCGGGGACAGCCCGGCACGGGGAAGGTGGCACGGGATCGCTTTCCTCTGAACGCTTCTCACTGCTCTTTGAG

[0239] CCTGCAGACACCTGGGGGGATACGGGGAAAAAGCTTTATCGCTCTGTCGCA

[0240] SEQ ID NO: 15 INS1 5' to 3' (bases 2189-2739 of SEQ ID NO: 19) 551 bp

[0241] CACTTAGACAGACTCATATCCGCGGGAGCTCACGGGGACAGCCCCCCCCCAAAGCCCCCAGGGCTGTAATTGCGTTCCTCTCCCGCTAGGGGGCAGCAGC

[0242] GAGCCGCCCGGGGCTCCGCTCCGGCCCGACGCTCCCCCCGCATCCCCGAGCCGGAGCCCGCAGCGTGCGGGGACAGCCCGGCACGGGGAAGGTGGCACGG

[0243] GATCGCTTTCCTCTGAACGCTTCTCGCTGCTCTTTGAGCCTGCAGACACCTGGGGGGATACGGGGAAAAAGCTTTATCTAGATCCGCGGGAGCTCACGGG

[0244] GACAGCCCCCCCCCAAAGCCCCCAGGGCTGTAATTGCGTTCCTCTCCCGCTAGGGGGCAGCAGCGAGCCGCCCGGGGCTCCGCTCCGGCCCGACGCTCCC

[0245] CCCGCATCCCCGAGCCGGAGCCCGCAGCGTGCGGGGACAGCCCGGCACGGGGAAGGTGGCACGGGATCGCTTTCCTCTGAACGCTTCTCACTGCTCTTTG

[0246] AGCCTGCAGACACCTGGGGGGATACGGGGAAAAAGCTTTATCGCTCTGTCCGCCAGTAGG

[0247] SEQ ID NO: 16 INS2 5' to 3' (bases 8577-8018 of SEQ ID NO: 19) 560bp

[0248] TGCTTTCTCTGACCAGCATTCTCTCCCCTGGGCCTGTGCCGCTTTCTGTCTGTAGCTTGTGGCCTGGGTCACCTCTACGGCTGGCCCAG

[0249] ATCCTTCCCTGCCGCCTCCTTCAGGTTCCGTCTTCCTCCACTCCCTCTTCCCCTTGCTCTCTGCTGTGTTGCTGCCCAAGGATGCTCTT

[0250] TCCGGAGCACTTCCTTCTCGGCGCTGCACCACGTGATGTCCTCTGAGCGGATCCTCCCCGTGTCTGGGTCCTCTCCGGGCATCTCTCCT

[0251] CCCTCACCCAACCCCATGCCGTCTTCACTCGCTGGGTTCCCTTTTCCTTCTCCTTCTGGGGCCTGTGCCATCTCTCGTTTCTTAGGATG

[0252] GCCTTCTCCGACGGATGTCTCCCTTGCGTCCCGCCTCCCCTTCTTGTAGGCCTGCATCATCACCGTTTTTCTGGACAACCCCAAAGTAC

[0253] CCCGTCTCCCTGGCTTTAGCCACCTCTCCATCCTCTTGCTTTCTTTGCCTGGACACCCCGTTCTCCTGTGGATTCGGGTCACCTCTCAC

[0254] TCCTTTCATTTGGGCAGCTCCCCTACCCCCCTTACCTCTCTAGTCTGTGCTAGCTCTTCCAGCCCCCTGTCATGGCATCTTCCAGGGGT

[0255] CCGAGAGCTCAGCTAGTCTTCTTCCTCCAACCCGGGCCCCTATGTCCACTTCAGGACAGCATGTTTGCTGCCTCCAGGGATCCTGTGTC

[0256] CCCGAGCTGGGACCACCTTATATTCCCAGGGCCGGTTAATGTGGCTCTGGTTCTGGGTACTTTTATCTGTCCCCTCCACCCCACAGTGG GGC

[0257] SEQ ID NO: 17 5’HAR Homology Arm (bases 402-1205 of SEQ ID NO: 19) 804bp

[0258] ACTAGGGACAGGATTGGTGACAGAAAAGCCCCATCCTTAGGCCTCCTCCTTCCTAGTCTCCTGATATTGGGTCTAACCCCCACCTCCTG

[0259] TTAGGCAGATTCCTTATCTGGTGACACACCCCCATTTCCTGGAGCCATCTCTCTCCTTGCCAGAACCTCTAAGGTTTGCTTACGATGGA

[0260] GCCAGAGAGGATCCTGGGAGGGAGAGCTTGGCAGGGGGTGGGAGGGAAGGGGGGGATGCGTGACCTGCCCGGTTCTCAGTGGCCACCCT

[0261] GCGCTACCCTCTCCCAGAACCTGAGCTGCTCTGACGCGGCTGTCTGGTGCGTTTCACTGATCCTGGTGCTGCAGCTTCCTTACACTTCC

[0262] CAAGAGGAGAAGCAGTTTGGAAAAACAAAATCAGAATAAGTTGGTCCTGAGTTCTAACTTTGGCTCTTCACCTTTCTAGTCCCCAATTT

[0263] ATATTGTTCCTCCGTGCGTCAGTTTTACCTGTGAGATAAGGCCAGTAGCCAGCCCCGTCCTGGCAGGGCTGTGGTGAGGAGGGGGGTGT

[0264] CCGTGTGGAAAACTCCCTTTGTGAGAATGGTGCGTCCTAGGTGTTCACCAGGTCGTGGCCGCCTCTACTCCCTTTCTCTTTCTCCATCC

[0265] TTCTTTCCTTAAAGAGTCCCCAGTGCTATCTGGGACATATTCCTCCGCCCAGAGCAGGGTCCCGCTTCCCTAAGGCCCTGCTCTGGGCT

[0266] TCTGGGTTTGAGTCCTTGGCAAGCCCAGGAGAGGCGCTCAGGCTTCCCTGTCCCCCTTCCTCGTCCACCATCTCATGCCCCTGGCTCTC

[0267] CTGCCCCTTCCCTACAGGGGTTCCTGGCTCTGCTCT

[0268] SEQ ID NO: 18 3 HAR 3’ Homology Arm (bases 11296-12132 of SEQ ID NO: 19)

[0269] TCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGC

[0270] AGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGT

[0271] GCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCCATTCGCCATTCAGGCTGCGCAACTGTT

[0272] GGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCA

[0273] GGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAATTGTGCTTTCTCTGACCAGCATTCTCTCCCCTGGGCCTGTGCCGCTT

[0274] TCTGTCTGTAGCTTGTGGCCTGGGTCACCTCTACGGCTGGCCCAGATCCTTCCCTGCCGCCTCCTTCAGGTTCCGTCTTCCTCCACTCC CTCTTCCCCTTGCTCTCTGCTGTGTTGCTGCCCAAGGATGCTCTTTCCGGAGCACTTCCTTCTCGGCGCTGCACCACGTGATGTCCTCT

[0275] GAGCGGATCCTCCCCGTGTCTGGGTCCTCTCCGGGCATCTCTCCTCCCTCACCCAACCCCATGCCGTCTTCACTCGCTGGGTTCCCTTT

[0276] TCCTTCTCCTTCTGGGGCCTGTGCCATCTCTCGTTTCTTAGGATGGCCTTCTCCGACGGATGTCTCCCTTGCGTCCCGCCTCCCCTTCT

[0277] TGTAGGCCTGCATCATCACCGTTTTTCTGGACAACCCCAAAGTACCCCGTCTCCCTGGCTTTAGCCACCTCTCCATCCTCTTGCTTTCT

[0278] TTGCCTGGACACCCCGTTCTCCTGTGGATTCGGGTCACCTCTCACTCCTTTCATTTGGGCAGCTCCCCTACCCCCCTTACCTCTCTAGT

[0279] CTGTGCTAGCTCTTCCAGCCCCCTGTCATGGCATCTTCCAGGGGTCCGAGAGCTCAGCTAGTCTTCTTCCTCCAACCCGGGCCCCTATG

[0280] TCCACTTCAGGACAGCATGTTTGCTGCCTCCAGGGATCCTGTGTCCCCGAGCTGGGACCACCTTATATTCCCAGGGCCGGTTAATGTGG

[0281] CTCTGGTTCTGGGTACTTTTATCTGTCCCCTCCACCCCACAGTGGGGCAAGCTTCTGACCTCTTCTCTTCCTCCCACAGGGCCTCGAGA

[0282] GATCTGGCAGCGGAGAGGGCAGAGGAAGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCCGGCCCTAGGCTCGAGATGACCGAGTAC

[0283] AAGCCCACGGTGCGCCTCGCCACCCGCGACGACGTCCCCAGGGCCGTACGCACCCTCGCCGCCGCGTTCGCCGACTACCCCGCCACGCG

[0284] CCACACCGTCGATCCGGACCGCCACATCGAGCGGGTCACCGAGCTGCAAGAACTCTTCCTCACGCGCGTCGGGCTCGACATCGGCAAGG

[0285] TGTGGGTCGCGGACGACGGCGCCGCGGTGGCGGTCTGGACCACGCCGGAGAGCGTCGAAGCGGGGGCGGTGTTCGCCGAGATCGGCCCG

[0286] CGCATGGCCGAGTTGAGCGGTTCCCGGCTGGCCGCGCAGCAACAGATGGAAGGCCTCCTGGCGCCGCACCGGCCCAAGGAGCCCGCGTG

[0287] GTTCCTGGCCACCGTCGGCGTCTCGCCCGACCACCAGGGCAAGGGTCTGGGCAGCGCCGTCGTGCTCCCCGGAGTGGAGGCGGCCGAGC

[0288] GCGCCGGGGTGCCCGCCTTCCTGGAGACCTCCGCGCCCCGCAACCTCCCCTTCTACGAGCGGCTCGGCTTCACCGTCACCGCCGACGTC

[0289] GAGGTGCCCGAAGGACCGCGCACCTGGTGCATGACCCGCAAGCCCGGTGCCTGATCTAGAGGGCCCGTTTAAACCCGCTGATCAGCCTC

[0290] GACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTT

[0291] CCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAG

[0292] GATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGTCAGTAGACAGACTCATATCCGCGGGAGCTCACGGGGACA

[0293] GCCCCCCCCCCAAAGCCCCCAGGGCTGTAATTGCGTTCCTCTCCCGCTAGGGGGCGGCAGCGAGCCGCCCAGGGCTCCGCTCCGGCCCG

[0294] ACGCTCCCCCCGCATCCCCGAGCCGGAGCCCGCAGCGTGCGGGGACAGCCCGGCACGGGGAAGGTGGCACGGGATCGCTTTCCTCTGAA

[0295] CGCTTCTCGCTGCTCTTTGAGCCTGCAGACACCTGGGGGGATACGGGGAAAAAGCTTTATCTAGATCCGCGGGAGCTCACGGGGACAGC

[0296] CCCCCCCCAAAGCCCCCAGGGCTGTAATTGCGTTCCTCTCCCGCTAGGGGGCAGCAGCGAGCCGCCCGGGGCTCCGCTCCGGCCCGACG

[0297] CTCCCCCCGCATCCCCGAGCCGGAGCCCGCAGCGTGCGGGGACAGCCCGGCACGGGGAAGGTGGCACGGGATCGCTTTCCTCTGAACGC

[0298] TTCTCACTGCTCTTTGAGCCTGCAGACACCTGGGGGGATACGGGGAAAAAGCTTTATCGCTCTGTCGCACTCGAGTTTACTCCCTATCA

[0299] GTGATAGAGAACGTATGAAGAGTTTACTCCCTATCAGTGATAGAGAACGTATGCAGACTTTACTCCCTATCAGTGATAGAGAACGTATA

[0300] AGGAGTTTACTCCCTATCAGTGATAGAGAACGTATGACCAGTTTACTCCCTATCAGTGATAGAGAACGTATCTACAGTTTACTCCCTAT

[0301] CAGTGATAGAGAACGTATATCCAGTTTACTCCCTATCAGTGATAGAGAACGTATGTCGAGGTAGGCGTGTACGGTGGGCGCCTATAAAA

[0302] GCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGCAATTCCACAACACTTTTGTCTTATACTTACTAGTGCCACCATGGTGAGCAA

[0303] GGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGG

[0304] GCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACC

[0305] ACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTA

[0306] CGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACC

[0307] GCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTAT

[0308] ATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCA

[0309] CTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACC

[0310] CCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTCCGGA

[0311] CTCAGATCTCGACTAGCTAGTAGCTACCCCGGGGCGGCCGCTGGAAGCGGACAGTGTACTAATTATGCTCTCTTGAAATTGGCTGGAGA

[0312] TGTTGAGAGCAACCCTGGACCTTTCGAAATGGAGTTCCCTGGCCTGGGGTCCCTGGGGACCTCAGAGCCCCTCCCCCAGTTTGTGGATC

[0313] CTGCTCTGGTGTCCTCCACACCAGAATCAGGGGTTTTCTTCCCCTCTGGGCCTGAGGGCTTGGATGCAGCAGCTTCCTCCACTGCCCCG

[0314] AGCACAGCCACCGCTGCAGCTGCGGCACTGGCCTACTACAGGGACGCTGAGGCCTACAGACACTCCCCAGTCTTTCAGGTGTACCCATT

[0315] GCTCAACTGTATGGAGGGGATCCCAGGGGGCTCACCATATGCCGGCTGGGCCTACGGCAAGACGGGGCTCTACCCTGCCTCAACTGTGT

[0316] GTCCCACCCGCGAGGACTCTCCTCCCCAGGCCGTGGAAGATCTGGATGGAAAAGGCAGCACCAGCTTCCTGGAGACTTTGAAGACAGAG

[0317] CGGCTGAGCCCAGACCTCCTGACCCTGGGACCTGCACTGCCTTCATCACTCCCTGTCCCCAATAGTGCTTATGGGGGCCCTGACTTTTC

[0318] CAGTACCTTCTTTTCTCCCACCGGGAGCCCCCTCAATTCAGCAGCCTATTCCTCTCCCAAGCTTCGTGGAACTCTCCCCCTGCCTCCCT

[0319] GTGAGGCCAGGGAGTGTGTGAACTGCGGAGCAACAGCCACTCCACTGTGGCGGAGGGACAGGACAGGCCACTACCTATGCAACGCCTGC

[0320] GGCCTCTATCACAAGATGAATGGGCAGAACAGGCCCCTCATCCGGCCCAAGAAGCGCCTGATTGTCAGTAAACGGGCAGGTACTCAGTG

[0321] CACCAACTGCCAGACGACCACCACGACACTGTGGCGGAGAAATGCCAGTGGGGATCCCGTGTGCAATGCCTGCGGCCTCTACTACAAGC TACACCAGGTGAACCGGCCACTGACCATGCGGAAGGATGGTATTCAGACTCGAAACCGCAAGGCATCTGGAAAAGGGAAAAAGAAACGG

[0322] GGCTCCAGTCTGGGAGGCACAGGAGCAGCCGAAGGACCAGCTGGTGGCTTTATGGTGGTGGCTGGGGGCAGCGGTAGCGGGAATTGTGG

[0323] GGAGGTGGCTTCAGGCCTGACACTGGGCCCCCCAGGTACTGCCCATCTCTACCAAGGCCTGGGCCCTGTGGTGCTGTCAGGGCCTGTTA

[0324] GCCACCTCATGCCTTTCCCTGGACCCCTACTGGGCTCACCCACGGGCTCCTTCCCCACAGGCCCCATGCCCCCCACCACCAGCACTACT

[0325] GTGGTGGCTCCGCTCAGCTCAGGCGGCCGCACGCGTGGAAGCGGAGCTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGA

[0326] GAACCCTGGACCTACTAGTTGCGATCGCATGACCGAAAGACCCCCTAGCGAGGCCGCCAGAAGCGACCCTCAGCTGGAAGGCAGAGATG

[0327] CCGCCGAGGCCTCTATGGCCCCTCCTCATCTGGTGCTGCTGAACGGCGTGGCCAAAGAGACAAGCAGAGCCGCCGCTGCCGAGCCCCCT

[0328] GTGATTGAACTGGGAGCTAGAGGCGGACCTGGCGGAGGACCTGCAGGCGGAGGCGGAGCTGCTAGAGATCTGAAGGGAAGGGATGCCGC

[0329] CACAGCCGAGGCCAGACACAGAGTGCCTACCACCGAGCTGTGCAGACCTCCAGGACCTGCTCCAGCTCCTGCCCCTGCTTCTGTGACAG

[0330] CTGAACTGCCTGGCGACGGCCGGATGGTGCAGCTGTCTCCTCCTGCTCTGGCTGCTCCTGCTGCACCTGGCAGAGCCCTGCTGTACTCT

[0331] CTGTCTCAGCCTCTGGCCAGCCTGGGCAGCGGCTTTTTTGGAGAGCCCGACGCCTTCCCCATGTTCACCACCAACAACAGAGTGAAGCG

[0332] GAGGCCCAGCCCCTACGAGATGGAAATCACCGACGGCCCCCACACCAAGGTCGTGCGGAGAATCTTCACCAACTCCAGAGAGCGGTGGC

[0333] GGCAGCAGAATGTGAATGGCGCCTTCGCCGAGCTGCGGAAGCTGATCCCTACCCACCCCCCCGACAAGAAGCTGAGCAAGAACGAGATC

[0334] CTGCGGCTGGCTATGAAGTACATCAACTTTCTGGCCAAGCTGCTGAATGACCAGGAAGAGGAAGGCACCCAGCGGGCCAAGACCGGCAA

[0335] AGATCCTGTCGTGGGAGCTGGCGGCGGAGGGGGGGGAGGGGGAGGCGGCGCTCCTCCTGATGATCTGCTGCAGGATGTGCTGAGCCCCA

[0336] ACAGCAGCTGTGGCAGCAGTCTGGATGGCGCCGCTAGCCCCGATAGCTACACAGAGGAACCCGCCCCTAAGCACACCGCCAGATCTCTG

[0337] CATCCCGCCATGCTGCCTGCTGCCGATGGCGCTGGACCTAGAGCGATCGCGATAGTCGACTTGCGTACGGGAAGCGGAGAGGGCAGAGG

[0338] AAGTCTGCTAACATGCGGTGACGTCGAGGAGAATCCTGGACCTATGGACGGCACCATCAAAGAGGCCCTGAGCGTCGTCAGCGACGACC

[0339] AGAGCCTGTTCGACAGCGCCTACGGCGCTGCCGCCCATCTGCCCAAGGCCGATATGACAGCCAGCGGCAGCCCCGACTACGGCCAGCCC

[0340] CACAAGATCAACCCCCTGCCCCCCCAGCAGGAATGGATCAACCAGCCCGTGCGCGTGAACGTGAAGCGCGAGTACGACCACATGAACGG

[0341] CAGCCGCGAGAGCCCCGTGGACTGCAGCGTGTCCAAGTGCAGCAAGCTCGTGGGCGGAGGCGAGAGCAACCCCATGAACTACAACAGCT

[0342] ACATGGACGAGAAGAACGGCCCTCCCCCCCCTAACATGACCACCAACGAGCGGAGAGTGATCGTGCCCGCCGACCCCACCCTGTGGACA

[0343] CAGGAACATGTGCGGCAGTGGCTGGAATGGGCCATCAAAGAATACTCCCTGATGGAAATCGATACCAGCTTCTTCCAGAACATGGACGG

[0344] CAAAGAACTGTGCAAGATGAACAAAGAGGACTTCCTGCGGGCCACCACCCTGTACAACACCGAGGTGCTGCTGAGCCACCTGAGCTACC

[0345] TGAGAGAGAGCAGCCTGCTGGCCTACAACACCACCAGCCACACCGACCAGAGCAGCCGGCTGAGCGTGAAAGAGGACCCCAGCTACGAC

[0346] AGCGTGCGGAGAGGCGCCTGGGGCAACAACATGAACAGCGGCCTGAACAAGAGCCCCCCACTGGGCGGAGCCCAGACCATCAGCAAGAA

[0347] CACCGAGCAGAGGCCCCAGCCCGACCCCTACCAGATCCTGGGCCCTACCAGCAGCAGACTGGCCAACCCTGGCAGCGGCCAGATCCAGC

[0348] TGTGGCAGTTTCTGCTGGAACTGCTGAGCGACAGCGCCAACGCCAGCTGCATCACCTGGGAGGGCACCAACGGCGAGTTCAAGATGACC

[0349] GACCCCGACGAGGTGGCCAGACGCTGGGGCGAGAGAAAGTCCAAGCCCAACATGAACTACGACAAGCTGAGCAGAGCCCTGCGGTACTA

[0350] CTACGATAAGAACATCATGACCAAGGTGCACGGCAAGCGCTACGCCTACAAGTTCGACTTCCACGGAATCGCCCAGGCCCTGCAGCCCC

[0351] ACCCTACCGAGAGCAGCATGTACAAGTACCCCAGCGACATCAGCTACATGCCCAGCTACCACGCCCACCAGCAGAAAGTGAACTTCGTG

[0352] CCCCCCCACCCCAGCAGCATGCCCGTGACCTCCAGCAGCTTCTTCGGAGCCGCCAGCCAGTACTGGACCAGCCCCACCGGCGGCATCTA

[0353] CCCCAACCCCAACGTGCCCAGACACCCCAATACCCACGTGCCCTCCCACCTGGGCAGCTACTACTGAGTCGACTCCTCGAGGGAATTCG

[0354] AGCTCGGTACCCGGGGATCCTCTAGTCAGCTGACGCGTGCTAGCGCGGCCGCATCGATAAGCTTGTCGACGATATCTCTAGAGGATCAT

[0355] AATCAGCCATACCACATTTGTAGAGGTTTTACTTGCTTTAAAAAACCTCCCACACCTCCCCCTGAACCTGAAACATAAAATGAATGCAA

[0356] TTGTTGTTGTTAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCA

[0357] CTGCCTTCCTACTGGCGGACAGAGCGATAAAGCTTTTTCCCCGTATCCCCCCAGGTGTCTGCAGGCTCAAAGAGCAGTGAGAAGCGTTC

[0358] AGAGGAAAGCGATCCCGTGCCACCTTCCCCGTGCCGGGCTGTCCCCGCACGCTGCGGGCTCCGGCTCGGGGATGCGGGGGGAGCGTCGG

[0359] GCCGGAGCGGAGCCCCGGGCGGCTCGCTGCTGCCCCCTAGCGGGAGAGGAACGCAATTACAGCCCTGGGGGCTTTGGGGGGGGGCTGTC

[0360] CCCGTGAGCTCCCGCGGATCTAGATAAAGCTTTTTCCCCGTATCCCCCCAGGTGTCTGCAGGCTCAAAGAGCAGCGAGAAGCGTTCAGA

[0361] GGAAAGCGATCCCGTGCCACCTTCCCCGTGCCGGGCTGTCCCCGCACGCTGCGGGCTCCGGCTCGGGGATGCGGGGGGAGCGTCGGGCC

[0362] GGAGCGGAGCCCCGGGCGGCTCGCTGCTGCCCCCTAGCGGGAGAGGAACGCAATTACAGCCCTGGGGGCTTTGGGGGGGGGCTGTCCCC

[0363] GTGAGCTCCCGCGGATATGAGTCTGTCTAAGTGGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACAT

[0364] AACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCA

[0365] ATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTAC

[0366] GCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACA

[0367] TCTACGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCA

[0368] ATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGG CGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGC

[0369] GGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGGAGTCGCTGCGACGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCG

[0370] CCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAG

[0371] CGCTTGGTTTAATGACGGCTTGTTTCTTTTCTGTGGCTGCGTGAAAGCCTTGAGGGGCTCCGGGAGGGCCCTTTGTGCGGGGGGAGCGG

[0372] CTCGGGGGGTGCGTGCGTGTGTGTGTGCGTGGGGAGCGCCGCGTGCGGCTCCGCGCTGCCCGGCGGCTGTGAGCGCTGCGGGCGCGGCG

[0373] CGGGGCTTTGTGCGCTCCGCAGTGTGCGCGAGGGGAGCGCGGCCGGGGGCGGTGCCCCGCGGTGCGGGGGGGGCTGCGAGGGGAACAAA

[0374] GGCTGCGTGCGGGGTGTGTGCGTGGGGGGGTGAGCAGGGGGTGTGGGCGCGTCGGTCGGGCTGCAACCCCCCCTGCACCCCCCTCCCCG

[0375] AGTTGCTGAGCACGGCCCGGCTTCGGGTGCGGGGCTCCGTACGGGGCGTGGCGCGGGGCTCGCCGTGCCGGGCGGGGGGTGGCGGCAGG

[0376] TGGGGGTGCCGGGCGGGGCGGGGCCGCCTCGGGCCGGGGAGGGCTCGGGGGAGGGGCGCGGCGGCCCCCGGAGCGCCGGCGGCTGTCGA

[0377] GGCGCGGCGAGCCGCAGCCATTGCCTTTTATGGTAATCGTGCGAGAGGGCGCAGGGACTTCCTTTGTCCCAAATCTGTGCGGAGCCGAA

[0378] ATCTGGGAGGCGCCGCCGCACCCCCTCTAGCGGGCGCGGGGCGAAGCGGTGCGGCGCCGGCAGGAAGGAAATGGGCGGGGAGGGCCTTC

[0379] GTGCGTCGCCGCGCCGCCGTCCCCTTCTCCCTCTCCAGCCTCGGGGCTGTCCGCGGGGGGACGGCTGCCTTCGGGGGGGACGGGGCAGG

[0380] GCGGGGTTCGGCTTCTGGCGTGTGACCGGCGGCTCTAGAGCCTCTGCTAACCATGTTCATGCCTTCTTCTTTTTCCTACAGCTCCTGGG

[0381] CAACGTGCTGGTTATTGTGCTGTCTCATCATTTTGGCAAAGAATTAATTCGGATCCACCATGTCTAGACTGGACAAGAGCAAAGTCATA

[0382] AACTCTGCTCTGGAATTACTCAATGGAGTCGGTATCGAAGGCCTGACGACAAGGAAACTCGCTCAAAAGCTGGGAGTTGAGCAGCCTAC

[0383] CCTGTACTGGCACGTGAAGAACAAGCGGGCCCTGCTCGATGCCCTGCCAATCGAGATGCTGGACAGGCATCATACCCACTCCTGCCCCC

[0384] TGGAAGGCGAGTCATGGCAAGACTTTCTGCGGAACAACGCCAAGTCATACCGCTGTGCTCTCCTCTCACATCGCGACGGGGCTAAAGTG

[0385] CATCTCGGCACCCGCCCAACAGAGAAACAGTACGAAACCCTGGAAAATCAGCTCGCGTTCCTGTGTCAGCAAGGCTTCTCCCTGGAGAA

[0386] CGCACTGTACGCTCTGTCCGCCGTGGGCCACTTTACACTGGGCTGCGTATTGGAGGAACAGGAGCATCAAGTAGCAAAAGAGGAAAGAG

[0387] AGACACCTACCACCGATTCTATGCCCCCACTTCTGAAACAAGCAATTGAGCTGTTCGACCGGCAGGGAGCCGAACCTGCCTTCCTTTTC

[0388] GGCCTGGAACTAATCATATGTGGCCTGGAGAAACAGCTAAAGTGCGAAAGCGGCGGGCCGACCGACGCCCTTGACGATTTTGACTTAGA

[0389] CATGCTCCCAGCCGATGCCCTTGACGACTTTGACCTTGATATGCTGCCTGCTGACGCTCTTGACGATTTTGACCTTGACATGCTCCCCG

[0390] GGTAAACGCGTAGCTCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGAC

[0391] CCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGG

[0392] GTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGAGAATAGCAGGCATGCTGGGGGTAGTACTCTTAAGTCGACTTACTAGGGA

[0393] CAGGATTGGTGACAGAAAAGCCCCATCCTTAGGCCTCCTCCTTCCTAGTCTCCTGATATTGGGTCTAACCCCCACCTCCTGTTAGGCAG

[0394] ATTCCTTATCTGGTGACACACCCCCATTTCCTGGAGCCATCTCTCTCCTTGCCAGAACCTCTAAGGTTTGCTTACGATGGAGCCAGAGA

[0395] GGATCCTGGGAGGGAGAGCTTGGCAGGGGGTGGGAGGGAAGGGGGGGATGCGTGACCTGCCCGGTTCTCAGTGGCCACCCTGCGCTACC

[0396] CTCTCCCAGAACCTGAGCTGCTCTGACGCGGCTGTCTGGTGCGTTTCACTGATCCTGGTGCTGCAGCTTCCTTACACTTCCCAAGAGGA

[0397] GAAGCAGTTTGGAAAAACAAAATCAGAATAAGTTGGTCCTGAGTTCTAACTTTGGCTCTTCACCTTTCTAGTCCCCAATTTATATTGTT

[0398] CCTCCGTGCGTCAGTTTTACCTGTGAGATAAGGCCAGTAGCCAGCCCCGTCCTGGCAGGGCTGTGGTGAGGAGGGGGGTGTCCGTGTGG

[0399] AAAACTCCCTTTGTGAGAATGGTGCGTCCTAGGTGTTCACCAGGTCGTGGCCGCCTCTACTCCCTTTCTCTTTCTCCATCCTTCTTTCC

[0400] TTAAAGAGTCCCCAGTGCTATCTGGGACATATTCCTCCGCCCAGAGCAGGGTCCCGCTTCCCTAAGGCCCTGCTCTGGGCTTCTGGGTT

[0401] TGAGTCCTTGGCAAGCCCAGGAGAGGCGCTCAGGCTTCCCTGTCCCCCTTCCTCGTCCACCATCTCATGCCCCTGGCTCTCCTGCCCCT

[0402] TCCCTACAGGGGTTCCTGGCTCTGCTCTAAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATT

[0403] CCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACT

[0404] GCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCT

[0405] CTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTA

[0406] TCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGC

[0407] GTT T T T CCATflGGC T C CGCC CC CC TGflc GAGCATCACAAAAATC GACGCTCAAG TCAGflGG TGGCGflAAC CC GACAGGAC TATAAAGAT

[0408] ACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCG

[0409] GGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACC

[0410] CCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAG

[0411] CCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGA

[0412] ACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGG

[0413] TAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTG

[0414] ACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAA

[0415] TGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGAT CTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTG CAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCT GCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGT TGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACAT GATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATG GTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTG AGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCA TCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAAC TGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGAC ACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTG AATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATG ACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTC

[0416] SEQ ID NO: 19 Nucleic acid construct pAAVS1_HS4_TET_eGFP_PC3_rTTAv7 14372bp (Aimv7) (single underlined - Tet-responsive promoter (SEQ ID NO: 9); double underlined rtTA coding sequence (SEQ ID NO: 13); dotted underline INS1 and INS2 (SEQ ID NOs 15 and 16);

[0417] ATGTCTAGACTGGACAAGAGCAAAGTCATAAACTCTGCTCTGGAATTACTCAATGAAGTCGGTATCGAAGGCCTGACGACAAGGAAACT CGCTCAAAAGCTGGGAGTTGAGCAGCCTACCCTGTACTGGCACGTGAAGAACAAGCGGGCCCTGCTCGATGCCCTGGCAATCGAGATGC TGGACAGGCATCATACCCACTTCTGCCCCCTGGAAGGCGAGTCATGGCAAGACTTTCTGCGGAACAACGCCAAGTCATTCCGCTGTGCT CTCCTCTCACATCGCGACGGGGCTAAAGTGCATCTCGGCACCCGCCCAACAGAGAAACAGTACGAAACCCTGGAAAATCAGCTCGCGTT CCTGTGTCAGCAAGGCTTCTCCCTGGAGAACGCACTGTACGCTCTGTCCGCCGTGGGCCACTTTACACTGGGCTGCGTATTGGAGGATC AGGAGCATCAAGTAGCAAAAGAGGAAAGAGAGACACCTACCACCGATTCTATGCCCCCACTTCTGAGACAAGCAATTGAGCTGTTCGAC CATCAGGGAGCCGAACCTGCCTTCCTTTTCGGCCTGGAACTAATCATATGTGGCCTGGAGAAACAGCTAAAGTGCGAAAGCGGCGGGCC GGCCGACGCCCTTGACGATTTTGACTTAGACATGCTCCCAGCCGATGCCCTTGACGACTTTGACCTTGATATGCTGCCTGCTGACGCTC TTGACGATTTTGACCTTGACATGCTCCCCGGGTAA

[0418] SEQ ID NO: 20 Coding sequence for rTA-Advanced for TET-OFF

[0419] MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHVKNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCA LLSHRDGAKVHLGTRPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTTDSMPPLLRQAIELFD HQGAEPAFLFGLELI ICGLEKQLKCESGGPADALDDFDLDMLPADALDDFDLDMLPADALDDFDLDMLPG*

[0420] SEQ ID NO: 21 amino acid sequence for rTA-Advanced for TET-OFF 248 AA

Claims

Claims:1 . A nucleic acid construct comprising;(i) a first nucleotide sequence for expression,(ii) a first promoter operably linked to the first nucleotide sequence, said first promoter being inducible,(iii) a first insulating sequence located upstream of the first nucleotide sequence and first promoter,(iv) a second insulating sequence located downstream of the first nucleotide sequence and first promoter,(v) a second nucleotide sequence encoding a transcription regulator protein that induces expression from the first promoter, and(vi) a second promoter operably linked to the second nucleotide sequence; wherein the second nucleotide sequence and second promoter are located either (a) upstream of the first insulating sequence or (b) downstream of the second insulating sequence.

2. A nucleic acid construct according to claim 1 wherein the first nucleotide sequence comprises one or more coding sequences.

3. A nucleic acid construct according to claim 2 wherein the first nucleotide sequence comprises coding sequences for a set of transcription factors that program a pluripotent stem cell (PSC) into a somatic cell.

4. A nucleic acid construct according to claim 3 wherein;(i) the set of transcription factors comprises GATA1 , FLI1 and TAL1 and the somatic cell is a haematopoetic cell, preferably a megakaryocyte(ii) the set of transcription factors comprises MYOD1 and the somatic cell is a myocyte,(iii) the set of transcription factors comprises Gata4, Mef2c, Baf60c and Tbx5 and the somatic cell is a cardiomyocyte(iv) the set of transcription factors comprises L-Myc (RXOL) Runx2, and Osterix. and the somatic cell is a osteocyte,(v) the set of transcription factors comprises c-Myc Klf4, and SOX9 and the somatic cell is a chondrocyte,(vi) the set of transcription factors comprises C / EBP-p and c-Myc and the somatic cell is an adipocyte,(vii) the set of transcription factors comprises SOX-10, OLIG2, NKX2.2, and NKX6.2 and the somatic cell is a oligodendrocyte,(viii) the set of transcription factors comprises NFIA, NFIB, and SOX9 and the somatic cell is a astrocyte,(ix) the set of transcription factors comprises Ascii , neurogenin, and NeuroD or Pax6, Neurog2, Ascii , Dlx2, and NeuroD and the somatic cell is a neuron,(x) the set of transcription factors comprises HNF6, HNF1A, FOXA3, RORc, and optionally ERa and the somatic cell is a hepatocyte.(xi) the set of transcription factors comprises PU.1 , and m-CSFR, and optionally RARp, GATA6, and mCSF and the somatic cell is a macrophage,(xii) the set of transcription factors comprises MEF2A and MEF2D; or SPI1 / PU.1 , CEBPA and MTF, and the somatic cell is a macrophage,(xiii) the set of transcription factors comprises Neurogenin 2 (NEUROG2, Ngn-2) and the somatic cell is a neuronal cell, or(xiv) the set of transcription factors comprises SOX10 and OLIG2 and the somatic cell is an oligodendrocyte.

5. A nucleic acid construct according to any one of claims 2 to 4 wherein the first nucleotide sequence further comprises a coding sequence for a marker gene.

6. A nucleic acid construct according to any one of claims 2 to 5 wherein the coding sequences of the first nucleotide sequence are polycistronic.

7. A nucleic acid construct according to claim 6 wherein the coding sequences of the first nucleotide sequence are separated by sequences encoding self-cleaving oligopeptides.

8. A nucleic acid construct according to any one of claims 1 to 7 wherein the transcription regulator protein is activated by an inducer compound.

9. A nucleic acid construct according to claim 8 wherein the inducer compound is tetracycline or doxycycline.

10. A nucleic acid construct according to claim 8 or 9 wherein the transcription regulator protein is a reverse tetracycline-controlled transactivator (rtTA).

11. A nucleic acid construct according to claim 9 or claim 10 wherein the first promoter sequence comprises a tetracycline response element (TRE).

12. A nucleic acid construct according to any one of claims 1 to 1 1 wherein second promoter sequence is a constitutive promoter.

13. A nucleic acid construct according to claim 12 wherein the constitutive promoter is a CAG promoter14. A nucleic acid construct according to any one of claims 1 to 13 wherein the first and second insulating sequences are in opposite orientations in the construct15. A nucleic acid construct according to any one of claims 1 to 14 wherein the first and second insulating sequences comprise the p-globin chicken hypersensitivity site 4 (HS4).

16. A nucleic acid construct according to any one of claims 1 to 15 wherein the construct further comprises 5’ and 3’ homology arms for targeted insertion into a GSH locus.

17. A nucleic acid construct according to claim 16 wherein the 5’ and 3’ homology arms comprise a sequence of 30 to 1000 nucleotides that is complementary or identical to the sequence of a GSH locus in a mammalian cell.

18. A vector comprising a nucleic acid construct according to any one of claims 1 to 17.

19. A method of producing a cell for controlled expression of a nucleotide sequence comprising; inserting a nucleic acid construct according to any one of claims 1 to 17 into a genomic safe harbour(GSH) locus within the genome of a mammalian cell.

20. A method according to claim 19 wherein the GSH is the Adeno- Associated Virus Integration Site 1 (AAVS1) locus.

21. A method according to claim 19 or claim 20 wherein the construct is inserted into the GSH locus by introducing a DNA DSB into a site within the GSH locus, such that repair of the DNA DSB by the mammalian cell inserts the construct into the site.

22. A method according to claim 21 wherein the DNA DSB is introduced into the site by a Zinc Finger Nuclease (ZFN).

23. A method according to any one of claims 19 to 22 wherein the mammalian cell is a human cell.

24. A method according to any one of claims 19 to 23 wherein the mammalian cell is a pluripotent stem cell.

25. A mammalian cell comprising a nucleic acid construct according to any one of claims 1 to 17, said construct being located at a GSH locus within the genome of the cell.

26. A mammalian cell according to claim 25 wherein the GSH is the Adeno- Associated Virus Integration Site 1 (AAVS1) locus.

27. A mammalian cell according to claim 25 or 26, wherein the mammalian cell is a human cell.

28. A mammalian cell according to any one of claims 25 to 27 wherein the mammalian cell is a pluripotent stem cell.

29. A mammalian cell according to any one of claims 25 to 28 wherein the first nucleotide sequence comprises coding sequences for a set of transcription factors that forward program a PSC into a somatic cell.

30. A method of expressing a nucleotide sequence comprising;a) providing a mammalian cell according to any one of claims 25 to 29; wherein the second nucleotide sequence is expressed in the mammalian cell to produce the transcription regulator protein in the mammalian cell, b) activating the transcriptional regulator protein to induce expression from the first promoter in the mammalian cell, such that the first nucleotide sequence is expressed.

31. A method according to claim 30 wherein the transcriptional regulator protein is activated by introducing an inducer compound into the mammalian cell.

32. A method of forward programming a pluripotent stem cell into a somatic cell comprising; a) providing a pluripotent stem cell (PSC) comprising a nucleic acid construct according to any one of claims 1 to 18 at a genomic safe harbour locus within its genome, wherein the first nucleotide sequence comprises coding sequences for a set of transcription factors that forward program a PSC into a somatic cell, and the first second nucleotide sequence is expressed in the PSC to produce the transcription regulator protein, b) activating the transcriptional regulator protein to induce the expression from the first promoter, such that the first nucleotide sequence is expressed to produce the set of transcription factors in the PSC and said transcription factors convert the PSC into the somatic cell.

33. A method according to claim 32 wherein the somatic cell is a megakaryocyte.

34. A method according to claim 33 wherein the set of transcription factors comprises GATA1 , TAL1 and FLI1.

35. A method according to claim 32 wherein;(i) the set of transcription factors comprise MYOD1 and the somatic cell is a myocyte,(ii) the set of transcription factors comprise Gata4, Mef2c, BafBOc and Tbx5 and the somatic cell is a cardiomyocyte(iii) the set of transcription factors comprise L-Myc (RXOL) Runx2, and Osterix. and the somatic cell is an osteocyte,(iv) the set of transcription factors comprise c-Myc Klf4, and SOX9 and the somatic cell is a chondrocyte,(v) the set of transcription factors comprise C / EBP-p and c-Myc and the somatic cell is an adipocyte,(vi) the set of transcription factors comprise SOX-10, OLIG2, NKX2.2, and NKX6.2 and the somatic cell is a oligodendrocyte,(vii) the set of transcription factors comprise NFIA, NFIB, and SOX9 and the somatic cell is a astrocyte,(viii) the set of transcription factors comprise Ascii , neurogenin, and NeuroD or Pax6, Neurog2, Ascii , Dlx2, and NeuroD and the somatic cell is a neuron,(x) the set of transcription factors comprises HNF6, HNF1A, FOXA3, RORc, and optionally ERa and the somatic cell is a hepatocyte.(xi) the set of transcription factors comprises PU.1 , and m-CSFR, and optionally RARp, GATA6, and mCSF and the somatic cell is a macrophage,(xii) the set of transcription factors comprises MEF2A and MEF2D; or SPI1 / PU.1 , CEBPA and MTF, and the somatic cell is a macrophage, (xiii) the set of transcription factors comprises Neurogenin 2 (NEUROG2, Ngn-2) and the somatic cell is a neuronal cell, or(xiv) the set of transcription factors comprises SOX10 and OLIG2 and the somatic cell is an oligodendrocyte.

36. A method according to any one of claims 32 to 35 wherein the method comprises isolating or purifying the somatic cell.

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