Controllable transcription

Abstract

The present invention relates to a stable method for introducing at least one induction cassette into a cell and enabling controllable transcription from within the induction cassette. The method can be used for any cell type from any eukaryotic organism, but has particular application in the introduction of an induction cassette into pluripotent stem cells, such as animal pluripotent stem cells or human pluripotent stem cells (hPSCs). The induction cassette is controllably inserted to ensure that the genetic material it contains is not silenced or negatively affected from the insertion site, and that transcription of the genetic material is controlled. [Selection diagram] Figure 4

Description

[Technical Field]

[0001] The present invention relates to a stable method for introducing at least one induction cassette into a cell and enabling controllable transcription from within the induction cassette. The method can be used for any cell type from any eukaryotic organism, but is particularly applicable to the introduction of an induction cassette into pluripotent stem cells, such as animal pluripotent stem cells or human pluripotent stem cells (hPSCs). The induction cassette is controllably inserted to ensure that the genetic material it contains is not silenced or negatively affected by the insertion site, and that transcription of the genetic material is controlled. [Background technology]

[0002] BACKGROUND OF THE INVENTION Stem cell research holds great promise for the study of human development, regenerative medicine, disease modeling, drug discovery, and cell transplantation. Stem cell-derived cells also enable the study of physiological and pathological responses of human cell populations that are not readily accessible. This often involves the study of genes (and other forms of regulatory mechanisms encoded within non-protein-coding RNA—ncRNA). Unfortunately, the controllable transcription or expression of genetic information in human cells has proven particularly challenging.

[0003] Furthermore, several important aspects of regenerative medicine, disease modeling, drug discovery, and cell transplantation require the manipulation and production of mature human cell types from easily accessible sources. Controlling transgene expression in human cells is a cornerstone of biological research. However, this has proven challenging in human cells. Furthermore, there is a real need for the in vitro derivation of many highly desirable human cell types in quantities and quality suitable for drug discovery and regenerative medicine purposes. Because directed differentiation of stem cells into desired cell types is often difficult, other approaches have emerged, including direct reprogramming of cells to the desired cell type. In particular, forward programming, a method for directly converting pluripotent stem cells, including hPSCs, into mature cell types, has been recognized as a powerful strategy for the derivation of human cells. This reprogramming involves the forced expression of key lineage transcription factors (or non-coding RNAs, including IncRNAs and microRNAs) to convert stem cells into specific mature cell types. This context also poses a challenge to the controllable expression of genetic information in human cells. Currently, available forward programming protocols are primarily based on lentiviral transduction of cells, which results in variable expression or complete silencing of randomly inserted inducible cassettes. This necessitates additional purification steps to isolate subpopulations expressing the desired transcription factors. Therefore, further refinement of these methods is clearly needed.

[0004] The ability to control the knockdown and knockout of genes or other coding sequences in cells is highly desirable, allowing for loss-of-function studies to be performed independently of the inducible expression of transgenes. Loss-of-function studies in stem cells and mature cell types offer unique opportunities for studying mechanisms regulating human development, disease, and physiology. However, current technologies do not allow for simple and efficient manipulation of gene expression. Current techniques for introducing substances such as inducible short hairpin RNAs (shRNAs) into stem cells to induce gene knockdown suffer from many of the drawbacks seen in forward reprogramming described above, such as transgene silencing and position-effect-limited activity. Therefore, there is a need for inducible gene knockout and knockdown in stem cells that would enable loss-of-function studies in stem cells.

[0005] Any improvement to the above methods must ensure stable transcription of the genetic material contained within the inducible cassette, such as a transgene, that is resistant to silencing and other negative effects associated with the integration site. Silencing can be caused by multiple epigenetic mechanisms, including DNA methylation or histone modification. Using prior art methods based on lentiviral transduction, the resulting cells are heterogeneous populations in which the transgene is fully expressed, partially expressed, or silenced. Clearly, this is undesirable for many applications. Viral vectors integrate their genetic material into transcriptionally active regions of the genome, thus increasing the likelihood of oncogenic events due to insertional mutagenesis.

[0006] For many applications, it is desirable for the induction cassette to control transcription of the genetic material inserted into the cell so that it can be switched on and transcribed at specific levels, including high levels, when needed, which cannot be achieved if insertion of the induction cassette is random within the genome.

[0007] Therefore, the present inventors have developed a method that allows stable introduction of an induction cassette into the genome of a cell while controlling the transcription of the induction cassette. This is particularly advantageous for any cell type in which it is desired to introduce the induction cassette into pluripotent stem cells to control the transcription of the inserted genetic material. The induction cassette can contain any genetic material that can be transcribed, such as a transgene or non-coding RNA (ncRNA). The material included in the induction cassette is determined by the effect desired from the stem cell, including expression of a transgene or knockdown or knockout of a gene. Summary of the Invention [Problem to be solved by the invention]

[0008] (Summary of the Invention) The inventors have found that by using the dual genomic safe harbor targeting system described herein, it is possible to insert an induction cassette and control the transcription of the genetic material within the induction cassette. Such a method is highly desirable because it reduces the risk of epigenetic silencing of the inserted genetic material and allows for the generation of a homogenous population of cells that transcribe the induction cassette. [Means for solving the problem]

[0009] Accordingly, the present invention provides a method for regulating transcription of a gene sequence in a cell, comprising the steps of: a) targeted insertion of a transcriptional regulatory protein-encoding gene into a first gene safe harbor site; and b) targeted insertion of an inducible cassette into a second genetic safe harbor site, said inducible cassette comprising said genetic sequence operably linked to an inducible promoter, said promoter being regulated by said transcriptional regulatory protein; Including, The first and second genetic safe harbor sites are different.

[0010] Integration of the guided cassette into specific genomic safe harbor sites (GSH) is preferable to random insertion into the genome. GSHs have previously been defined as intragenic or extragenic regions of the human genome that can accommodate predictable expression of newly integrated DNA without adversely affecting the host cell or organism. A useful safe harbor must allow sufficient transcription of the inserted gene sequence to obtain the desired levels of protein (through further translation) or non-coding RNA. The GSH must also not predispose cells to malignant transformation or alter cellular function (Sadelain et al., 2012, Nature Reviews Cancer, 12(1), 51-8. doi:10.1038 / nrc3179).

[0011] The first gene safe harbor site is utilized to introduce at least one gene encoding a transcriptional regulatory protein. Transcriptional regulatory proteins (or transcription factors) increase gene transcription of a gene. Most transcriptional regulatory factors are DNA-binding proteins that bind to enhancer or promoter-proximal elements operably linked to a gene. [Effects of the Invention]

[0012] In some embodiments, the transcriptional regulatory protein is constitutively expressed and permanently expressed in cells. Thus, the transcriptional regulatory protein can be operably linked to a constitutive promoter. A constitutive promoter directs gene expression uniformly in most tissues and cells at all stages of growth and development. When used in the methods of the present invention, a constitutive promoter confers high levels of gene expression.

[0013] Additional genetic material, including a gene, can be inserted into the first GSH containing a transcriptional regulatory protein. Such a gene can include one or more markers, such as green fluorescent protein (GFP), which can be used to indicate that the transcriptional regulatory protein has been successfully inserted. Other options include genes that enable gene editing, such as Cas9 and its derivatives or CasL and its derivatives, and reporter sequences that can be used to assay the endogenous or exogenous expression of specific genes in cells.

[0014] The second GSH is used to introduce an inducible cassette in which the desired gene sequence is operably linked to an inducible promoter. Such a promoter allows transcription only when properly induced by a transcriptional regulatory protein. The transcriptional regulatory protein can be controlled by substances supplied externally to the cell. Thus, the presence of an exogenous substance can allow or prevent expression from the inducible promoter. Examples of such controllable expression include the Tet-ON system, which is further described herein.

[0015] The further induction cassette(s) may be inserted into a further GSH, said GSH being separate from the first and second GSH described above.

[0016] One or more gene sequences may be controllably transcribed from within the second and / or further GSH. Indeed, the induction cassette may contain 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 gene sequences that it is desired to insert into the GSH and whose transcription is to be controllably induced.

[0017] The gene sequence or sequences desired to be inserted into the GSH or GSHs are operably linked to an inducible promoter and are present in an inducible cassette. These gene sequences may be any suitable sequences that can be transcribed into RNA after the promoter's activity is induced. Suitable gene sequences include, but are not limited to, transgenes (protein-coding genes whose RNA is translated into a polypeptide, i.e., messenger RNA (mRNA)), non-coding RNA (ncRNA - including, but not limited to, shRNA, antisense RNA (asRNA), guide RNA (gRNA), microRNA (miRNA), small interfering RNA (siRNA), trans-acting RNA (tasiRNA), antagomir, aptamer, miRNA sponge, and any other functional RNA).

[0018] The inducible cassette may contain additional genetic material inserted into a second or further GSH. Such additional genetic material may include one or more markers, such as green fluorescent protein (GFP), to indicate that transcription is occurring. Alternatively, or in addition, a gene, such as an antibiotic or drug resistance gene, may allow for selection of successfully inserted inducible cassettes. Also, inducible expression of a particular gene to study its function or a sequence that interferes with its function may be desirable. Similarly, expression of genes to enhance or interfere with the biological function of a cell or to affect cells in other parts of an organism may be desirable, including expression of peptide hormones, including growth factors, insulin, etc.

[0019] Technically, the first and / or second GSH insertions can occur on one chromosome or on both chromosomes. GSH is present at the same locus on both chromosomes in diploid organisms. Insertion into both chromosomes is advantageous because it allows for increased transcription levels from the genetic material inserted into the induction cassette, thereby achieving particularly high levels of transcription.

[0020] The insertion into GSH can be based on the customized site-specific generation of DNA double-strand break (DSB) at GSH, and the specific insertion of genetic material into specific GSH can be controlled.Then, this genetic material can be introduced by any suitable mechanism, such as homologous recombination.Any method for generating specific DSB in genome can be used, but preferred systems include CRISPR / Cas9 and its modified version, ZFN and TALEN system.

[0021] Furthermore, the insertion of the transcriptional regulator and / or induction cassette can be designed to be reversible, and the inserted genetic material can be removed and / or replaced with an alternative transcriptional regulator / induction cassette, as needed. Methods for replacing transcriptional regulators and / or induction cassettes are part of the present invention. Such replacements can be useful when a cell culture has been successfully modified with one transcriptional regulator and / or one induction cassette, and it is desirable to replace the transcriptional regulator and / or induction cassette. This can allow for larger insertions to be made, leveraging already successful insertions. To implement this aspect of the present invention, the insert can include a cleavage sequence that allows for removal of all or a portion of the insert from GSH, such as a portion of the insert. Preferred methods of removal or replacement include recombinant methods.

[0022] Furthermore, the present invention relates to vectors suitable for the insertion of transcriptional regulators and / or induction cassettes into GSH.

[0023] In one aspect, the present invention provides a method for regulating expression of a transgene in a cell, comprising: a) targeted insertion of a transcriptional regulatory protein-encoding gene into a first gene safe harbor site; and b) targeted insertion of a transgene operably linked to an inducible promoter into a second genetic safe harbor site, wherein the inducible promoter is regulated by the transcriptional regulatory protein; Including, The method is provided wherein the first and second genetic safe harbor sites are different.

[0024] In this embodiment of the present invention, the aforementioned inducible cassette comprises a transgene operably linked to an inducible promoter. In this embodiment of the present invention, the desired gene sequence contained within the inducible cassette is a transgene, preferably a protein-coding gene. Thus, transcription and translation (expression) of the transgene can be controlled within the cell. An advantage of this method is that it allows for overexpression of the transgene, if necessary.

[0025] Furthermore, in this aspect of the invention, additional identical or different transgenes can be inserted into the additional GSH that are different from the first and second GSH, such transgenes being operably linked to an inducible promoter as described above.

[0026] In one aspect, the present invention provides a method for regulating transcription of a non-coding RNA in a cell, comprising: a) targeted insertion of a transcriptional regulatory protein-encoding gene into a first gene safe harbor site; and b) targeted insertion of an inducible cassette into a second genetic safe harbor site, said inducible cassette comprising DNA encoding a non-coding RNA sequence operably linked to an inducible promoter, said promoter being regulated by said transcriptional regulatory protein; Including, The method is provided wherein the first and second genetic safe harbor sites are different.

[0027] Furthermore, in this aspect of the invention, additional identical or different inducible cassettes can be inserted into additional GSHs that are different from the first and second GSHs. Such inducible cassettes can comprise DNA encoding a non-coding RNA sequence or any other gene sequence operably linked to an inducible promoter, which promoter is regulated by the transcriptional regulatory protein.

[0028] More specifically, this method allows for the knockdown of an endogenous gene in a cell. Accordingly, the present invention provides a method for reducing the transcription and / or translation of an endogenous gene in a cell, comprising: a) targeted insertion of a transcriptional regulatory protein-encoding gene into a first gene safe harbor site; and b) targeted insertion of an inducible cassette into a second genetic safe harbor site, said inducible cassette comprising DNA encoding a non-coding RNA sequence operably linked to an inducible promoter, said promoter being regulated by said transcriptional regulatory protein, and said non-coding RNA sequence repressing the transcription or translation of an endogenous gene; Including, The method is provided wherein the first and second genetic safe harbor sites are different.

[0029] Furthermore, in this aspect of the invention, additional identical or different inducible cassettes can be inserted into additional GSHs that are different from the first and second GSHs. Such inducible cassettes can comprise DNA encoding a non-coding RNA sequence or any other gene sequence operably linked to an inducible promoter, which promoter is regulated by the transcriptional regulatory protein.

[0030] In any aspect or embodiment, the endogenous gene may encode a protein or a non-coding RNA.

[0031] In the above two aspects of the present invention, the induction cassette(s) comprise DNA encoding non-coding RNA, i.e., functional but not translated into protein. This non-coding RNA can be any suitable RNA, such as those previously described, but is preferably a short hairpin RNA (shRNA). In the latter aspect of the present invention, the non-coding RNA can generally achieve gene knockdown in any suitable manner by blocking the transcription or translation or preventing the expression of the gene. Ultimately, the expression of the gene is reduced or blocked, but the gene itself remains intact.

[0032] Alternatively, the non-coding RNA contained within the sequence of the induction cassette may comprise an RNA that can be used to knock out an endogenous gene in a cell, particularly to replace or disrupt the gene itself. Suitable non-coding RNAs that can be used in this aspect of the invention include elements of the CRISPR / Cas9 platform, more specifically, guide RNAs (gRNAs) that are directed to target endogenous genes.

[0033] Thus, in one aspect, the present invention provides a method of knocking out an endogenous gene in a cell, comprising: a) targeted insertion of a transcriptional regulatory protein-encoding gene and a gene encoding Cas9 or a derivative thereof into a first gene safe harbor site; and b) targeted insertion of an inducible cassette into a second genetic safe harbor site, said inducible cassette comprising a guide RNA operably linked to an inducible promoter, said promoter being regulated by said transcriptional regulatory protein, and said gRNA sequence targeting an endogenous gene; Including, The method is provided wherein the first and second genetic safe harbor sites are different.

[0034] Furthermore, in this aspect of the invention, additional, identical or different, inducible cassettes can be inserted into additional GSHs that are different from the first and second GSHs. Such inducible cassettes can include any gene sequence operably linked to an inducible promoter, which promoter is regulated by the transcriptional regulatory protein.

[0035] Thus, in the above-described aspects of the invention, transcription of the gRNA is controllably induced.

[0036] In a further aspect, the present invention provides a method for reducing the transcription and / or translation of an endogenous gene in a cell, comprising the steps of: a) targeted insertion of a transcriptional regulatory protein-encoding gene into a first allele of a genetic safe harbor site; and b) targeted insertion of an inducible cassette into a second allele of the same genetic safe harbor site, wherein the inducible cassette comprises DNA encoding a non-coding RNA sequence operably linked to an inducible promoter, the promoter being regulated by the transcriptional regulatory protein, and the non-coding RNA sequence represses the transcription or translation of the endogenous gene; The method includes:

[0037] The present invention further provides a method for knocking out an endogenous gene in a cell, comprising: a) targeted insertion of a transcriptional regulatory protein-encoding gene and a gene encoding Cas9 or a derivative thereof into a first allele of a genetic safe harbor site; and b) targeted insertion of an inducible cassette into a second allele of the same genetic safe harbor site, wherein the inducible cassette comprises a guide RNA operably linked to an inducible promoter, the promoter being regulated by a transcriptional regulatory protein, and the gRNA sequence targets an endogenous gene; The method includes:

[0038] Such a single step knockout or knockdown is novel and may form part of the present invention.

[0039] In one aspect, the present invention provides a method for forward programming of pluripotent stem cells, comprising: a) targeted insertion of a transcriptional regulatory protein-encoding gene into a first gene safe harbor site; and b) targeted insertion of an inducible cassette into a second genetic safe harbor site, said inducible cassette comprising a transcription factor-encoding gene sequence of a lineage of interest operably linked to an inducible promoter, said inducible promoter being regulated by said transcription regulatory protein; Including, The method is provided wherein the first and second genetic safe harbor sites are different.

[0040] Further or additional induction cassette(s) may be inserted into further GSH apart from the first and second GSH.

[0041] Forward programming of pluripotent stem cells into specific mature cell types is highly desirable and can be achieved using the dual targeting platform of the present invention. Specific methods for several cell types are described below.

[0042] In one aspect, the present invention provides a method for generating muscle cells from pluripotent stem cells, comprising: a) targeted insertion of a transcriptional regulatory protein-encoding gene into a first gene safe harbor site; and b) targeted insertion of the MYOD1 gene operably linked to an inducible promoter into a second gene safe harbor site, wherein the inducible promoter is regulated by the transcriptional regulatory protein; wherein the first and second genetic safe harbor sites are different; The method further comprises culturing the cells in the presence of retinoic acid.

[0043] The MYOD1 gene is a gene encoding myogenic differentiation 1 protein. Preferably, the retinoic acid (RA) is all-trans RA.

[0044] In another aspect, the present invention provides a method for generating muscle cells from pluripotent stem cells that express MYOD1, the method comprising culturing the cells in the presence of retinoic acid.

[0045] Preferably, the RA is all-trans RA. Preferably, the cells overexpress MYOD1.

[0046] In a further aspect, the present invention provides a method for generating oligodendrocytes from pluripotent stem cells, comprising the steps of: a) targeted insertion of a transcriptional regulatory protein-encoding gene into a first gene safe harbor site; and b) targeted insertion of any combination of SOX10, OLIG2, NKX2.2, and NKX6.2 genes operably linked to an inducible promoter into a second gene safe harbor site, wherein the inducible promoter is regulated by the transcriptional regulatory protein; wherein the first and second genetic safe harbor sites are different.

[0047] The SOX10, OLIG2, NKX2.2, and NKX6.2 genes encode the transcription factors SOX10, OLIG2, NKX2.2, and NKX6.2, respectively. [Brief explanation of the drawings]

[0048] [Figure 1](a-d): Validation of the optimized dual genomic safe harbor targeted overexpression system. Figure 1(a) Design of gene targeting vectors for the hROSA26 and AAVS1 loci. HAR: homology arm; SA: splice acceptor; T2A: T2A ribosomal skipping signal; Neo: neomycin resistance gene; Puro: puromycin resistance gene; pA: polyadenylation signal; CAG: constitutively active CAG promoter; rtTA: third-generation rtTA; TRE: inducible Tet response element; EGFP: enhanced green fluorescent protein. Figure 1(b) shows the EGFP induction and rescue kinetics in EGFP-expressing hESCs (Figure 1(c)) detected by flow cytometry (median fluorescence intensity, MFI). Results are from two biological replicates per time point and are presented as mean ± SEM. All values ​​were normalized to the maximum fluorescence intensity 5 days after doxycycline (referred to as day 0 in the figure). Figure 1(d) shows the doxycycline dose response for EGFP overexpression in EGFP-expressing hESCs after 5 days of doxycycline induction. Results are from two biological replicates per condition and are presented as mean ± SEM. All values ​​were normalized to the maximum fluorescence intensity measured in the experiment. EGFP expression levels in GSH-targeted constitutive CAG-EGFP hPSCs and in dual GSH-targeted inducible TRE-EGFP hPSCs after induction with doxycycline. Wild-type hPSCs and uninduced TRE-EGFP cells were included as negative controls. [Figure 2](a-d): Overview of the experimental approach and results of rapid, single-step conversion of hPSCs into neural cells (i-neurons) after doxycycline (dox) treatment. Figure 2(a) is a schematic diagram of this conversion. Cells transformed with NGN2 according to the present invention are induced to differentiate into neurons after Dox treatment. Figure 2(b) shows the forward programming time course of i-neuron generation from hESCs as demonstrated by quantitative RT-PCR analysis, showing the temporal expression patterns of pan-neuronal marker genes (MAP2, SYP), forebrain marker genes (BRN2, FOXG1), and glutamatergic neuron marker genes (VGLUT2, GRIA4). Cells were analyzed on the indicated days of doxycycline treatment. Values ​​are shown relative to the endogenous housekeeping gene PBGD and normalized to the pluripotent state. Results are from three biological replicates per time point and are expressed as mean ± SEM. Figure 2(c) shows quantification of βIII-tubulin (TUBB3)-positive neurons by immunostaining in i-neurons derived from hESCs after 1 week of induction. Undifferentiated cells were used as a negative control (control), and figures are reported for i-neuron generation after 25 passages (+P25) in freshly isolated NGN2-expressing hESCs. Figure 2(d) shows a photograph of cells depicting the forward programming time course of i-neuron generation from hESCs via a series of phase-contrast images showing morphological changes. [Figure 3](a–d): Forward programming of hPSCs into skeletal muscle cells. Figure 3a shows a schematic diagram of the rapid, single-step conversion of hPSCs into skeletal muscle cells by inducible overexpression of MYOD1 and treatment with retinoic acid. Figure 3b shows quantitative RT-PCR analysis of the temporal expression patterns of myocyte marker genes during myocyte generation from hESCs. All values ​​are shown relative to hPSCs. Results are from three biological replicates per time point and are expressed as mean ± SEM. Figures 3(c) and (d) show quantification of MHC-positive cells by flow cytometry at 10 days postinduction, demonstrating that OPTi-MYOD1 hPSCs retain their myogenic potency even after extended culture periods and passages (p) after targeted integration of the MYOD1 system. Undifferentiated cells were used as a negative control (control), and figures for myocyte generation in freshly isolated OPTi-MYOD1 hESCs or in the same cells after 50 passages (+P50) are reported. [Figure 4](a-f): Targeting strategy of the dual GSH-targeted Tet-ON overexpression system. Figure 4(a) shows the experimental workflow for sequential targeting of the hROSA26 and AAVS1 loci in hPSCs. Keywords: Cas9n: D10A nickase mutant Cas9 endonuclease from S. pyogenes; ZFN: zinc finger nuclease; Neo: neomycin; Puro: puromycin; rtTA: third-generation reverse tetracycline transactivator. This shows an inducible EGFP expression system (i-EGFP). Figure 4(b) shows a schematic diagram of the hROSA26 targeting strategy. Figure 4(c) shows the AAVS1 targeting strategy. Keywords in Figures 4(b) and (c): R26-prom: ROSA26 locus promoter (THUMPD3-AS1 gene); AAV-prom: AAVS1 locus promoter (PPP1R12C gene); ZFN: zinc finger nuclease; 5'-HAR / 3'-HAR: upstream / downstream homology arms; SA: splice acceptor; T2A: T2A peptide; pA: polyadenylation signal; CAG: CMV early enhancer, chicken β-actin, and rabbit β-globin hybrid promoter; TRE: Tet-responsive element; EGFP: enhanced green fluorescent protein. Figure 4(d) shows a schematic diagram of the genotyping strategy used to identify correctly targeted hROSA26- and AAVS1-targeted hPSC lines; GSH-prom: GSH promoter (hROSA26 and AAVS1, respectively); WT: wild-type; and inducible cassette: all exogenous sequences integrated after targeting. Locus PCR: PCR spanning the target locus using both primers that exclusively bind to genomic DNA outside the genomic sequence corresponding to the homology arms. Note that due to its high GC content, the CAG promoter cannot be amplified by conventional PCR. Therefore, correct insertion of the CAG-containing expression cassette results in the disappearance of the PCR amplicon. The presence of a wild-type band indicates the presence of a non-targeted allele; the disappearance of the wild-type band indicates homozygous targeting. 5'-INT / 3'-INT: PCR: PCR spanning the 5' and 3' insertion sites, respectively. A PCR amplicon of the correct size indicates correct integration.3'BB PCR: PCR spanning the homology arm / targeting vector backbone junction. The presence of a PCR product indicates off-target, nonspecific integration of the donor plasmid. Figure 4(e) is a photograph of a gel showing the results of genotyping for selected hROSA26-CAG-rtTA-targeted heterozygous (HET) and homozygous (HOM) H9 hESCs. Figure 4(f) is a photograph of a gel showing the results of genotyping for selected AAVS1-TRE-EGFP-targeted heterozygous (HET) and homozygous (HOM) H9 hESCs. 1kb+: 1kb plus DNA ladder; WT: wild-type hESCs; PL: targeting plasmid; HO: water control. [Figure 5](a-e): Development of an optimized inducible overexpression platform (OPTi-OX) based on hPSC dual GSH targeting. Figure 5(a) shows that dual GSH-targeted inducible EGFP H9 hESCs were pooled into four experimental groups depending on whether one or both alleles of the hROSA26 and AAVS1 loci were successfully targeted. Figure 5(b) shows detection of rtTA protein by Western blot in successfully targeted heterozygous and homozygous H9 hROSA26-CAG-rtTA hESCs. Human ESCs carrying second-generation rtTA at random genomic locations were included as control samples. α-Tubulin: Loading control. Figure 5(c) shows flow cytometry analysis of representative examples of various dual GSH-targeted inducible EGFP hESCs described in Figure 5(a). Figure 5(d) shows the median fluorescence intensity (MFI) of EGFP expression in various dual GSH-targeted inducible EGFP hESCs described in Figure 5(a). Cells were analyzed by flow cytometry under control conditions (no doxycycline, CTR) or after 5 days of doxycycline treatment (DOX). Each data point represents an individual clonal line. CAG-EGFP hESCs and wild-type (WT) hESCs were included for comparison. Statistical analysis of the doxycycline-treated groups (n = 4–5, as indicated) showed that EGFP expression levels were highest in the double homozygous clones (one-way ANOVA with post-hoc Dunnett's test; F(2, 10) = 25.34, p = 0.0001; **** p < 0.0001; ** p = 0.0026). This condition was selected for further experiments. Figure 5(e) shows the percentage of EGFP+ cells in the various double GSH-targeted i-EGFP hESCs described in Figure 5(a). [Figure 6](a-d): Characterization of the OPTi-OX platform in hPSCs and during germ layer differentiation. Figure 6(a) shows flow cytometry analysis of EGFP levels in successfully targeted live hPSCs and their differentiation into the three germ layers after treatment with doxycycline for 5 days. Acquisition settings were configured to include high levels of induced EGFP expression (DOX). The uninduced control population (CTR) is located immediately adjacent to the left y-axis. Figures 6(b) and 6(c) show an overview of the flow cytometry plots in 6(a), including median fluorescence intensity (MFI) and percentage of EGFP+ cells. Figure 6(d) shows bar graphs of quantitative RT-PCR results for EGFP mRNA expression levels in homozygous pluripotent stem cells and after differentiation into the three germ layers. WT: wild type. [Figure 7] Characterization of human i-neurons. Quantitative RT-PCR results show rapid downregulation of pluripotency factors NANOG and OCT4 upon treatment with doxycycline. [Figure 8] RA signaling during myocyte induction. This figure shows qPCR analysis of six retinoids and retinoid receptors during myocyte induction, demonstrating that RARα, RARβ, and all three RXR isoforms, but not RARγ, are expressed throughout the course of i-myocyte induction. A is α, B is β, and G is γ. [Figure 9]Figures 9(a)-9(c): Characterization of OPTi-MYOD1 hESC development into human i-myocytes. Figure 9(a) shows a time course of forward programming of OPTI-MYOD1 hPSCs into induced myocytes. Morphological changes were demonstrated by automated phase-contrast images acquired every 30 minutes using a Nikon Biostation IM time-lapse system. Scale bar: 200 μm. Figure 9(b) shows qPCR results demonstrating rapid downregulation of the pluripotency factors NANOG and OCT4 upon treatment of OPTi-NGN2 hESCs with doxycycline (left graph). All five major human skeletal muscle cell-specific myocyte heavy chain isoforms (encoded by the MYH gene family) are strongly upregulated during myocyte forward programming (right graph). These include two isoforms expressed during embryonic and postnatal muscle development (embryonic isoform MYH3; neonatal isoform MYH8), and three isoforms normally expressed in adult skeletal muscle [MYH7 in slow-twitch (type I) fibers; MYH2 in fast-twitch, fatigue-resistant (type IIa) fibers; and MYH1 in fast-fatigable (type IIx) fibers]. In contrast, MYH4, which represents the MHC isoform constitutive in fast-twitch, fast-fatigable myocyte fibers in cats, is not expressed in significant amounts (<1%) in humans and is not induced during the forward programming time course. Figure 9(c) shows induced skeletal muscle cells expressing a wide range of typical marker proteins, including F-actin (visualized via AlexaFluor488-conjugated phalloidin toxin), neural cell adhesion molecule (NCAM), desmin (DES), myosin heavy chain (MYH), titin (TTN), α-actinin (ACTN2), and troponin T (TNNT), but not the myoblast progenitor markers PAX3 and PAX7. All samples were counterstained with myogenin (MYOG). Scale bar: 50 μm. DAPI: nuclear stain. [Figure 10]These three graphs show qPCR results for total MYOD1, endogenous MYOD1, and MYOG at 2 days after induction of OPTi-MYOD1 hPSCs with different concentrations of doxycycline. qPCR results are shown at 48 hours after induction with different concentrations of doxycycline. Expression is plotted against the endogenous housekeeping gene PBGD. [Figure 11] Diagram of the Tet-ON system. Tet-ON consists of two components: at the top, a constitutive promoter (cP) drives the expression of the activator cassette (rtTA) (reverse tetracycline transactivator). rtTA is a fusion protein consisting of a mutant form of the prokaryotic Tet repressor (TetR) and the transcriptional transactivator domain VP16 (derived from herpes simplex virus). At the bottom, the response domain is shown. It consists of an inducible promoter (TRE, Tet response element) and the gene of interest. The TRE is an artificial promoter that responds to rtTA. It consists of seven consecutive tet operons (tetO7) and a strong minimal CMV promoter (mCMV), which is inactive by itself and only recruits the transcription machinery upon binding of rtTA to the seven tet operons. Doxycycline, a tetracycline derivative, is required for binding of the mutant TetR to the TRE, resulting in expression of the inducible cassette, in this case, EGFP (pA: polyadenylation signal). [Figure 12](a-d) Forward programming of hPSCs into oligodendrocytes. Figure 12(a) shows a schematic diagram of the experimental approach for rapid conversion of OPTi-OLIG2-SOX10 hPSCs into oligodendrocyte-lineage cells (i-OPCs and i-OLs). Figure 12(b) shows quantification of BrdU-positive cells after three consecutive passages every four days and the accompanying BrdU pulses, each lasting four days (P = passage number). Figure 12(c) shows quantitative RT-PCR analysis of the temporal expression patterns of genes encoding myelin-associated proteins (CNP, MAG, MBP, MOG, and PLP) during i-oligodendrocyte generation from hPSCs. OPTi-OLIG2-SOX10 hPSCs were induced in oligodendrocyte medium supplemented with PDGF-aa and FGF2. One week after induction, mitogens were removed to allow terminal differentiation. All values ​​are shown normalized to the pluripotent state relative to the endogenous housekeeping gene PBGD. Results are from two to three biological replicates per time point and are expressed as mean ± SEM. Figure 12(d) shows quantification of CNP- and PLP-positive cells by immunostaining in i-oligodendrocytes derived from OPTi-OLIG2-SOX10 hPSCs after 20 days of induction. Undifferentiated cells were used as a negative control; the figure shows i-oligodendrocytes after 50 passages (+P50) in freshly isolated OPTi-NGN2 hPSCs. [Figure 13]

[0023] Figure 1 is a schematic diagram of the principle of the present invention. Essentially, this shows insertion into two different gene safe harbor sites in the core of the present invention. One insertion controls the expression of the gene sequence in the induction cassette in the second insertion. As shown, additional genetic material can be included in the polycistronic vector construct. Furthermore, three or more gene safe harbor sites can be targeted so that multiple induction cassettes or other genetic material can be placed under the control of the regulatory element located in the first GSH site. [Figure 14](a–f) Schematic diagrams of results demonstrating the development of an inducible knockdown system based on dual GSH targeting of hSPCs. Figure 14a shows the experimental approach: H1 - H1 promoter, TO - tet operon, tetR - tetracycline repressor. Figure 14b is a schematic diagram of the transgenic alleles generated to obtain hESCs expressing the EFGP reporter transgene, which can be silenced using an inducible EGFP shRNA. Figure 14c shows EGFP expression in hESCs targeted with the indicated combinations of inducible EGFP shRNA and tetR in the absence or presence of tetracycline for 5 days (STD = wild-type standard, OPT = codon-optimized). Dual-targeted hESCs without EGFP shRNA were used as negative controls. ns = p > 0.05 (not significant), ** = p > 0.01, *** = p > 0.001 versus the same tetR line without tet or shRNA. Figure 14d shows a representative Western blot of tetR in ROSA26-targeted hESCs expressing STD or OPT tetR. HET = heterozygous targeting, HOM = homozygous targeting. hESCs with STD tetR random integration are shown as a positive reference, while WT h9 hESCs are a negative control. TUB4A4A is a loading control. Various protein amounts were added to facilitate quantitative comparisons. Figure 14(E): EGFP knockdown and rescue kinetics in EGFP OPTiKD hESCs measured by flow cytometry (MFI) and qPCR (mRNA). Results are from two independent cultures per time point. Figure 14(F): Tetracycline dose-response curve of EGFP knockdown in EGFP OPTiKD hESCs. Half-maximal inhibitory concentrations (IC50) are reported. Results are from two independent cultures per dose and represent the mean values. [Figure 15](a, b, and c) Validation of the ROSA26 and AAVS1 loci as bona fide GSH. Figure 15a shows the experimental approach for generating GSH EGFP reporter hPSCs to test GSH expression during differentiation. Neurons, oligodendrocytes, and astrocytes were obtained in bulk cultures containing a mixture of these cell lineages, while all other cell types were generated individually. Figure 15b is a schematic diagram of the ROSA26 and AAVS1 EGFP reporter transgenic alleles. R26-prom: ROSA26 locus promoter; AAV-prom: AAVS1 locus promoter; 5'-HAR / 3'-HAR: upstream / downstream homology arms; SA: splice acceptor; T2A: self-cleaving T2A peptide; Neo: neomycin resistance; Puro: puromycin resistance; pA: polyadenylation signal; CAG: CAG promoter; EGFP: enhanced green fluorescent protein. Figure 15(C): EGFP expression in hESCs targeted with the indicated combinations of inducible EGFP shRNA and tetR (wild-type standard tetR, STDtetR, or codon-optimized tetR, OPTtetR) in the absence or presence of tetracycline for 5 days. Dual-targeted hESCs harboring no EGFP shRNA were used as negative controls. Results are from 2-3 individual lines per condition (Table 1). ns = p > 0.05 (not significant), ** = p < 0.01, *** = p < 0.001 (ANOVA with post-hoc Holm-Sidak comparison) vs. the same tetR line without tet or shRNA. [Figure 16](a-d) Generation of ROSA26 and AAVS1 EGFP reporter hESCs. Figure 16(A): Schematic of the ROSA26 targeting approach and genotyping strategy used to accurately identify targeted lines. Cas9n: D10A nickase mutant Cas9 endonuclease from S. pyogenes. R26-prom: ROSA26 locus promoter (THUMPD3-AS1 gene); 5'-HAR / 3'-HAR: upstream / downstream homology arms; transgene: region integrated after gene targeting; locus PCR: PCR product of wild-type ROSA26 locus (indicating the non-targeted allele); locus PCR / loss of allele: PCR product of targeted allele / PCR that fails when the transgene contains a GC-rich CAG promoter (indicating expected transgene targeting); 5'INT / 3'INT PCR: PCR product of the integration region at the 5' / 3' end of the transgene (indicating expected transgene targeting); 5'BB / 3'BB PCR: PCR product of the 5' / 3' end of the vector backbone (indicating off-target, nonspecific plasmid integration). Note that a similar targeting and genotyping strategy was applied to AAVS1 gene targeting. Figure 16(B): Schematic diagram of ROSA26 transgenic alleles generated to test the best strategy for constitutive EGFP (enhanced green fluorescent protein) expression. ENDO-EGFP: EGFP driven by the endogenous ROSA26 promoter (R26-prom; targeting vector pR26-Puro_ENDO-EGFP); EF1α-EGFP: EGFP driven by the elongation factor 1α promoter (targeting vector pR26-Neo_EF1α-EGFP); CAG-EGFP: EGFP driven by the CAG promoter (targeting vector pR26-Neo_CAG-EGFP); SA: splice acceptor; Puro: puromycin resistance (puromycin N-acetyltransferase); Neo: neomycin resistance (neomycin phosphotransferase II); pA: polyadenylation signal.Figure 16(C): Flow cytometry quantification of the percentage of EGFP-positive cells (EGFP+; gates shown) and median EGFP fluorescence intensity (MFI) in representative ROSA26-EGFP reporter hESC clonal lines, or wild-type H9 hESCs. Figure 16(D): Percentage of EGFP-positive cells in ROSA26-EGFP reporter hESCs. Results are for three clones with heterozygous ROSA26 targeting per condition. [Figure 17] Validation of the optimized inducible knockdown platform after hPSC differentiation. Plots show EGFP expression measured by qPCR in the indicated cell types derived from EGFP OPTiKD (iKD) and sOPTiKD (siKD) hESCs in the absence (CTR) or presence (TET) of tetracycline for 5 days. EGFP levels are reported for each individual lineage relative to the control condition in the same strain. Abbreviations indicate the lineages listed in Figure 15 (pluri: undifferentiated). Results are from two independent cultures per condition. [Figure 18] (a-d) Development of an optimized inducible CRISPR / Cas9 knockout platform in hPSCs. Figure 18a shows the experimental approach for generating inducible knockout (iKO) hPSCs. Figure 18b shows a schematic diagram of the cloning procedure for generating an AAVS1 targeting vector carrying an inducible gRNA cassette. Figure 18c shows the transgenic alleles generated to obtain hESCs expressing an EGFPd2 reporter transgene that can be knocked out by CRISPR / Cas9 using an inducible EGFP gRNA (EGFP sOPTiKO hESCs). Bsd: blasticidin resistance; EGFPd2: unstable EGFP. Figure 18(d): Flow cytometry quantification of EGFPd2-induced knockout kinetics in sOPTiKO cells from Figures 19c (gRNA 2-TO) and b (gRNA 3-2TO). The percentage of EGFP-positive cells was monitored daily after the addition of tetracycline. Results are from two independent cultures. [Figure 19](a-e): Development of an optimized inducible CRISPR / Cas9 knockout platform in hESCs. (A-D) Representative flow cytometry analysis of EGFPd2 expression in EGFPd2 homozygous sOPTiKO hESCs harboring the indicated combinations of gRNA (2 or 3) and inducible promoter (TO or 2TO, see Figure 19e). Targeting vectors: pAAV-Puro_siKO-EGFP-2 (19a), pAAV-Puro_siKO-2TO-EGFP-2 (19b), pAAV-Puro_siKO-EGFP-3 (19c), and pAAV-Puro_siKO-2TO-EGFP-3 (19d). Cells were cultured in the presence of tetracycline (TET) or maintained in control (CTR) conditions in the absence of tetracycline for 5 days. Note that to facilitate direct visual comparison, histograms were normalized so that the area under the curve equaled 1 (100%) for all samples presented. Figure 19(e): Nucleotide sequence of the inducible H1 Pol III promoter for the sOPTiKO system containing one or two tet operons (H1-TO and H1-2TO, respectively). Important sequence features are highlighted. Restriction enzyme cleavage sites used for gRNA cloning are indicated (Figure 18B). DSE: distal sequence element. PSE: proximal sequence element; TETO2: tet operon; +1: start of RNA transcription. [Figure 20] 1 is a depiction of the plasmid map used within the Examples of the present application. pSpCas9n(BB)_R26-R. [Figure 21] 1 is a depiction of the plasmid map used within the Examples of the present application. pSpCas9n(BB)_R26-L. [Figure 22] 1 is a depiction of the plasmid map used within the examples of the present application. pR26_CAG_EGFP. [Figure 23] 1 is a depiction of the plasmid map used within the examples of the present application. pR26_CAG_rtTA. [Figure 24] 1 is a depiction of the plasmid map used within the Examples of the present application: pZFN-AAVS1-L-ELD (Zinc Finger Nuclease Left). [Figure 25] 1 is a depiction of the plasmid map used within the examples of the present application: pZFN-AAVS1-R-KKR (zinc finger nuclease right). [Figure 26] 1 is a depiction of the plasmid map used within the Examples of the present application. pAAV_CAG_EGFP (donor). [Figure 27] 1 is a depiction of the plasmid map used within the examples of the present application. pR26-Neo_CAG-OPTtetR (codon-optimized tetR targeting hROSA26). [Figure 28] 1 is a depiction of the plasmid map used within the examples of the present application. pAAV-Puro_iKD (inducible shRNA targeting AAVS1). [Figure 29] 1 is a depiction of the plasmid map used within the Examples of the present application. pAAV-Neo_CAG-Cas9 (AAVS1 targeting of Cas9). [Figure 30] 1 is a depiction of the plasmid map used within the Examples of the present application. pAAV-Puro_siKO (inducible gRNA targeting AAVS1). [Figure 31] 1 is a depiction of the plasmid map used within the Examples of this application. pAAV-Puro_siKO-2TO (AAVS1-targeting inducible gRNA, version with two tet operons within the promoter). [Figure 32] 1 is a depiction of the plasmid map used within the examples of the present application. pAAV_TRE-EGFP (EGFP-inducible overexpression, attached). [Figure 33] 1 is a depiction of the plasmid map used within the examples of the present application. pAAV_TRE-MYOD1 (MYOD1 inducible overexpression for muscle). DETAILED DESCRIPTION OF THE INVENTION

[0049] (Detailed explanation) The present inventors have developed a method that is useful for the inducible transcription of genetic sequences contained within an inducible cassette in eukaryotic cells, particularly pluripotent stem cells and their progeny.

[0050] The method is particularly applicable to the forward programming of pluripotent stem cells via overexpression of an induction cassette within said stem cells that promotes the development of specific mature cell types. It is also applicable to the knockdown or knockout of endogenous functions within cells to study loss of function or alter cellular function or behavior in these cells. Knockdown or knockout can be applied to protein-coding genes or DNA sequences encoding non-coding RNA. Either can be targeted by the method of the present invention through knockout or knockdown.

[0051] This method is based on at least dual targeting of safe harbor sites within the genome of stem cells using a system for inducible transcription that is divided into two or more GSH loci. However, this method is not limited to stem cells and can be used to modify the genome of any cell type, for example, in research or gene therapy. In the method of the present invention, one GSH locus is modified to contain the transcriptional regulatory factors necessary to induce transcription of a gene sequence contained within an inducible cassette that is inserted into a different GSH locus elsewhere in the genome. The transcriptional regulatory factors are preferably constitutively expressed. An exogenous substance / drug must preferably be provided to control the activity of the transcriptional regulatory protein and therefore the expression of the inducible cassette. Because at least two separate GSH loci are used in the method of the present invention, each GSH locus is present on both chromosomes of a diploid organism, resulting in a total of four potential insertion loci. This increases the amount of potential transcription from a cell if all four loci are modified using the method of the present invention. An example of various outcomes of targeted insertion is shown in Figure 5a. Furthermore, the method of the present invention uses at least two different GSH loci. It will be appreciated that the additional GSH sites can be used to introduce any other genetic material, including, but not limited to, additional transcriptional regulators, inducible cassettes or selectable markers, antibiotic or drug resistance genes, genes associated with the CRISPR / Cas9 system or genes of unknown function.

[0052] Accordingly, the present invention provides a method for controlling expression of an inserted gene sequence in a cell, comprising the steps of: a) targeted insertion of a transcriptional regulatory protein-encoding gene sequence into a first gene safe harbor site; and b) targeted insertion of an inducible cassette into a second genetic safe harbor site, said inducible cassette comprising said genetic sequence operably linked to an inducible promoter, said promoter being regulated by said transcriptional regulatory protein; Including, The first and second genetic safe harbor sites are different.

[0053] Furthermore, in this aspect of the invention, additional, identical or different, induction cassettes may be inserted into additional GSHs that are different from the first and second GSHs, such induction cassettes being as described herein.

[0054] Specific insertion within a genetic safe harbor site is preferred over random genomic integration because it is expected to be a safer modification of the genome and less likely to result in unwanted side effects such as silencing natural gene expression or causing mutations that lead to cancerous cell types.

[0055] A gene safe harbor (GSH) site is a locus within the genome where a gene or other genetic material can be inserted without any adverse effects on the cell or the inserted genetic material. GSH sites are most beneficial because the expression of the inserted gene sequence is not disrupted by any read-through expression from adjacent genes and the expression of the induction cassette minimizes interference with the endogenous transcription program. More formal criteria have been proposed to help determine whether a particular locus is a GSH site in the future (Papapetrou et al., 2011, Nature Biotechnology, 29(1), 73-8. doi:10.1038 / nbt.1717). These criteria include: (i) sites that are more than 50 kb from the 5' end of any gene; (ii) sites that are more than 300 kb from any cancer-related gene; (iii) sites that are more than 300 kb from any microRNA (miRNA); (iv) sites that are located outside of transcription units; and (v) sites that are outside of ultraconserved regions (UCRs). It may not be necessary to meet all of these proposed criteria, as previously identified GSHs do not meet all of the criteria. A suitable GSH would likely meet at least two, three, four, or all of these criteria.

[0056] Additional sites can be identified by searching for sites where the virus naturally integrates without disrupting native gene expression.

[0057] Any suitable GSH site can be used in the methods of the present invention, provided that it allows for the insertion of genetic material without adverse effects on the cell and allows for transcription of the inserted genetic material. One of skill in the art can use this simplified criteria to identify a suitable GSH, and / or the more formal criteria described above.

[0058] Several GSH loci have been identified in the human genome, including the AAVS1 locus, the hROSA26 locus, and the CLYBL gene. The CCR5 gene and the HPRT gene have also been discussed as possible GSH loci, and further investigation may identify one or more of these as GSH loci in the human genome.

[0059] The adeno-associated virus integration site 1 locus (AAVS1) is located within the protein phosphatase 1, regulatory subunit 12C (PPP1R12C) gene on human chromosome 19 and is uniformly and ubiquitously expressed in human tissues. This site functions as a specific integration locus for AAV serotype 2 and has therefore been identified as a potential GSH. AAVS1 has been shown to be a favorable transcriptional environment because it contains a natural chromosomal insulator that allows for an open chromatin structure and resistance to silencing of the induction cassette. There are no known side effects to cells resulting from disruption of the PPP1R12C gene. Furthermore, induction cassettes inserted at this site remain transcriptionally active in many diverse cell types. Therefore, AAVS1 is considered a GSH and has been widely used for targeted gene recombination in the human genome.

[0060] The hROSA26 locus was identified based on sequence similarity to mouse GSH (ROSA26-reverse splice acceptor site #26). Although an orthologous site has been identified in humans, this site is not commonly used for directed cassette insertion. We developed a targeting system specific to the hROSA26 locus, allowing us to insert genetic material into this locus. The hROSA26 locus is located on chromosome 3 (3p25.3) and can be found in the Ensembl database (GenBank: CR624523). The precise genomic coordinates of the integration site are 3:9396280-9396303: Ensembl. The integration site is within the open reading frame (ORF) (reverse strand) of the THUMPD3 long non-coding RNA. Because the hROSA26 locus has an endogenous promoter, the inserted genetic material can utilize the endogenous promoter or can be inserted so that it is operably linked to the promoter.

[0061] Intron 2 of the citrate lyase beta-like (CLYBL) gene on the long arm of chromosome 13 was identified as a suitable GSH because it is one of the identified integration hotspots for phage derived from phiC31 integrase. Studies have demonstrated that an induction cassette randomly inserted into this locus is stably expressed. Insertion of an induction cassette at this GSH has been shown not to disrupt local gene expression (Cerbibi et al., 2015, PLOS One, DOI:10.1371). Thus, CLYBL provides a GSH that may be suitable for use in the present invention.

[0062] CCR5, located on chromosome 3 (location 3p21.31), is the gene encoding the major co-receptor for HIV-1. The idea of ​​using this site as a GSH arises from null mutations in this gene that predispose to resistance to HIV-1 infection, although they are unlikely to have adverse effects. Zinc finger nucleases that target the third exon have been developed, allowing for the insertion of genetic material at this locus. Given that the native function of CCR5 has yet to be elucidated, this site remains a putative GSH that may be useful for the present invention.

[0063] The hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene encodes a transferase enzyme that plays a central role in the creation of purine nucleotides via the purine salvage pathway. Therefore, further work is needed to ensure that insertion at this site does not disrupt normal cellular function. However, it is controversial as the GSH site. Insertion at this site may be more applicable to mature cell types, such as for gene therapy.

[0064] GSH loci have been identified in other organisms, including the ROSA26, HRPT, and Hipp11 (H11) loci in mice. Mammalian genomes can contain GSH loci based on pseudo attP sites. For such sites, hiC31 integrase, a recombinase derived from a Streptomyces phage, has been developed as a non-viral insertion tool because it has the ability to integrate an inducible cassette-containing plasmid carrying an attB site into the pseudo attP site.

[0065] GSH is also present in the genome of plants, and the modification of plant cells can be part of the present invention. GSH has been identified in the rice genome (Cantos et al., Front. Plant Sci., 26 June 2014, Volume 5, Article 302, http: / / dx.doi.org / 10.3389 / fpls.2014.00302 ).

[0066] In the method of the present invention, the insertion occurs in different GSHs, so at least two GSHs are required for the method of the present invention. The first GSH is modified by inserting a transcriptional regulatory protein. The second GSH is modified by inserting an inducible cassette containing a gene sequence operably linked to an inducible promoter. Other genetic material can also be inserted with either or both of these elements. The gene sequence operably linked to the inducible promoter in the inducible cassette is preferably a DNA sequence. The gene sequence(s) of the inducible cassette encodes an RNA molecule and is therefore transcribable. Transcription is controlled using an inducible promoter. The RNA molecule can be of any sequence, but is preferably an mRNA, shRNA, or gRNA that encodes a protein.

[0067] The first GSH can be any suitable GSH site.Optionally, it is a GSH with a constitutively expressed endogenous promoter.This results in the constitutive expression of the inserted transcriptional regulatory protein.Suitable GSH is the hROSA26 site for human cells.Alternatively, the inserted transcriptional regulatory protein is operably linked to a promoter, preferably a constitutive promoter.A constitutive promoter can be used in combination with the insertion at the hROSA26 site.

[0068] Transcriptional regulatory proteins are proteins that bind in a sequence-specific manner to DNA, preferably to a DNA site located in or near a promoter, and either promote the binding of the transcriptional apparatus to the promoter and thus the transcription of the DNA sequence (transcriptional activators) or block this process (transcriptional repressors). Such entities are also known as transcription factors.

[0069] DNA sequences to which transcriptional regulatory proteins bind are called transcription factor binding sites or response elements, and they are found in or near the promoter of the regulatory DNA sequence.

[0070] Transcriptional activator proteins bind to response elements and promote gene expression. Such proteins are preferred in the methods of the present invention for controlling expression of an inducible cassette.

[0071] Transcriptional repressor proteins bind to response elements and prevent gene expression.

[0072] Transcriptional regulatory proteins can be activated or inactivated by several mechanisms, including binding of substances, interaction with other transcription factors (e.g., homo- or heterodimerization) or co-regulatory proteins, phosphorylation, and / or methylation. Transcriptional regulatory factors can be controlled by activation or inactivation.

[0073] When the transcriptional regulatory protein is a transcriptional activator protein, it is preferred that the transcriptional activator protein requires activation. This activation can be via any suitable means, but it is preferred that the transcriptional regulatory protein is activated through the addition of an exogenous substance to a cell. The supply of the exogenous substance to the cell can be controlled, thereby controlling the activation of the transcriptional regulatory protein. Alternatively, the exogenous substance can be supplied to inactivate the transcriptional regulatory protein, and then removed to activate the transcriptional regulatory protein.

[0074] When the transcriptional regulatory protein is a transcriptional repressor protein, it is preferred that the transcriptional repressor protein requires inactivation, and therefore a substance is provided to prevent the transcriptional repressor protein from repressing transcription, thus allowing transcription to occur.

[0075] Any suitable transcriptional regulatory protein can be used, preferably one that can be activated or inactivated. Preferably, an exogenous substance can be supplied to control the transcriptional regulatory protein. Such transcriptional regulatory proteins are also called inducible transcriptional regulatory proteins.

[0076] Tetracycline-controlled transcriptional activation is a method of inducible gene expression in which transcription is reversibly turned on or off in the presence of the antibiotic tetracycline or one of its derivatives (e.g., the more stable doxycycline). In this system, the transcriptional activator protein is the tetracycline-responsive transcriptional activator protein (rtTA) or its derivatives. The rtTA protein can bind to DNA at specific TetO operator sequences. Several repeats of such TetO sequences are placed upstream of a minimal promoter (such as the CMV promoter), together forming a tetracycline response element (TRE). This system exists in two forms, depending on whether the addition of tetracycline or a derivative activates (Tet-On) or inactivates (Tet-Off) the rtTA protein.

[0077] In the Tet-Off system, tetracycline or its derivatives bind to rtTA, inactivating it and rendering it unable to bind to the TRE sequence, thereby preventing transcription of the TRE-controlled gene. This system was first described by Bujard et al. (1992). Proc. Natl. Acad. Sci. USA 89(12):5547-51.

[0078] The Tet-On system consists of two components: (1) a constitutively expressed tetracycline-responsive transcription activator protein (rtTa) and an rtTA-sensitive inducible promoter (Tet response element, TRE). It binds tetracycline or its more stable derivatives, including doxycycline (dox), resulting in activation of rtTa, allowing it to bind to the TRE sequence and induce expression of the TRE-controlled gene. This use may be preferred in the methods of the present invention. This system is illustrated in Figure 11.

[0079] Thus, the transcriptional regulatory protein can be a tetracycline-responsive transcriptional activator protein (rtTa) protein that can be activated or inactivated by an exogenously supplied antibiotic, tetracycline, or one of its derivatives. When the transcriptional regulatory protein is rtTA, the inducible promoter inserted into the second GSH site contains a tetracycline response element (TRE). The exogenously supplied substance is the antibiotic, tetracycline, or one of its derivatives.

[0080] Mutant and modified rtTa proteins can be used in the methods of the invention, including Tet-On high transactivator (also known as rtTA2S-M2) and Tet-On 3G (also known as rtTA-V16, derived from rtTA2S-S2).

[0081] The tetracycline response element (TRE) generally consists of seven repeats of a 19-bp bacterial TetO sequence separated by a spacer sequence along with a minimal promoter. The minimal promoter can be any suitable promoter, allowing for mutations and modifications of the TRE sequence. Preferably, the minimal promoter exhibits zero or minimal expression levels in the absence of rtTa binding. Therefore, the inducible promoter inserted into the second GSH gene can include a TRE.

[0082] A modified system based on tetracycline regulation is the T-REx™ system (Thermofisher Scientific), in which the transcriptional regulatory protein is the transcriptional repressor protein TetR. Components of this system include (i) an inducible promoter containing the strong human cytomegalovirus immediate-early (CMV) promoter and two tetracycline operator 2 (TetO2) sites, and a Tet repressor (TetR). The TetO2 sequence is a 19-nucleotide sequence separated by a 2-base pair spacer. [ka] In the absence of tetracycline, the Tet repressor forms a homodimer that binds with very high affinity to each TetO2 sequence in the inducible promoter, preventing transcription from the promoter. When tetracycline is added, it binds with high affinity to each Tet repressor homodimer, preventing it from binding to the Tet operator. The Tet repressor:tetracycline complex then dissociates from the Tet operator, allowing expression to be induced. In this case, the transcriptional regulatory protein is TetR, and the inducible promoter contains two TetO2 sites. The exogenously supplied substance is tetracycline or a derivative thereof.

[0083] The present invention further relates to a codon-optimized tetR (OPTtetR), which can be used in any of the methods described herein or for any additional use where enhanced induction is desired. This entity was generated using multiparameter optimization of the bacterial tetR cDNA sequence. OPTtetR allows for a 10-fold increase in tetR expression when compared to the standard sequence (STDtetR). Homozygous OPTtetR expression of tetR is sufficient to prevent shRNA leakage and, in the examples, maintain knockdown induction. The sequence of OPTtetR is included herein, along with the standard sequence shown for comparison. Sequences with at least 75%, 80%, 85%, or 90% homology to this sequence, more specifically, 91, 92, 93, 94, 95, 96, 97, or 99% homology, are claimed herein. Residues shown to be altered between STDtetR and OPTtetR are indicated in the sequence, and these residues are believed to be important for improved properties and are therefore preferably not altered in any derivative of OPTtetR, which optionally retains these modifications at the indicated positions.

[0084] Other inducible expression systems are known and can be used in the methods of the present invention. These include the Complete Control Inducible system from Agilent Technologies, which is based on the insect hormone ecdysone or its analog ponasterone A (ponA), which can activate transcription in mammalian cells transfected with both the Drosophila melongaster ecdysone receptor (EcR) gene and an inducible promoter containing a binding site for the ecdysone receptor. EcR is a member of the retinoid X receptor (RXR) family of nuclear receptors. In humans, EcR forms a heterodimer with RXR, which binds to the ecdysone response element (EcRE). In the absence of PonA, transcription is repressed by the heterodimer.

[0085] Thus, the transcriptional regulatory protein can be a repressor protein, such as the ecdysone receptor or its derivatives. Examples of the latter include Agilent Technologies' VgEcR synthetic receptor, which is a fusion of the DNA-binding domain of the EcR, glucocorticoid receptor, and the transcriptional activation domain of herpes simplex virus VP16. The inducible promoter includes a minimal promoter along with an EcRE sequence or a modified version thereof. The modified version includes Agilent Technologies' E / GRE recognition sequence, which has been mutated. The E / GRE recognition sequence contains the inverted half-site recognition element and GR-binding domain of the retinoid X receptor (RXR). In all permutations, the exogenously supplied substance is ponasterone A, which removes the repressive effect of EcR or its derivatives on the inducible promoter, allowing transcription to occur.

[0086] Alternatively, an inducible system can be based on the synthetic steroid mifepristone as an exogenously supplied substance. In this situation, a hybrid transcriptional regulatory protein based on the DNA-binding domain of the yeast GAL4 protein, a truncated ligand-binding domain (LBD) of the human progesterone receptor, and the activation domain (AD) of human NF-κB is inserted. This hybrid transcriptional regulatory protein is available from Thermofisher Scientific (Gene Switch™). Mifepristone activates the hybrid protein, allowing transcription from an inducible promoter containing GAL4 upstream of an activation sequence (UAS) and an adenovirus E1b TATA box. This system is described in Wang, Y. et al. (1994) Proc. Natl. Acad. Sci. USA 91, 8180-8184.

[0087] Thus, the transcriptional regulatory protein can be any suitable regulatory protein, either an activator protein or a repressor protein. Suitable transcriptional activator proteins include tetracycline-responsive transcriptional activator protein (rtTa) or gene switch hybrid transcriptional regulatory proteins. Suitable repressor proteins include Tet-Off versions of rtTA, TetR, or EcR. The transcriptional regulatory protein can be modified or derivatized as needed.

[0088] Inducible promoters can contain elements suitable for binding to or interacting with transcriptional regulatory proteins, the interaction of which is preferably controlled by an exogenously supplied substance.

[0089] The exogenously supplied substance can be any suitable substance that binds to or interacts with the transcriptional regulatory protein. Suitable substances include tetracycline, ponasterone A, and mifepristone.

[0090] Thus, insertion of a transcriptional regulatory protein-encoding gene into the first GSH site is operably linked to an inducible promoter, providing a control mechanism for expression of an inducible cassette inserted into a second, different GSH site.

[0091] The transcriptional regulatory protein gene can be provided for insertion with other genetic material. Such materials include genes for marker or reporter molecules, such as genes that induce visually identifiable characteristics, including fluorescent and luminescent proteins. Examples include the jellyfish green fluorescent protein (GFP), which causes expressing cells to glow green under blue / UV light; luciferase, which catalyzes a reaction with luciferin to produce light; and the gene encoding the red fluorescent protein dsRed. Such marker or reporter genes are useful because the presence of the reporter protein confirms protein expression from the first GSH, indicating successful insertion. Selectable markers may also include antibiotic or other drug resistance genes. Marker or reporter gene sequences that allow for the study of endogenous (or exogenous) gene expression can also be introduced. These include Cas proteins, including CasL and Cas9 proteins, which allow for excision of the gene of interest, and Cas fusion proteins, which mediate changes in the expression of other genes, for example, by acting as transcriptional enhancers or repressors. Additionally, non-inducible expression of molecular tools may be desirable, including optogenetic tools, nuclear receptor fusion proteins such as ERT in tamoxifen-inducible systems, and designer receptors activated exclusively by designer drugs. Furthermore, sequences encoding signaling factors that alter the function of the same cell, adjacent cells, or even more distant cells within an organism, including hormonal autocrine or paracrine factors, can be co-expressed from the same GSH as transcriptional regulatory proteins.

[0092] The additional genetic material may also include sequences that encode non-coding RNAs as described herein. Examples of such genetic material include genes for miRNAs that can function as gene switches.

[0093] Preferably, the transcriptional regulatory protein-encoding gene is operably linked to a constitutive promoter. Alternatively, the first GSH can be selected so that it already has a constitutive promoter capable of driving the expression of the transcriptional regulatory protein gene and any associated genetic material. The constitutive promoter ensures sustained, high-level gene expression. Commonly used constitutive promoters include the human β-actin promoter (ACTB), cytomegalovirus (CMV), elongation factor 1α (EF1α), phosphoglycerate kinase (PGK), and ubiquitin C (UbC). The CAG promoter is a strong synthetic promoter frequently used to drive high-level gene expression and was constructed from the following sequences: (C) the cytomegalovirus (CMV) early enhancer element, (A) the promoter, first exon, and first intron of the chicken β-actin gene, and (G) the splice acceptor of the rabbit β-globin gene.

[0094] Furthermore, the transcriptional regulator and any additional genetic material may be provided with a cleavage sequence. Such sequences are sequences recognized by entities capable of specifically cleaving DNA, and include restriction sites that are target sequences for restriction enzymes or sequences for recognition by other DNA-cleaving entities, such as nucleases, recombinases, ribozymes, or artificial constructs. At least one cleavage sequence may be included, but preferably two or more are present. These cleavage sequences may be at any suitable point within the insert, allowing selective removal of selected portions of the insert, or all of the insert, from GSH. Thus, the method can be extended to remove and / or replace an insert or portions thereof from GSH. Thus, the cleavage site may flank part or all of the insert that may be desired to be removed. The transcriptional regulator and / or additional genetic material may be removed using this method.

[0095] A portion of the insert can be any portion up to 99% of the insert, i.e., 1-99%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10% or less than 10%.

[0096] It may be preferred that the portion of the insert flanked by the cleavage site contains a constitutive promoter. Alternatively, a constitutive promoter is not included in the portion flanked by the cleavage sequence.

[0097] A preferred cleavage sequence is a loxP site for Cre recombinase, to allow direct replacement of the removed insert. Alternatively or additionally, the cleavage sequence is a rox site for Dre recombinase.

[0098] Preferably, the insertion at the first GSH occurs at both loci of the genome so that each allele is modified by the insertion, allowing for greater expression from the gene encoding the transcriptional regulator and any associated genetic material.

[0099] The second GSH site can be any suitable GSH site. It may be preferable that the second GSH site is not associated with an endogenous promoter so that expression of the inserted inducible cassette is solely under the control of a transcriptional regulatory protein.

[0100] The induction cassette contains a desired gene sequence, preferably a DNA sequence, to be introduced into a cell. Introduction of the induction cassette into the genome has the potential to alter the phenotype of the cell by adding a gene sequence that allows gene expression or by knocking down / knocking out endogenous expression. The method of the present invention provides for the controllable transcription of the gene sequence(s) within the induction cassette in the cell.

[0101] The desired gene sequence for insertion is preferably a DNA sequence encoding an RNA molecule. The RNA molecule can be any sequence but is preferably coding RNA or non-coding RNA. Coding RNA or messenger RNA encodes a polypeptide sequence, and transcription of such RNA results in the expression of a protein within a cell. Non-coding RNA can be functional and include, but are not limited to, microRNA, small interfering RNA, Piwi-interacting RNA, antisense RNA, small nuclear RNA, small nucleolar RNA, small Cajal body RNA, Y RNA, enhancer RNA, guide RNA, ribozyme, small hairpin RNA, small temporal RNA, trans-acting RNA, small interfering RNA, and subgenomic messenger RNA. Non-coding RNA may also be known as functional RNA. Some types of RNA are naturally regulated and can down-regulate gene expression, for example, by being complementary to a portion of mRNA or the DNA of a gene. MicroRNAs (miRNAs; 21–22 nucleotides) are found in eukaryotes and act through RNA interference (RNAi), in which miRNA and enzyme effector complexes cleave complementary mRNAs, prevent them from being translated, or promote their degradation. Another type of RNA, small interfering RNAs (siRNAs; 20–25 nucleotides), act through RNAi in a manner similar to miRNAs. Some miRNAs and siRNAs can methylate targeted genes, thereby decreasing or increasing their transcription. Animals possess Piwi-interacting RNAs (piRNAs; 29–30 nucleotides), which are active in germline cells and are thought to act as a defense against transposons. Many prokaryotes possess CRISPR RNAs, a regulatory system similar to RNA interference, which includes guide RNAs (gRNAs). Antisense RNAs are widespread; most downregulate genes, but some are also transcriptional activators. Antisense RNAs can act by binding to mRNAs to form double-stranded RNAs that are then degraded by enzymes.There are many long non-coding RNAs that regulate genes in eukaryotes, and one such RNA is Xist, which coats and inactivates one X chromosome in female mammals. Therefore, there are many functional RNAs that can be used in the methods of the present invention.

[0102] Thus, the induction cassette may comprise a gene sequence that is a protein-coding gene. This gene may not naturally occur in the cell, or may naturally occur in the cell, but controllable expression of the gene is required. Alternatively, the induction cassette may be a mutant, modified, or correct version of a gene present in the cell, particularly for gene therapy purposes or for the derivation of a disease model. Thus, the induction cassette may comprise a transgene from a different organism of the same species (i.e., a disease / mutant version of a gene from human, or a wild-type gene from human), or may be from a different species.

[0103] In any aspect or embodiment, the gene sequence contained within the induction cassette may be a synthetic sequence.

[0104] The induction cassette may comprise any suitable gene sequence that is desired to be inserted into the genome of a cell. Thus, the gene sequence may be a gene encoding a protein product or a sequence that is transcribed into functional ribonucleic acid (RNA), such as small nuclear RNA (snRNA), antisense RNA, microRNA (miRNA), small interfering RNA (siRNA), transfer RNA (tRNA), and other non-coding RNA (ncRNA), including CRISPR-RNA (crRNA) and guide RNA (gRNA).

[0105] Thus, the inducible cassette may contain any gene sequence whose transcription it is desired to control in a cell. As explained further below, the gene sequence chosen will depend on the cell type and the use of the cell after modification.

[0106] For example, for gene therapy, it may be desirable to provide a wild-type gene sequence as a component of an induction cassette. In this context, the gene sequence can be any human or animal protein-coding gene. Examples of protein-coding genes include the human β-globin gene, human lipoprotein lipase (LPL) gene, human Rab escort protein 1, encoded by the CHM gene and many more genes. Alternatively, the induction cassette can express growth factors, including BDNF, GDF, NGF, IGF, FGF, and / or enzymes capable of cleaving propeptides to form active forms. Gene therapy can also be achieved by expressing an induction cassette containing a gene sequence encoding an antisense RNA, miRNA, siRNA, or any type of RNA that interferes with the expression of another gene in a cell.

[0107] Alternatively, if the cell is a stem cell, the induction cassette can include a gene sequence encoding an important lineage-specific master regulator, herein abbreviated as master regulator. A master regulator can be one or more of a transcription factor, a transcription regulator, a cytokine receptor, or a signaling molecule, etc. A master regulator is an expressed gene that influences the lineage of the cell that expresses it. A network of master regulators may be required for the lineage of a cell to be determined. As used herein, a master regulator gene expressed at the initiation of a developing lineage or cell type is responsible for that lineage specificity by regulating multiple downstream genes directly or through a cascade of gene expression changes. Upon expression, a master regulator has the ability to redirect the fate of cells destined to form other lineages. Examples of master regulators include the myogenic transcription factor MyoD and the hematopoietic transcription factor SCL. In particular, master regulators include: Neural lineage: Oligodendrocytes: SOX10, OLIG2, NKX2.2, NKX6.2; Astrocytes: NFIA, NFIB, and SOX9; Neurons: Ascl1, Neurogenin, and NeuroD, Pax6, Neurog2, Ascl1, Dlx2, and NeuroD1; Hematopoietic cells including erythrocytes and megakaryocytes: GATA1, FLI1, and TAL1 Mesenchymal lineages: skeletal muscle: MYOD; cardiomyocytes: Gata4, Mef2c, Baf60c, and Tbx5; bone: L-Myc (RXOL), Runx2, Osterix, and Oct4; cartilage: c-Myc, Klf4, and SOX9; and brown adipocytes: C / EBP-β and c-Myc endoderm Pancreatic cell types: PDX1 and GATA6 Stem cells: epiblast SC: Oct4, Sox2, Klf4 and c-Myc These include, but are not limited to:

[0108] Alternatively or additionally, the gene sequence or further genetic material may be a gene whose function needs to be investigated, such that controllable expression allows the effect of expression on the cells to be investigated, the gene may comprise a growth factor and / or cytokine, for the cells to be used in cell transplantation, and / or the gene may be a component of a reporter assay.

[0109] Furthermore, the gene sequence may encode a non-coding RNA whose function is to knock down the expression of an endogenous gene, or may be a DNA sequence that encodes a non-coding RNA in a cell. Alternatively, the gene sequence may encode a guide RNA for a CRISPR-Cas9 system that results in the knockout of an endogenous gene.

[0110] Thus, the method of the present invention encompasses a method for knocking down the expression of an endogenous gene in a cell. The method is as described above, wherein the inducible cassette comprises a gene sequence encoding a non-coding RNA operably linked to an inducible promoter, and the non-coding RNA suppresses the expression of the endogenous gene. The non-coding RNA can suppress gene expression by any suitable means, including RNA interference and antisense RNA. Thus, the gene sequence can encode an shRNA that can interfere with the messenger RNA of the endogenous gene.

[0111] The reduction in endogenous gene expression can be partial or complete - i.e., expression can be reduced by 50, 55, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% compared to the cell before induction of transcription of the non-coding RNA.

[0112] Although any other suitable system for gene knockout can be used, the method of the present invention also extends to a method for knocking out endogenous genes in cells using the CRIPSR-Cas9 system. In this context, the Cas9 gene is preferably contained in a first GSH containing a transcriptional regulator gene for constitutive expression. The gRNA-encoding gene sequence may be contained in an induction cassette inserted into a second GSH. The gRNA is a short synthetic RNA composed of a scaffold sequence required for Cas9 binding and an approximately 20-nucleotide target sequence that defines the genomic target to be modified. Therefore, the genomic target of Cas9 can be changed simply by modifying the target sequence present in the gRNA. The primary use of such a system is to design gRNAs that target endogenous genes to knock out genes, but it can also be modified to selectively activate or suppress target genes, purify specific DNA regions, and even image DNA. All possible applications are envisioned.

[0113] The inducible cassette contains a gene sequence operably linked to an inducible promoter. A "promoter" is a nucleotide sequence that initiates and regulates transcription of a polynucleotide. An "inducible promoter" is a nucleotide sequence in which expression of a gene sequence operably linked to a promoter is controlled by an analyte, cofactor, regulatory protein, etc. In the present invention, such control is exerted by a transcriptional regulatory protein. The terms "promoter" or "control element" are intended to include full-length promoter regions and functional (e.g., transcriptional or translational) segments of these regions. "Operably linked" refers to the arrangement of elements such that the components described are configured to perform their normal function. Thus, a given promoter operably linked to a gene sequence is capable of effecting expression of that sequence when the appropriate enzymes are present. The promoter need not be contiguous with the sequence, so long as it functions to direct the expression of the sequence. Thus, for example, intervening non-translated but transcribed sequences can be present between the promoter sequence and the gene sequence, and the promoter sequence can still be considered "operably linked" to the gene sequence. Thus, the term "operably linked" is intended to encompass any spacing or orientation of the promoter element and gene sequence within the induction cassette that allows initiation of transcription of the induction cassette upon recognition of the promoter element by a transcription complex.

[0114] Additionally, other genetic material can be operably linked to the inducible promoter. The additional genetic material can include genes, such as marker or reporter genes, RNA coding sequences, and other genetic material. Such additional genetic material has been previously described. In some situations, it may be desirable to include a suicide gene in the inducible cassette, but the gene sequence itself should not be a suicide gene for cancer gene therapy. The suicide gene can use the same inducible promoter in the inducible cassette or can be a separate inducible promoter to allow for separate control. Such genes can be useful in gene therapy situations where it is desirable to be able to destroy donor / transfected cells when certain conditions are met. A suicide gene is a gene that expresses a protein that can cause apoptosis in cells or that may require externally supplied cofactors or co-drugs to function. The cofactors or co-drugs can be converted into highly cytotoxic entities by the product of the suicide gene.

[0115] Furthermore, the derivative cassette may contain a cleavage sequence. Such a sequence is a sequence recognized by an entity capable of specifically cleaving DNA, and includes a restriction site, which is a target sequence for a restriction enzyme, or a sequence for recognition by other DNA-cleaving entities, such as nucleases, recombinases, ribozymes, or artificial constructs. At least one cleavage sequence may be included, but preferably two or more are present. These cleavage sequences may be at any suitable point within the cassette, allowing selective removal of selected portions of the cassette, or the entire cassette, from GSH. Thus, the method can extend to the removal and / or replacement of the cassette or portions thereof from GSH. Thus, the cleavage site may flank some or all of the gene sequence that may be desired to be removed. The method can result in the removal of the derivative cassette and / or additional genetic material.

[0116] The portion of the cassette can be any portion up to 99% of the cassette, i.e., 1-99%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10% or less than 10%.

[0117] Preferably, the portion of the insert flanked by the cleavage site may include a promoter operably linked to the gene sequence, or alternatively, the portion flanked by the cleavage sequence does not include a promoter operably linked to the gene sequence.

[0118] A preferred cleavage sequence is a loxP site for Cre recombinase, to allow direct replacement of the removed insert. Alternatively or additionally, the cleavage site may be a roxP site for Dre recombinase.

[0119] The transcriptional regulatory protein and induction cassette, along with any associated genetic material, are inserted into the genome of the cell at a different GSH.

[0120] The insertion into GSH is preferably particularly within the aforementioned GSH sequence. Any suitable technique for inserting a polynucleotide into a specific sequence can be used, and some have been described in the art. Suitable techniques include any method that introduces a break at a desired position and allows recombination into the gap of the vector. Therefore, the first step important for target site-specific genome modification is to create a double-stranded DNA break (DSB) at the genomic locus to be modified. Different cellular repair mechanisms can be used to repair DSB and introduce desired sequences, including non-homologous end joining repair (NHEJ), which is more error-prone, and homology-directed repair (HR), which is mediated by a donor DNA template that can be used to insert a guide cassette.

[0121] Several technologies exist to enable customized site-specific DSB generation within genomes. Many of these involve the use of customized endonucleases, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), or clustered regularly interspaced short palindromic repeats / CRISPR-associated protein (CRISPR / Cas9) systems (Gaj, T et al., "ZFN, TALEN, and CRISPR / Cas-based methods for genome engineering," Trends Biotechnol, 31:397-405, July 2013).

[0122] Zinc finger nucleases are artificial enzymes created by fusing the nuclease domain of the restriction enzyme FokI with a zinc finger DNA-binding domain. The latter has a nonspecific cleavage domain that must dimerize to cleave DNA. This means that two ZFN monomers are required to allow the FokI domain to dimerize and cleave DNA. The DNA-binding domain can be designed to target any genomic sequence of interest and is a tandem array of Cys2His2 zinc fingers, each of which recognizes three adjacent nucleotides in the target sequence. The two binding sites are separated by 5–7 bp to allow optimal dimerization of the FokI domain. Thus, the enzyme can cleave DNA at specific sites, and target specificity is increased by ensuring that two adjacent DNA-binding events must occur to achieve a double-stranded break.

[0123] Transcription activator-like effector nucleases, or TALENs, are dimeric transcription factors / nucleases. They are created by fusing a TAL effector DNA-binding domain to a DNA-cleavage domain (nuclease). Transcription activator-like effectors (TALEs) can be engineered to bind virtually any desired DNA sequence, and when combined with a nuclease, DNA can be cleaved at specific locations. TAL effectors are proteins secreted by Xanthomonas bacteria, and their DNA-binding domains contain highly conserved 33-34 amino acid repeats that differ at the 12th and 13th amino acids. These two positions are highly variable and show a strong correlation with specific nucleotide recognition. This direct relationship between amino acid sequence and DNA recognition allows the engineering of specific DNA-binding domains by selecting combinations of repeat segments containing appropriate residues at the two variable positions. TALENs are therefore constructed from an array of 33-35 amino acid modules, each of which targets a single nucleotide. By selecting an array of modules, almost any sequence can be targeted. Again, the nuclease used can be FokI or a derivative thereof.

[0124] Three types of CRISPR machinery have been identified, of which Type II is the most studied. The CRISPR / Cas9 system (Type II) utilizes the Cas9 nuclease to create double-stranded breaks in DNA at sites determined by a short guide RNA. The CRISPR / Cas system is a prokaryotic immune system that confers resistance to exogenous genetic elements. CRISPRs are segments of prokaryotic DNA containing short repeats of a base sequence. Each repeat is followed by a short segment of "protospacer DNA" from prior exposure to the exogenous genetic element. The CRISPR spacer recognizes and cleaves the exogenous genetic element using RNA interference. The CRISPR immune response occurs through two steps: CRISPR-RNA (crRNA) biogenesis and crRNA-induced interference. The crRNA molecule consists of a variable sequence and CRISPR repeats transcribed from the protospacer DNA. Each crRNA molecule then hybridizes with a second RNA known as a trans-activating CRISPR RNA (tracrRNA), and together, these two ultimately form a complex with the nuclease Cas9. The portion of the crRNA encoded by the protospacer DNA directs Cas9 to cleave the complementary target DNA sequence when it is adjacent to a short sequence known as a protospacer adjacent motif (PAM). This natural system has been engineered and utilized to introduce DSB breaks at specific sites in genomic DNA, among many other applications. In particular, the CRISPR type II system from Streptococcus pyogenes can be used. In its simplest form, the CRISPR / Cas9 system contains two components that are delivered into cells to provide genome editing: the Cas9 nuclease itself and a small guide RNA (gRNA). The gRNA is a fusion of a customized site-specific crRNA (directed to the target sequence) and a standardized tracrRNA.

[0125] After a DSB has occurred, a donor template with homology to the target locus is provided and the DSB can be repaired by the homology-directed repair (HDR) pathway, which allows precise insertion to be made.

[0126] Derivatives of this system are also possible. Mutant forms of Cas9, such as Cas9D10A, which only have nickase activity, are also available. This means that it only cleaves one DNA strand and does not activate NHEJ. Instead, when a homologous repair template is provided, DNA repair occurs exclusively through the high-fidelity HDR pathway. Cas9D10A (Cong L. et al., (2013) Science, 339, 819-823) is used in a pair of Cas9 complexes designed to create adjacent DNA nicks with two sgRNAs complementary to adjacent regions on opposite strands of the target site, which may be particularly advantageous.

[0127] The elements for generating double-stranded DNA breaks can be introduced into one or more vectors, such as plasmids, for expression in cells.

[0128] Thus, any method for creating a specific targeted double-stranded break in the genome for inserting a gene / inducer cassette can be used in the methods of the present invention. Preferably, the method for inserting the gene / inducer cassette utilizes any one or more of the ZFN, TALEN and / or CRISPR / Cas9 systems or any derivatives thereof.

[0129] After a DSB has been created by any suitable means, the gene / induction cassette for insertion can be provided in any suitable manner, as described below. The gene / induction cassette and associated genetic material form the donor DNA for DNA repair at the DSB and are inserted using standard cellular repair mechanisms / pathways. The manner in which the break is initiated will depend on which pathway is used for damage repair, as described above.

[0130] The transcriptional regulatory protein and the induction cassette can be provided on separate vectors for the methods of the present invention. A "vector" is a nucleic acid molecule, such as a DNA molecule, used as a vehicle for artificially transporting genetic material into cells. A vector is generally a nucleic acid sequence consisting of an insert (such as an induction cassette or a transcriptional regulatory protein gene) and a larger sequence that serves as the "backbone" of the vector. The vector can be in any suitable configuration, including a plasmid, minicircle, or linear DNA. The vector contains at least a transcriptional regulator gene or an induction cassette operably linked to an inducible promoter, along with minimal sequences to allow insertion of the gene into the relevant GSH. Optionally, the vector also possesses an origin of replication (ori) that allows amplification of the vector, for example, in bacteria. Additionally or alternatively, the vector can contain a selectable marker, such as an antibiotic resistance gene, a coloration marker gene, and a suicide gene.

[0131] Examples of vectors used in the examples are illustrated in Figures 20 to 33.

[0132] The cells used in the method of the present invention can be any human or animal cells.Preferably, they are mammalian cells, such as rodents such as mice and rats; marsupials such as kangaroos and koalas; non-human primates such as bonobos, chimpanzees, lemurs, gibbons and apes; camelids such as camels and llamas; livestock animals such as horses, pigs, cows, buffalo, bison, goats, sheep, deer, reindeer, donkeys, banteng, yaks, chickens, ducks and turkeys; domesticated animals such as cats, dogs, rabbits and guinea pigs.These cells are preferably human cells.In certain embodiments, the cells are preferably derived from livestock animals.

[0133] The type of cells used in the methods of the present invention will depend on the application of the cells once insertion of genetic material into the GSH site is complete.

[0134] When the goal is to generate mature cell types from progenitor cells, the cells to be modified are stem cells, preferably pluripotent stem cells. Pluripotent stem cells have the ability to differentiate into almost any cell type in the body. There are several sources of pluripotent stem cells. Embryonic stem cells (ES cells) are pluripotent stem cells derived from the inner cell mass of the blastocyst, an early preimplantation embryo. Induced pluripotent stem cells (iPSCs) are adult cells genetically reprogrammed to an embryonic stem cell-like state by forcing them to express genes and factors important for maintaining the defining characteristics of embryonic stem cells. In 2006, it was shown that the introduction of four specific genes encoding transcription factors can convert adult cells into pluripotent stem cells (Takahashi, K; Yamanaka, S (2006), Cell 126 (4): 663-76), but subsequent studies have reduced / altered the number of genes required. Several members of the Oct-3 / 4 and Sox gene families have been identified as potentially important transcriptional regulators involved in the induction process. Additional genes, including some members of the Klf family, Myc family, Nanog, and LIN28, may improve induction efficiency. Examples of genes that may be contained in the reprogramming factors include Oct3 / 4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, ERas, ECAT15-2, Tcl1, beta-catenin, Lin28b, Sall1, Sall4, Esrrb, Nr5a2, Tbx3, and Glis1, and these reprogramming factors may be used alone or in combination of two or more thereof.

[0135] If the goal is to generate stem cells with a knocked-down or knocked-out gene for further study, such as developmental or gene function studies, the modified cells can be stem cells, preferably pluripotent stem cells, or mature cell types. Sources of pluripotent stem cells are described above.

[0136] When cells modified by the insertion of an induction cassette are used in a human patient, it may be preferable that the cells are iPSCs derived from that individual. The use of such autologous cells obviates the need to match cells to the recipient. Alternatively, commercially available iPSCs, such as those available from WiCell™ (WiCell Research Institute, Inc., Wisconsin, US), can be used. Alternatively, the cells can be tissue-specific stem cells, which can be autologous or donated. Suitable cells include epiblast stem cells, induced neural stem cells, and other tissue-specific stem cells.

[0137] In certain embodiments, the cells used may preferably be embryonic stem cells or stem cell lines. Numerous embryonic stem cell lines are now available, for example, WA01 (H1) and WA09 (H9) are available from WiCell, and KhES-1, KhES-2, and KhES-3 are available from the Institute for Frontier Medical Sciences, Kyoto University (Kyoto, Japan).

[0138] Because such techniques are readily available, it may be preferable to derive embryonic stem cells without destroying the embryo, particularly when the cells are human (Chung, Young et al., Cell Stem Cell, Vol. 2, Issue 2, 113-117). Stem cell lines derived without destroying the embryo are also available. In one aspect, the present invention does not extend to methods involving the destruction of a human embryo.

[0139] A preferred embodiment of the present invention is the forward programming of pluripotent stem cells into mature cell types. Thus, the methods of the present invention can be used to produce mature cell types from pluripotent stem cells. As mentioned above, in this embodiment of the present invention, the induction cassette for insertion into the second GSH is preferably one or more master regulators. These induction cassettes can enable cells to be programmed into a specific lineage, and a different induction cassette is used to direct differentiation into mature cell types. Any type of mature cell is contemplated, including, but not limited to, nerve cells, muscle cells, bone cells, chondrocytes, epithelial cells, secretory cells, and / or blood cells.

[0140] The inventors of the present application have developed a rapid, efficient, and scalable method for generating virtually any mature cell type. Such a simple, inexpensive method has particular value for regenerative medicine. Previous forward programming techniques utilize the Tet-On system, but attempts have been made to include all materials in one vector / site (all-in-one Tet-On) or to insert the induction cassette into one AAVS1 allele and the regulatory system into the other AAVS1 allele (DeKelver et al., 2010, Genome Res., 20, 1133-43 and Qian et al., 2014, Stem Cells, 32, 1230-8). Surprisingly, the dual GSH targeting method developed and described herein has many unexpected advantages. There is no potential promoter interference between the gene inserted into the first GSH and the gene sequence of the induction cassette inserted into the second GSH. Second, it allows for the insertion of larger cargo from the vector, as less material needs to be inserted at each site. Third, it maximizes the number of copies safely inserted. Fourth, it allows for greater design flexibility. Finally, it allows for additional genetic material to be inserted, including reporter genes and miRNA switches. The method of the present invention has been demonstrated to be a robust and efficient method for producing mature cells from pluripotent cells.

[0141] Once a gene is inserted into the first GSH and an induction cassette containing the transgene is inserted into the second GSH, the pluripotent stem cells can be cultured to allow forward programming to occur. These culture conditions can be specific to the type of pluripotent stem cell used or can depend on the final mature cell type. Whatever culture conditions are used, exogenous substances can be continuously supplied to control the expression of the gene sequence in the induction cassette and induce transcription, and then removed, or can be supplied when transcription is required, depending on its mode of action, as described above.

[0142] If the goal is to program stem cells, it may be advantageous to provide the cells with extracellular stimuli to support differentiation in conjunction with the delivery of an induction cassette encoding a master regulator. Cell reprogramming strategies can be enhanced by combining extracellular signaling cues with overexpression of master regulators or transcription factors. Thus, it may be possible to systematically screen for differentiation-promoting factors by modulating key signaling cascades involved in the development of that particular mature cell type. An example of this is shown in Example 3.

[0143] In one aspect, the present invention provides a method for generating muscle cells from pluripotent stem cells, comprising: a) targeted insertion of a transcriptional regulatory protein-encoding gene into a first gene safe harbor site; and b) targeted insertion of the MYOD1 gene operably linked to an inducible promoter into a second gene safe harbor site, wherein the inducible promoter is regulated by the transcriptional regulatory protein; wherein the first and second genetic safe harbor sites are different; The method further comprises culturing the cells in the presence of retinoic acid.

[0144] The MYOD1 gene is a gene encoding myogenic differentiation 1 protein. Preferably, the retinoic acid (RA) is all-trans RA.

[0145] In another aspect, the present invention provides a method for generating muscle cells from pluripotent stem cells that express MYOD1, the method comprising culturing the cells in the presence of retinoic acid.

[0146] Preferably, the RA is all-trans RA. Preferably, the cells overexpress MYOD1.

[0147] In a further aspect, the present invention provides a method for generating oligodendrocyte muscle cells from pluripotent stem cells, comprising the steps of: a) targeted insertion of a transcriptional regulatory protein-encoding gene into a first gene safe harbor site; and b) targeted insertion of a SOX10 gene operably linked to an inducible promoter into a second gene safe harbor site, wherein the inducible promoter is regulated by the transcriptional regulatory protein; wherein the first and second genetic safe harbor sites are different; The method further comprises culturing the cells in the presence of retinoic acid.

[0148] The cells used for this purpose may be animal or human cells. If the cells are animal, the animal is preferably a domestic animal as defined above.

[0149] The SOX 10 gene encodes the transcription factor SOX 10. Preferably, the retinoic acid (RA) is all-trans RA.

[0150] The cells used in the methods of the present invention are pluripotent, and the resulting cells can be lineage-restricted specific stem cells, progenitor cells, or mature cell types with desired properties due to the expression of master regulatory factors. These lineage-specific stem cells, progenitor cells, or mature cells can be used in any appropriate manner. For example, the mature cells can be used directly for transplantation into the human or animal body, depending on the cell type. Alternatively, the cells can serve as test materials for research involving the effects of drugs on gene expression and the interaction of specific genes with drugs. Cells for research can involve the use of induction cassettes carrying gene sequences of unknown function to investigate the controllable expression of the gene sequence. Furthermore, this can enable the cells to be used to produce large quantities of desired materials, such as growth factors or cytokines.

[0151] In a different embodiment, the cells can be used in tissue engineering. Tissue engineering involves the creation of tissues that can be used to replace human or animal tissues or even entire organs. Tissue engineering methods, known to those skilled in the art, include the use of scaffolds (extracellular matrices) onto which cells are applied to create tissues / organs. These methods can be used to create "artificial" tracheas, bladders, livers, pancreases, stomachs, intestines, blood vessels, cardiac tissue, bone, bone marrow, mucosal tissue, nerves, muscles, skin, kidneys, or any other tissue or organ. Alternatively, methods of creating tissues may involve additive manufacturing, also known as three-dimensional (3D) printing, which may involve directly printing cells to create tissues. Accordingly, the present invention provides methods of creating tissues using cells generated as described in any embodiment of the present invention.

[0152] Tissues produced using cells produced according to the methods of the present invention can be used for transplantation into the human or animal body. Alternatively, if the cells are derived from an animal, the tissue can be used for in vitro / cultured meat. The primary cell type for cultured meat is myocytes. However, such tissues can involve the use of a combination of cell types produced according to the methods of the present invention. These can be myocytes (muscle cells), vascular cells, blood cells, and adipocytes (fat cells). If the purpose of the engineered tissue is for cultured meat, the cells can be harvested from livestock animals.

[0153] The methods of the present invention may also be performed on cells that are not pluripotent stem cells for a variety of reasons, including research, gene therapy, including genetic vaccines, generation of in vitro disease models, and generation of non-human in vivo models.

[0154] Therefore, the cells used in the methods of the present invention can be any type of adult stem cell. These are undifferentiated cells that can develop into many, but not all, cell types. Adult stem cells are undifferentiated cells found throughout the body that divide to replace dead cells and regenerate damaged tissues. Also known as somatic stem cells, they are not pluripotent. Adult stem cells have been identified in many organs and tissues, including the brain, bone marrow, peripheral blood, blood vessels, skeletal muscle, skin, teeth, heart, intestine, liver, ovarian epithelium, and testes. To identify somatic stem cells, one skilled in the art must demonstrate that a single adult stem cell can generate genetically identical cell lines that give rise to all appropriate differentiated cell types of the tissue. To experimentally confirm that a putative adult stem cell is indeed a stem cell, the cell must give rise to these genetically identical cells in culture, or a purified population of these cells must repopulate the tissue after transplantation into an animal. Suitable cell types include, but are not limited to, neural, mesenchymal, and endodermal stem and progenitor cells.

[0155] Alternatively, the cells used can be mature cell types. Such cells are differentiated and specialized and cannot develop into different cell types. Mature cell types include, but are not limited to, nerve cells, muscle cells, bone cells, chondrocytes, epithelial cells, secretory cells, and / or blood cells. Mature cell types can be any cell derived from the human or animal body.

[0156] Somatic stem cells and mature cell types can be modified according to the present invention and then used in applications such as gene therapy or gene vaccination. Gene therapy can be defined as the intentional insertion of foreign DNA into a cell nucleus for therapeutic purposes. Such definition includes the provision of a gene or genes to a cell to provide a wild-type version of a defective gene, the addition of an RNA molecule (which may be defective) that interferes with the expression of a target gene, suicide genes (such as the enzymes herpes simplex virus thymidine kinase (HSV-tk) and cytosine deaminase (CD) that convert the harmless prodrug ganciclovir (GCV) into a cytotoxic drug), the provision of DNA vaccines (including adoptive immunotherapy) for immune or cancer therapy, and the provision of any other gene to a cell for therapeutic purposes.

[0157] Typically, the method of the present invention can be used to insert desired gene sequence for intracellular transcription, preferably expression, particularly in DNA vaccine.DNA vaccine typically encodes modified form of DNA of infectious organism.DNA vaccine is administered to subject expressing selected protein of infectious organism, and typically initiates immune response against protein that is protective.DNA vaccine can also encode tumor antigen in cancer immunotherapy approach.

[0158] DNA vaccines may contain nucleic acid sequences encoding antigens for the treatment or prevention of several conditions, including, but not limited to, cancer, allergies, virulence, and infections caused by pathogens such as, but not limited to, fungi, human papillomavirus (HPV), HIV, HSV2 / HSV1, influenza viruses (types A, B, and C), poliovirus, RSV, rhinovirus, rotavirus, hepatitis A virus, measles virus, parainfluenza virus, mumps virus, varicella-zoster virus, cytomegalovirus, Epstein-Barr virus, adenovirus, rubella virus, human T-cell lymphoma virus type I (HTLV-I), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus, poxvirus, Zika virus, Marburg virus, and Ebola; bacteria, including Neisseria meningitidis, Haemophilus influenzae (type b); and parasitic pathogens. DNA vaccines can comprise nucleic acid sequences encoding antigens from any suitable pathogen, which can be derived from a pathogen involved in human or animal disease, and in particular from a viral pathogen.

[0159] The DNA vaccine inserted into GSH may also contain a nucleic acid sequence encoding a tumor antigen. Examples of tumor-associated antigens include, but are not limited to, cancer antigens such as members of the MAGE family (e.g., MAGE 1, 2, 3), NY-ESO-I and SSX-2, differentiation antigens such as tyrosinase, gplOO, PSA, Her-2 and CEA, mutated autoantigens, and viral tumor antigens such as E6 and / or E7 from oncogenic HPV types. Further examples of specific tumor antigens include MART-I, Melan-A, p97, beta-HCG, GaINAc, MAGE-I, MAGE-2, MAGE-4, MAGE-12, MUCl, MUC2, MUC3, MUC4, MUC18, CEA, DDC, PIA, EpCam, melanoma antigen gp75, Hker 8, high molecular weight melanoma antigen, Kl 9, Tyrl, Tyr2, members of the pMel 17 gene family, c-Met, PSM (prostate mucin antigen), PSMA (prostate specific membrane antigen), prostate secretory protein, alpha-fetoprotein, CA 125, CA 19.9, TAG-72, BRCA-I and BRCA-2 antigens.

[0160] The inserted gene sequence can generate other types of therapeutic DNA molecules. For example, such DNA molecules can be used to express functional genes in subjects with genetic disorders caused by dysfunctional versions of those genes. Examples of such disorders include Duchenne muscular dystrophy, cystic fibrosis, Gaucher disease, and adenosine deaminase (ADA) deficiency. Other diseases for which gene therapy may be useful include inflammatory, autoimmune, chronic, and infectious diseases, including AIDS, cancer, neurological disorders, cardiovascular disease, hypercholesterolemia, various anemias, thalassemia, and hemophilia, and various blood disorders including emphysema. For the treatment of solid tumors, genes encoding toxic peptides (i.e., chemotherapeutic agents such as ricin, diphtheria toxin, and cobra venom factor), tumor suppressor genes such as p53, genes encoding mRNA sequences that are antisense to transforming oncogenes, antitumor peptides such as tumor necrosis factor (TNF) and other cytokines, or transdominant-negative mutants of transforming oncogenes can be expressed.

[0161] Other types of therapeutic DNA molecules are also contemplated. For example, DNA molecules that are transcribed into active non-coding RNA forms, such as small interfering RNA (siRNA), can be inserted. Thus, the methods of the present invention encompass methods for knocking down the expression of endogenous genes or knocking out endogenous genes using non-coding RNA in an induction cassette.

[0162] Thus, the method of the present invention can be used to specifically and stably insert gene sequences into an induction cassette that can be controllably transcribed. This has numerous advantages in somatic stem cells and mature cell types. It allows for a more tightly controlled gene therapy approach, ensuring that important genes are not disrupted and allowing the expression of the induction cassette to be turned off if side effects occur. It also allows for tightly regulated knockdown or knockout of endogenous genes to investigate gene function and development.

[0163] The present invention also encompasses cells produced by the methods of the present invention. The cells can be defined as modified with a first genomic safe harbor site for containing a transcriptional regulatory protein and a second genomic safe harbor site for containing a gene sequence operably linked to an inducible promoter regulated by the transcriptional regulatory protein. The two GSHs are different and distinct. Preferably, the cells are homozygous at both insertion sites. All elements are as described above.

[0164] Cells produced according to any of the methods of the invention have application in diagnostic and therapeutic methods. The cells may be used in vitro to study cell development, provide test systems for new drugs, enable the development of screening methods, refine treatment regimens, provide diagnostic tests, and the like. These uses form part of the present invention. Alternatively, the cells may be transplanted into a human or animal patient for diagnostic or therapeutic purposes. The use of cells in therapy is also included in the present invention. The cells may be allogeneic (i.e., mature cells removed, modified, and returned to the same individual) or derived from a donor (including stem cell lines).

[0165] All documents mentioned herein are incorporated herein by reference.

[0166] array AAVS1 - NCBI GenBank S51329.1 SEQ ID NO: 1: Tet02 19n sequence SEQ ID NO: 2: hROSA insertion site genomic sequence SEQ ID NO: 3: STDtetR-nls (nucleotide) and SEQ ID NO: 4: STDtetR-nls (amino acid) SEQ ID NO: 5: OPTtetR-nls (nucleotide) and SEQ ID NO: 6: OPTtetR-nls (amino acid) SEQ ID NOs: 7 to 80: Primers from Table 3 SEQ ID NO: 81: Figure 18B AAVS1 FWD; SEQ ID NO: 82: Figure 18B AAVS1 REV SEQ ID NO: 83: Figure 18B Tracer FWD; SEQ ID NO: 84: Figure 18B Tracer REV SEQ ID NO: 85: Figure 19E HI POL3 FWD; SEQ ID NO: 82: Figure 19E HI POL3 REV

[0167] This is the genomic sequence of the hROSA26 insertion site. It includes the 5' homology arm, the cleavage site (bold), and the 3' homology arm: (SEQ ID NO: 2). [ka]

[0168] STDtetR-nls (SEQ ID NOs: 3 and 4) Nucleotide and amino acid sequences of the tetracycline sensitivity inhibitor protein (tetR) containing the N-terminal SV40 nuclear localization signal (nls, highlighted in gray). Sequences are reported either before or after codon optimization (STDtetR and OPTtetR, respectively). Dots indicate synonymous mutations introduced into OPTtetR. [ka]

[0169] Optimized tetR: OPTtetR-nls (SEQ ID NOs: 5 and 6) [ka]

[0170] The invention will now be described with reference to the following non-limiting examples.

[0171] Example Materials and methods used in the examples: hPSC maintenance culture and germ layer differentiation hESCs (H9 line; WiCell) and hiPSCs (Cheung et al., Nat. Biotechnol. 30, 165-173 (2012)) were cultured in a feeder-free and serum-free environment. Briefly, cells were seeded onto gelatin / MEF medium-coated culture dishes [MEF medium consisted of Advanced DMEM / F12 (90%, Gibco), fetal bovine serum (10%, Gibco), L-glutamine (1 mM, Gibco), 2-mercaptoethanol (0.1 mM, Sigma-Aldrich), and penicillin / streptomycin (1%, Gibco)] supplemented with 10 ng / ml activin A and 12 ng / ml FGF2 in a chemically defined medium [CDM, IMDM (50%, Gibco), F12 (50%, Gibco), concentrated lipids (100x, Gibco), monothioglycerol (450 μM, Sigma-Aldrich), insulin (7 μg / ml, Roche), transferrin (15 μg / ml, The cells were cultured in a medium containing 1000kJ / ml of 1000kcal of 1000kJ / ml of 1000kcal of bovine serum albumin fraction V (Roche), bovine serum albumin fraction V (5 mg / ml), and penicillin / streptomycin (1%). Cells were passaged in small clumps using collagenase every 5–6 days.

[0172] Differentiation of hPSCs into germ layers was induced in adherent hESC cultures according to previously published directed differentiation protocols for endoderm, lateral plate mesoderm, and neuroectoderm (Touboul, T. et al., Hepatology 51, 1754-1765 (2010), Cheung et al., (2012) and Douvaras, P. et al., Stem Cell Reports 3, 250-259 (2014)). Briefly, definitive endoderm was obtained by culturing hPSCs for 3 days in CDM-PVA (without insulin) supplemented with FGF2 (20 ng / ml), activin A (100 ng / ml), BMP4 (10 ng / ml, Marko Hyvonen, Dept. of Biochemistry, University of Cambridge), and LY-294002 (10 μM, Promega). For neuroectoderm induction, SB-431542 (10 μM, Tocris), LDN-193189 (0.1 μM, Tocris), and RA (0.1 μM, hPSCs were cultured for 6 days in CDM-BSA supplemented with FGF2 (20 ng / ml), 10 ng / ml BMP4 (R&D), and LY294002 (10 μM). 4. Lateral plate mesoderm was obtained by culturing hPSCs for 36 hours in CDM-PVA supplemented with FGF2 (20 ng / ml), 10 ng / ml BMP4 (R&D), and LY294002 (10 μM), followed 3.5 days later by culturing them in CDM-PVA supplemented with FGF2 (20 ng / ml) and BMP4 (50 ng / ml).

[0173] hESC differentiation Differentiation was initiated in adherent cultures of hESCs 48 hours after passage. Medium changes were typically performed daily and adjusted for cell density. Mature cell types were obtained using methods previously described in the art. The resulting mature cell types included neurons, osteocytes, chondrocytes, smooth muscle, cardiac fibroblasts, cardiomyocytes, intestine, pancreas, hepatocytes, bile duct, and lung.

[0174] Gene targeting constructs and molecular cloning The design and construction of hROSA26 gRNA and Cas9n expression plasmids are described herein: A CRISPR / Cas9n-based strategy for specifically targeting the hROSA26 locus and inserting a guided cassette using homologous recombination. To induce a genomic DSB at the correct integration site, a CRISPR / Cas9 nickase system was designed. In contrast to the commonly used wild-type Cas9 nuclease, which is guided to its genomic target site by a single gRNA, the D10A mutant Cas9 nickase (Cas9n) is directed by a pair of appropriately designed gRNAs to simultaneously introduce single-strand breaks on both strands of the target DNA. This strategy increases specificity by effectively doubling the number of bases required for genome editing. Potential target sites for the crRNA-guided nuclease near the integration site were defined using the web-based software "CRISPR Design Tool." Within a 250 bp sequence stretch surrounding the target site (125 bp on each of the actual integration sites), the top hits were pairs of gRNAs that collectively achieved a "high quality" score of 97, which predicted no targeting effect. [ka] (PAM site in parentheses) were synthesized de novo and ligated into expression vectors. The final plasmids encode one of the two gRNAs and the Cas9n D10A mutant, respectively (Figures 20 and 21).

[0175] A donor plasmid was constructed to serve as template DNA to promote homology-directed repair of Cas9n-induced DSBs. Two hROSA26 homology arms were generated by high-fidelity PCR amplification. Genomic DNA isolated from H9 hESCs served as the template. The 5' and 3' homology arms were 904 bp and 869 bp in length, respectively. Both arms were then inserted into the multiple cloning site of the pUC19 vector. To target the hROSA26 locus, cells were transfected with the two gRNA / Cas9n constructs in the plasmid and the EGFP donor plasmid (Figure 22).

[0176] The pR26_CAG-rtTA targeting vector (Figure 23) was constructed by cloning the coding sequence of third-generation rtTA (PCR-amplified from pLVX-Tet3G) into the BamHI / MluI sites of pR26_CAG-EGFP, thus replacing the EGFP sequence. The AAVS1 ZFN expression plasmid was a generous gift from Dr. Kosuke Yusa (Wellcome-Trust Sanger Institute). The inducible EGFP AAVS1 targeting vector was constructed by Gibson Assembly (New England Biolabs), in which three inserts were ligated into the EcoRI / HindIII sites of the multiple cloning site of the pUC19 vector (Thermo Fisher Scientific). The first insert contained an upstream AAVS1 homology arm, splice acceptor, T2A site, and puromycin resistance cassette (PCR amplified from pTRE-EGFP; addgene 22074, deposited by Rudolf Jaenisch). The second insert contained an inducible TRE3G promoter (PCR amplified from pLVX-TRE3G). The third insert contained an EGFP expression cassette and an AAVS1 downstream homology arm (PCR amplified from pTRE-EGFP; addgene 22074, deposited by Rudolf Jaenisch). The resulting plasmid was designated pAAV_TRE-EGFP (Figure 32). The pAAV_TRE-NGN2 and pAAV_TRE-MYOD1 (Figure 33) targeting vectors were constructed by cloning the NGN2 and MYOD1 coding sequences, respectively (NGN2: PCR amplified from pLVX-TRE-NGN2, a gift from Oliver Brustle; MYOD1: PCR amplified from a commercially available cDNA plasmid (Open Biosystems MHS6278-202832821, Accession: BC064493, Clone ID: 5022419)) into the SpeI / EcoRI sites of pAAV_TRE-EGFP, thus replacing the EGFP sequence.

[0177] Additional plasmids were generated using similar methods, and all plasmids used are shown in Figures 20-33. These plasmids were either generated or generously donated. Plasmids used in the examples included (in the order of Figures 20-33): pSpCas9n(BB),_R26-R, pSpCas9n(BB) (the combination of these two plasmids is predicted to induce a specific double-strand break in the intron between exons 1 and 2 of THUMPDS3-AS1 (ROSA26 locus) on chromosome 3), R26-L, and pSpCas9n(BB). pR26_CAG_EGFP, pR26_CAG_rtTA, pZFN-AAVS1-L-ELD (zinc finger nuclease left), pZFN-AAVS1-R-KKR (zinc finger nuclease right), pAAV_CAG_EGFP (donor), pR26-Neo_CAG-OPTtetR (codon-optimized tetR targeting hROSA26), pAAV-Puro_iKD (inducible shRNA targeting AAVS1), pAAV-Neo_CAG-Cas9 (Cas9 targeting AAVS1), pAAV-Puro_siKO (inducible gRNA targeting AAVS1), pAAV-Puro_siKO-2TO (inducible gRNA targeting AAVS1, version with two tet operons in the promoter), pAAV_TRE-EGFP (EGFP-inducible overexpression, attached) and pAAV_TRE-MYOD1 (MYOD1-inducible overexpression for muscle).

[0178] Gene targeting Targeting of the hROSA26 locus and AAVS1 for gene knockdown and knockout was performed by nucleofection. Human pluripotent stem cells (PSCs) were dissociated into single cells using TrypLE Select (Gibco) and 2 × 10 6The cells were nucleofected using the Lonza P3 Primary Cell 4D-Nucleofector X Kit and cycle CA-137 of the Lonza 4D Nucleofector System (100 μl reaction volume; 12 μg total DNA, divided equally between the two gRNA / Cas9n plasmids and the targeting vector). Nucleofected hPSCs were plated onto irradiated multidrug-resistant (DR4) mouse embryonic fibroblasts and cultured in KSR medium (Advanced DMEM / F12 (80%), Knockout Serum Replacement (20%, Gibco), L-glutamine (1 mM), 2-mercaptoethanol (0.1 mM), and penicillin / streptomycin (1%)) supplemented with FGF2 (4 ng / ml, Department of Biochemistry, University of Cambridge). Y-27632 (5 μM, Tocris) was added 24 hours before and after nucleofection to promote cell survival. After 3–6 days, neomycin-resistant hPSCs were selected by adding G418 (50 μg / ml, Sigma-Aldrich) for 7–10 days. Individual clones were then picked, expanded in feeder-free conditions, and finally analyzed by genotyping.

[0179] Targeting of the AAVS1 locus was also performed by lipofection. Human PSCs were seeded in six-well plates under feeder-free conditions and transfected 48 hours after passage. Transfection was performed for 24 hours with a total of 4 μg of DNA (divided equally between the two AAVS1 ZFN plasmids and the targeting vector) in Opti-MEM (Gibco) supplemented with Lipofectamine 2000 (10 μl / well, Thermo Fisher Scientific). After 3–5 days, resistant hPSCs were selected by adding puromycin (1 μg / ml, Sigma-Aldrich) for 5–8 days. Individual clones were then picked, expanded, and analyzed by genotyping. Antibiotic resistance can be used to select clonal lines.

[0180] Drug-resistant hPSC clones from the targeting experiments were screened by genomic PCR to verify site-specific induced cassette integration, determine the number of targeted alleles, and exclude off-target integrations. PCR was performed using LongAmp Taq DNA polymerase (New England Biolabs). Table 2 reports the primer combinations used for the various targeting vectors. The results of all targeting experiments are summarized in Table 1. Karyotyping was performed by standard G-banding techniques (Medical Genetics Service, Cambridge University Hospitals). To prepare targeted human PSCs for karyotyping analysis, cells were incubated at +37°C for 4 hours in fresh culture medium supplemented with Y-27632 (5 μM, Tocris) and KaryoMAX Colcemid (100 ng / ml, Gibco). Cells were then harvested as single cells, washed, and pelleted. Nuclear swelling and chromosome spreading were achieved by treatment with hypotonic 0.055 M KCl solution for 5–10 min. Finally, cells were fixed with methanol and glacial acetic acid (ratio 3:1).

[0181] For OPTiKD, AAVS1 targeting was performed by lipofection as previously described. Briefly, hPSCs were seeded into feeder-free 6-well plates and transfected with 4 μg of DNA (divided equally between the two AAVS1 ZFN plasmids and the targeting vector) for 24 hours, 48 ​​hours after cell passage, using 10 μl of Lipofectamine 2000 per well in Opti-MEM medium (Gibco), all according to the manufacturer's instructions. After 4 days, 1 μg / ml puromycin was added to the medium, and individual clones were picked and expanded after 7–10 days of selection.

[0182] For single-site OPTiKO, AAVS1 targeting was performed by nucleofection. hESCs were pretreated with 10 μM Y-27632 (Tocris) for 16 h according to the manufacturer's instructions. They were dissociated into clumps of 2–8 cells using Accutase (Gibco) and transfected with 2 × 10 6 The cells were nucleofected with a total of 12 μg of DNA (4 μg each for the two ZFN plasmids and 2 μg each for the two targeting vectors) in 100 μl using the Lonza P3 Primary Cell 4D Nucleofector X Kit and Cycle CA-137 of the Lonza 4D Nucleofector System. Nucleofected hESCs were plated onto a feeder layer of irradiated DR4 (puromycin- and neomycin-resistant) mouse embryonic fibroblasts and cultured in KSR medium supplemented with 4 ng / ml FGF2 and 10 μM Y-27632 (only for the first 24 h). After 4 days, hPSC colonies carrying both the puromycin and neomycin resistance genes were selected with 25 μg / ml Geneticin (G418 sulfate, Gibco) and 0.5 μg / ml puromycin for 7–10 days. Individual clones were then picked and grown in feeder-free conditions.

[0183] AAVS1-EGFP, ROSA26-EGFP, ROSA26-STDtetR, ROSA26-OPTtetR, and ROSA26-EGFPd2 hESCs were generated by lipofection (AAVS1 locus) or nucleofection (ROSA26 locus) of targeting vectors carrying the AAVS1 ZFN or ROSA26 CRISPR / Cas9n pair (described above). 2 μg / ml Blasticidin S-HCl (Gibco) was used for the pR26-Bsd_CAG-EGFPd2 plasmid. The generation of inducible EGFP-overexpressing hESCs carrying the ROSA26-rtTA and AAVS1-TRE-EGFP transgenes has been described elsewhere. Briefly, cells were first sequentially gene targeted by nucleofection of pR26-Neo_CAG-rtTA with the ROSA26 CRISPR / Cas9n plasmid and then by lipofection of pAAVPuro_TRE-EGFP with the AAVS1 ZFN plasmid.

[0184] Gene-targeted hPSC clonal lines were screened by genomic PCR to confirm site-specific targeting, determine the number of targeted alleles, and rule out off-target integration of the targeting plasmid (see Figure 16A).

[0185] Overexpression of inducible cassettes Overexpression of the inducible cassettes (EGFP, NGN2, MYOD1, and OLIG2-SOX10, respectively) was induced by adding doxycycline hydrate (Sigma-Aldrich) to the culture medium. Unless otherwise noted, doxycycline was used at a final concentration of 1 μg / ml. The doxycycline-containing medium was replaced every 24 hours protected from light. EGFP-expressing cells are referred to herein as OPTi-EGFP, NGN2-expressing cells as OPTi-NGN2, MYOD1-expressing cells as OPTi-MYOD1, and OLIG2-SOX10-expressing cells as OPTi-OLIG2-SOX10.

[0186] Inducible gene knockout and knockdown Unless otherwise stated in the figure legends or examples, tetracycline hydrochloride (Sigma-Aldrich) was used at 1 μg / ml to induce gene knockdown or knockout. Neuronal induction. Pluripotent OPTi-NGN2 cells were dissociated into single cells with TrypLE and plated on Matrigel (35 μg / cm) at a density of 75,000 cells per well of a 12-well plate. 2 Cells were seeded onto (Scientific Laboratory Supplies) coated dishes. Forward programming was initiated 24–48 h after splitting. Unless otherwise noted, induction was performed in DMEM / F12 (Gibco) supplemented with GlutaMAX (100x, Gibco), non-essential amino acids (100x, Gibco), 2-mercaptoethanol (50 μM), penicillin / streptomycin (1%), and doxycycline (1 μg / ml). Two days after induction, the medium was switched to Neurobasal medium supplemented with GlutaMAX (100x), B27 (50x, Gibco), BDNF (10 ng / ml, Peprotech), NT3 (10 ng / ml, R&D Systems), penicillin / streptomycin (1%), and doxycycline (1 μg / ml).

[0187] Skeletal muscle cell induction Pluripotent OPTi-MYOD1 cells were dissociated into single cells using TrypLE and seeded onto gelatin / MEF medium-coated dishes at a density of 100,000 cells per well of a 12-well plate. Forward programming was initiated 24–48 h after splitting. Unless otherwise noted, induction was performed in DMEM (Sigma-Aldrich) supplemented with L-glutamine (2 mM), 2-mercaptoethanol (50 μM), penicillin / streptomycin (1%), insulin (7 μg / ml), all-trans retinoic acid (1 μM, Sigma-Aldrich), and doxycycline (1 μg / ml). Five days after induction, the medium was supplemented with CHIR99021 (3 μM, Tocris) and heat-inactivated horse serum (2%, Gibco) to enhance maturation.

[0188] Oligodendrocyte induction Pluripotent OLIG2-2A-SOX10 OPTi-OX hPSCs were grown in colonies on gelatin / MEF-coated culture dishes. They were treated with SB and LDN overnight before the start of induction. The next day, induction began in CDM supplemented with doxycycline (1 μg / ml) and RA (0.1 μM). One day after induction, cells were split onto PDL / laminin-coated culture dishes (100,000 cells per well of a 12-well plate) in CDM supplemented with RA (0.1 μM), PM (1 μM), and Y-27632 (5 μM), PDGFaa (20 ng / ml, Peprotech), and FGF2 (5 ng / ml). The next day, cells were switched to oligodendrocyte medium consisting of DMEM / F12 supplemented with Glutamax (100x), non-essential amino acids (100x), 2-mercaptoethanol (1000x), penicillin-streptomycin (100x), N2 supplement (100x), B27 supplement (50x), insulin 7 μg / ml (Marko Hyvonnen), T3 60 ng / ml (Sigma), biotin 100 ng / ml (Sigma), and db-cAMP 1 μM (Sigma). Oligodendrocyte medium was supplemented with dox (1 μg / ml), PDGFaa (20 ng / ml), FGF2 (5 ng / ml), RA (0.1 μM), and PM (1 μM). After 7 days of induction, RA and PM were removed. To maintain the induced cells in a proliferative state, cells were passaged every 4 days (75,000 cells per well of a 24-well plate) in the continuous presence of mitogens PDGFaa and FGF2. PDGFaa and FGF2 were removed for differentiation of proliferative oligodendrocyte precursors. Human recombinant NT3 (5 ng / μl, R&D Systems) was added to enhance cell survival.

[0189] Quantitative real-time PCR (qPCR) RNA was extracted using the GenElute Mammalian Total RNA Miniprep Kit and On-Column DNAse I Digestion Set (Sigma-Aldrich). cDNA synthesis was performed using the Maxima First Strand cDNA Synthesis Kit (Thermo Fisher Scientific). Applied Biosystems SYBR Green PCR Master Mix was used for qPCR. Samples were run on an Applied Biosystems 7500 Fast PCR machine. All samples were analyzed in technical duplicates and normalized to the housekeeping gene porphobilinogen deaminase 1 (PBGD). Results were analyzed using the ΔΔCt method. See Table 3 for primer sequences.

[0190] Flow cytometry For analysis of EGFP-expressing cells, cells were harvested using TrypLE Select (Gibco) at 37°C for 5–10 minutes to obtain a single-cell suspension. After washing with PBS, cells were resuspended in ice-cold PBS supplemented with DAPI (10 μg / ml) and incubated on ice for 5 minutes. Cells were analyzed using a cyan ADP flow cytometer to determine the level of EGFP expression in live cells (DAPI-negative). For staining and analysis of myosin heavy chain expression, cells were harvested using TrypLE Select (for EGFP expression analysis), washed once with PBS, fixed, and permeabilized with Cytofix / Cytoperm solution (BD Biosciences). Cells were then washed and blocked overnight at +4°C with Perm / Wash buffer (BD Biosciences) supplemented with 3% bovine serum albumin (BSA). Staining with a PE-conjugated anti-MYH antibody (Table 4) was performed in Perm / Wash buffer in the dark at +4°C for 1 hour. After washing three times with Perm / wash buffer, cells were analyzed on a Cyan ADP flow cytometer to determine the level of MHC expression. Data analysis was performed using FlowJo (v10) and GraphPad Prism (v6).

[0191] Western blot Total cellular proteins were extracted with CelLytic M (Sigma-Aldrich) supplemented with Complete Protease Inhibitor (Roche) and then quantified using the Protein Quantitation Kit-Rapid (Sigma-Aldrich). Protein electrophoresis was performed using NuPAGE LDS sample buffer and 4-12% NuPAGE Bis-Tris precast gels (Invitrogen). After protein transfer onto PVDF, the membrane was blocked for 1 hour at room temperature with PBS supplemented with 0.05% Tween-20 (PBST), 4% milk, and then incubated overnight with primary antibodies in PBST / 4% milk. The membrane was washed with PBST, incubated with HRP-conjugated secondary antibodies (Sigma-Aldrich) in PBST / 4% milk, incubated with Pierce ECL2 Western Blotting Substrate (Thermo Fisher Scientific), and exposed to X-ray Super RX film (Fujifilm).

[0192] immunocytochemistry Cells were fixed with 4% paraformaldehyde (diluted in PBS) at room temperature for 20 minutes and then washed three times with PBS. Cells were then blocked with 10% donkey serum (Sigma-Aldrich) and permeabilized with 0.3% Triton X-100 (diluted in PBS) at room temperature for 20 minutes. Cells were then incubated overnight at 4°C with appropriate dilutions of primary antibodies in 2% donkey serum and 0.1% Triton X-100 (diluted in PBS) (Supplemental Experimental Procedures). When staining for the surface antigens PDGFRA, A2B5, and O4, Triton X was omitted at all steps. After washing three times with PBS, cells were incubated with the corresponding donkey fluorophore-conjugated secondary antibodies (Alexa Fluor 488, 555, 568, and / or 647) in PBS supplemented with 1% donkey serum for 1 hour at room temperature. Nuclei were visualized using 4',6-diamidino-2-phenylindole (DAPI, Thermo Fisher Scientific). EGFP expression and immunostaining were imaged using a Zeiss LSM 700 confocal microscope (Leica). The percentage of βIII-tubulin-positive cells was calculated by determining βIII-tubulin expression in at least 50 randomly selected DAPI-positive cells within three fields of three biological replicates using an inverted Olympus IX71 fluorescence microscope.

[0193] Statistical analysis was performed using GraphPad Prism (v6). The number of replicates, statistical tests used, and test results are described in the figure legends. Data are presented as mean ± SEM unless otherwise stated.

[0194] Example 1: Dual targeting of EGFP To develop an inducible overexpression platform in hPSCs, we sequentially targeted two components of the Tet-ON system to two different GSH loci. Constitutively expressing third-generation rtTA was targeted to the human ROSA26 (hROSA26) locus using a CRISPR / Cas9n-based targeting strategy, and an inducible EGFP induction cassette was inserted into AAVS1 (Figure 1a; Figure 4a-c). Targeting of both hROSA26 and AAVS1 was highly efficient (Figure 4d-f, Table 1) and did not affect hPSC genome stability, self-renewal, or differentiation (data not shown), thus not counteracting rtTA-dependent cytotoxicity.

[0195] Next, we selected double GSH-targeted clones carrying either one or two copies of each of the two inducible cassettes (Figure 5a). Homozygous targeting of rtTA resulted in approximately twofold higher levels of rtTA protein (Figure 5b) and also significantly increased EGFP levels after induction compared to heterozygous rtTA expression (Figures 5c-5e). Furthermore, clones with homozygous targeting of the inducible EGFP cassette exhibited higher and more uniform EGFP levels compared to strains with heterozygous targeting (Figures 5c-5e). Importantly, all correctly targeted strains exhibited robust inducible EGFP expression, at least 20-fold higher than that of the strong constitutive CAG promoter (Figure 1b, Figures 5c-e). Collectively, these results support our initial hypothesis that targeting two copies of both elements of the Tet-ON system results in maximal expression after induction. Peak EGFP levels were reached approximately 4 days after induction, and expression quickly reversed upon removal of doxycycline (Figure 1c). Furthermore, EGFP expression could be titrated by adjusting the dose of doxycycline (Figure 1d). Importantly, inducible EGFP expression was not only highly efficient in hPSCs but also during differentiation into germ layers (color photographs not shown; data in Figures 6a–6d). Finally, consistent with the known tight transcriptional control of the third-generation Tet-ON system, there was no detectable background expression of EGFP mRNA or protein in the absence of doxycycline, as determined by flow cytometry and qPCR, respectively (Figure 1b, Figure 6d). Overall, these results establish that dual GSH targeting of the Tet-ON system is a powerful strategy for optimal expression of inducible cassettes in hPSCs and their derivatives.

[0196] Example 2: Induction of excitatory cortical neurons from hESCs and hiPSCs Previous studies have shown that these cells can be easily induced by lentiviral overexpression of any of the proneuronal bHLH factors (ASCL1, NGN2, or NEUROD1) in hPSCs. Therefore, we generated OPTi-NGN2 hPSCs (Figure 2a, Table 1). NGN2 induction resulted in rapid downregulation of pluripotency factors (Figure 7) and initiation of a neuronal transcriptional program (Figure 2b). As early as 3 days after induction, induced cells exhibited neurites (data not shown). After 1 week, all cells displayed neuronal morphology and expressed pan-neuronal marker proteins such as βIII-tubulin and MAP2 (Figure 2c). Quantitative RT-PCR revealed robust induction of typical forebrain markers such as BRN2 and FOXG1, as well as glutamatergic neurons containing GRIA4 and VGLUT2, indicative of excitatory cortical neuron identity (Figure 2b). Collectively, these results demonstrated dramatic improvements in both the speed and efficiency of neuronal generation compared to conventional hPSC differentiation protocols, as well as substantial increases in efficiency and purity for both transdifferentiation and lentiviral-based forward programming protocols. Similar results were obtained with OPTi-NGN2 hiPSCs, confirming the robustness of this method. Finally, we did not observe any decline in the efficiency of neural induction over extended culture periods of Opti-NGN2 hPSCs (more than 25 passages, Figure 2c). Overall, our results demonstrate that OPTi-NGN2 hPSCs can be used as an inexhaustible source for unlimited, scalable, rapid, single-step, virus-free, and near-definitive generation of neurons.

[0197] Example 3: Creation of skeletal muscle cells The transcription factor MYOD1 is known to induce myogenic transdifferentiation when overexpressed in various somatic cell types, but the ability of hPSCs to undergo MYOD1-induced myogenic forward programming is currently debated. We generated OPTi-MYOD1 hPSCs (Table 1) but noticed that induction of MYOD1 expression after doxycycline treatment resulted in nearly complete cell death within 3–5 days across a wide range of culture conditions previously suggested to promote hPSC conversion to skeletal muscle cells. Since it is widely established that cell reprogramming strategies can be improved by combining extracellular signaling cues with transcription factor overexpression, we conducted a systematic screen for pro-myogenic factors by modulating key signaling cascades involved in primitive streak formation, somitogenesis, and myogenesis. We found that the addition of all-trans retinoic acid (RA) in conjunction with MYOD1 overexpression resulted in rapid and nearly complete conversion to myogenin and myosin heavy chain (MHC) double-positive myocytes by 5 days after induction. The effect of RA was concentration-dependent and mediated through the RA receptor isoforms RARα and RARβ, consistent with the expression pattern of RA receptors during developmental myogenesis (Figure 8). This effect appears to be independent of the mechanism of MYOD1 overexpression. The induced skeletal muscle cells exhibited a typical spindle-shaped, elongated morphology, underwent extensive cell fusion, and showed strong myogenic marker expression at both the mRNA and protein levels (Figure 3b, Figures 9a-9c). Addition of nanomolar concentrations of acetylcholine (ACh) or the selective ACh receptor agonist carbachol resulted in complete myofiber contraction, demonstrating the functionality of the induced myocytes. Similar results were obtained with Opti-MYOD1 hiPSCs (data not shown). Importantly, myogenic induction efficiency did not decrease over extended culture periods (more than 50 passages, Figure 3d), thus demonstrating the robustness and reproducibility of this method. Finally, we noticed that the level of the MYOD1 induction cassette positively correlated with conversion efficiency, highlighting the importance of robust gene delivery and the superiority of this method over lentivirus-mediated reprogramming approaches (Figure 10).Overall, the OPTi-MYOD1 forward programming strategy is approximately 7 times faster and 5 times more efficient than current protocols for differentiating hPSCs into skeletal muscle cells. Compared to previous forward programming protocols (Tanaka, A. et al., PLoS One 8, e61540 (2013) and Abujarour, R. et al., Stem Cells Transl. Med. 3, 149-60 (2014)), it is more efficient (>95% vs. 30-80%), does not contain randomly inserted induction cassettes, is chemically defined, fully reproducible, and more scalable.

[0198] These findings demonstrate that this method of controlling inducible cassette expression in hPSCs can be used as an inexhaustible source for the high-throughput, large-scale production of homogenous cell populations. The speed of induction and purity of the desired target cells is currently unmatched by other methods.

[0199] Example 4: Generation of oligodendrocyte precursors and oligodendrocytes: OPTi-OX hPSCs harboring inducible SOX10, either alone or in combination with OLIG2 in the form of a bicistronic expression cassette. Cells induced with SOX10 alone robustly expressed the oligodendrocyte precursor (OPC) marker O4 10 days after induction, but these cells failed to further differentiate into myelin-expressing cells and gradually died. In contrast, OLIG2-SOX10 double-overexpressing cells readily progressed from the O4-positive progenitor stage to a mature CNP / MBP-positive phenotype 20 days after induction. Further marker protein expression analysis confirmed that OPTi-OLIG2-SOX10 hPSCs induced in oligodendrocyte medium supplemented with mitogens PDGFaa and FGF2 (Douvaras et al., 2014) initially passed through an OPC-like stage, where they were highly proliferative and co-expressed PDGFRA, A2B5, and O4. These cells were highly proliferative and could be maintained for at least three passages by culturing in the presence of mitogens (Figure 12b). Therefore, we named these cells i-OPCs, for induced OPCs. Notably, after mitogen withdrawal and in the continued presence of doxycycline, i-OPCs readily differentiated into mature oligodendrocytes capable of myelin sheath formation (data not shown) and expressing the major myelin proteins CNP, PLP, MAG, MOG, and MBP in approximately 1 week (Figures 12c-12d). Collectively, these results demonstrate that the present invention enables the development of a novel, robust, and rapid hPSC forward programming protocol for the generation of oligodendrocyte precursors and oligodendrocytes.

[0200] Table 1: Summary of genotyping results [Table 1] (a) Incomplete targeting: no evidence of targeting (absence of bands in 5' and 3'-integration PCR and presence of a WT band in locus PCR) or evidence of targeting but incomplete size in 5'- or 3'-integration PCR (b) Correct, targeted integration (3'-backbone PCR band) with further random integration of the plasmid. (c) Correct, on-target integration (HET, heterozygous; HOM, homozygous) (d) Percentage of clones with correct on-target integration (without further off-target integration) (e) Percentage of clones with correct on-target integration (with or without additional off-target integrations) *The three figures are from three different targeting experiments in hESCs.

[0201] Table 2: List of primers used for genotyping PCR. [Table 2]

[0202] Table 3: List of primers for quantitative PCR [Table 3] TIFF0007748174000009.tif236170TIFF0007748174000010.tif42170

[0203] Table 4: List of antibodies [Table 4] TIFF0007748174000012.tif134170

[0204] Example 5: TET-ON inducible knockdown system Development of an optimized inducible knockdown platform in hPSCs We generated hESC lines in which the EGFP transgene could be silenced in an inducible manner (Figure 14B). To do so, we targeted (1) a CAG-tetR expression cassette to the ROSA26 locus; and (2) a CAG-EGFP transgene plus an inducible EGFP shRNA cassette to the AAVS1 locus (Figures 14A and 14B). In the absence of tetracycline, expressing higher levels of tetR protein resulted in stronger suppression of shRNA expression. To this end, we performed multiparameter RNA and codon optimization of the bacterial tetR cDNA and used the resulting codon-optimized tetR (OPTtetR) to generate new EGFP-inducible knockdown hESC lines (Figure 14B). This modification enabled a 10-fold increase in tetR expression compared to the standard sequence (STDtetR; Figure 14D). Furthermore, homozygous expression of OPTtetR was sufficient to completely prevent shRNA leakage while maintaining efficient knockdown induction (Figure 14C). Notably, inducible knockdown was rapid, reversible, and dose-responsive (Figure 14E, F). Finally, inducible hESCs exhibited normal karyotypes (data not shown), demonstrating that the genome engineering required to generate these lines did not alter genetic stability.

[0205] Based on these promising results, we further validated this method in the context of endogenous genes by generating hESCs harboring inducible shRNAs against POU5F1 / OCT4 or B2M (data not shown). Remarkably, all analyzed sublines (six for each gene) showed robust inducible knockdown without significant shRNA leakage. Tetracycline titration identified the optimal concentration for partial or complete knockdown of OCT4. As expected, severe reduction of OCT4 specifically resulted in loss of pluripotency and induction of neuroectoderm and definitive endoderm markers. Similar results were obtained with 20 additional OCT4-inducible knockdown hESC sublines, confirming the robustness and reproducibility of this method. Importantly, the generation of hESCs with potent and tightly regulated knockdown was efficient enough to allow immediate phenotypic analysis after antibiotic selection in a mixed population of cells, thereby completely avoiding the need to pick individual colonies for clonal isolation. Overall, these results demonstrate that dual targeting of GSH with an optimized inducible knockdown system is a powerful method for controlling gene expression in hPSCs, an approach hereafter referred to as OPTiKD, for optimized inducible knockdown (Figure 14A).

[0206] Example 6 The ability to knockdown genes in a variety of differentiated cells is a significant advance over previous systems for inducible gene knockdown. To thoroughly test this potential, we analyzed the efficacy of the OPTiKD platform to knockdown the EGFP transgene in hPSCs differentiated into three germ layers, as well as in a panel of 13 fully differentiated cell types (Figure 15A). For both methods, qPCR analysis demonstrated potent and inducible knockdown of EGFP transcripts in all lineages tested (Figure 17). Microscopic observation confirmed a robust reduction in EGFP protein expression, and flow cytometry demonstrated a reduction in EGFP fluorescence of over 70% for most lineages (data not shown).

[0207] Example 7 Development of an optimized inducible CRISPR / Cas9 knockout platform in hPSCs We turned our attention to developing an inducible knockout approach. Current inducible CRISPR / Cas9 methods rely on conditional overexpression of Cas9 in the presence of a constitutively expressed gRNA. In this case, control of Cas9 overexpression is achieved through a TET-ON approach, in which the tetracycline-regulated reverse transactivator (rtTA) activates the Pol II-dependent tetracycline response element (TRE) promoter (a fusion of multiple TET operons and a minimal CMV promoter) after doxycycline treatment. While this TET-ON platform has been successfully applied to specific human cell types, we observed that this inducible system was silenced during differentiation of hPSCs into multiple lineages (including cardiomyocytes, hepatocytes, and smooth muscle cells) even after targeting the AAVS1 GSH (data not shown). We explored the possibility of developing another improved method by combining an inducible gRNA cassette based on that developed for inducible shRNA expression with a constitutively expressed CAG promoter-driven Cas9 (Figure 18A, B). Therefore, we generated hESC lines in which a fluorescent reporter gene could be knocked out in an inducible manner (Figure 18C). To this end, we targeted ROSA26-EGFPd2 reporter hESCs with both an inducible EGFP gRNA and a constitutive Cas9 into the AAVS1 locus, where each transgene is integrated into one of the two alleles. This dual-targeting approach was rapid (less than two weeks) and efficient (more than 90% of lines contained both transgenes). Remarkably, when individual clonal sublines were grown in the presence of tetracycline, we observed a decrease in EGFPd2 expression in all of the target lines, with EGFPd2 homozygous cells exhibiting near-homogeneous loss of at least one copy of the reporter gene (as indicated by a 50% decrease in EGFPd2 fluorescence) as early as 5 days after tetracycline induction. Long-term treatment with tetracycline resulted in a progressive complete loss of EGFPd2 fluorescence in up to 75% of EGFPd2 homozygous cells (data not shown).Interestingly, coexpression of either two or three copies of the same EGFP gRNA cassette from the same AAVS1 locus was sufficient to significantly improve the speed and efficiency of inducible EGFPd2 knockout in all analyzed clonal sublines. For example, coinduction of three copies of the same gRNA resulted in an exceptional 95% knockout efficiency after tetracycline treatment. Importantly, inducible EGFPd2 knockout hESCs showed no significant decrease in the percentage of EGFPd2-positive cells or their fluorescence after long-term culture in the absence of tetracycline, even when several gRNA copies were used. This demonstrated that inducible gRNA expression was tightly controlled. Finally, testing of additional gRNAs against EGFPd2 revealed that the speed and efficiency of inducible knockout were strongly gRNA-dependent. Indeed, the optimal sequence enabled up to 90% knockout after just two days of induction. Notably, the most efficient gRNA also resulted in uncontrolled EGFPd2 knockout, but this limitation was circumvented by simply adding a second TET operon to the inducible H1 promoter to ensure even more stringent transcriptional control. Collectively, these results demonstrate that the knockdown system can be easily repurposed to support inducible gRNA expression and enable tightly controlled RISPR / Cas9 activity over a wide range of gRNA efficacies. To our knowledge, this is the first conditional CRISPR / Cas9 approach based on inducible gRNA expression. The present application provides the following aspects of the invention. (Aspect 1) 1. A method for regulating transcription of a gene sequence in a cell, comprising: a) targeted insertion of a transcriptional regulatory protein-encoding gene into a first gene safe harbor site; and b) targeted insertion of an inducible cassette into a second genetic safe harbor site, said inducible cassette comprising said genetic sequence operably linked to an inducible promoter, said promoter being regulated by said transcriptional regulatory protein; wherein the first and second genetic safe harbor sites are different. (Aspect 2) 2. The method of embodiment 1, wherein the genetic sequence is a transgene. (Aspect 3) 2. The method of embodiment 1, wherein the gene sequence encodes a non-coding RNA. (Aspect 4) The method according to any one of aspects 1 to 3, wherein the activity of the transcriptional regulatory protein is controlled by an externally supplied substance. (Aspect 5) The method according to any one of aspects 1 to 4, wherein the transcriptional regulatory protein is constitutively expressed. (Aspect 6) 6. The method of any one of aspects 1 to 5, wherein the transcriptional regulatory protein is selected from any one of tetracycline-responsive transcriptional activator protein (rtTa), tetracycline repressor (TetR), VgEcR synthetic receptor or gene switch hybrid transcriptional regulatory protein, and any derivative thereof. (Aspect 7) The method according to any one of aspects 1 to 6, wherein the transcriptional regulatory protein is rtTA or any derivative thereof. (Aspect 8) 6. The method of embodiment 5, wherein the activity of said tTA is regulated by tetracycline or a derivative thereof, optionally doxycycline. (Aspect 9)

[0022] Embodiment 9. The method of embodiment 7 or embodiment 8, wherein the inducible promoter comprises a Tet response element (TRE). (Aspect 10) 10. The method of any one of aspects 1 to 9, wherein the first and second genomic safe harbor sites are selected from any two of the hROSA26 locus, the AAVS1 locus, the CLYBL gene, the CCR5 gene, or the HPRT gene. (Aspect 11) 11. The method of any one of embodiments 1 to 10, wherein insertion of the transcriptional regulatory protein-encoding gene into a first genetic safe harbor site occurs on both chromosomes of the cell, and / or insertion of the induction cassette into a second genetic safe harbor site occurs on both chromosomes of the cell. (Aspect 12) Additional genetic material is inserted into the first and / or second genomic safe harbor sites, optionally including: a) suicide gene; b) selection marker; c) a reporter gene; and / or d) Non-coding RNA genes The method according to any one of embodiments 1 to 11. (Aspect 13) 13. The method of any one of embodiments 1 to 12, wherein the method is performed ex vivo. (Aspect 14) The method according to any one of aspects 1 to 13, wherein the cells are selected from pluripotent stem cells, somatic stem cells, or mature cells. (Aspect 15) 3. The method of embodiment 1 or embodiment 2, wherein said method is for programming pluripotent stem cells into defined mature cells. (Aspect 16) 3. The method of embodiment 1 or embodiment 2, wherein said cell is a pluripotent stem cell and said genetic sequence is a transgene for one or more master regulators, optionally a transcription factor. (Aspect 17) 20. The method of embodiment 16, wherein transcription of said gene sequence results in forward programming of said cell to a defined mature cell type. (Aspect 18) 20. The method of embodiment 17, wherein said mature cell type is selected from any one of the following cell types: nerve cells, muscle cells, bone cells, chondrocytes, epithelial cells, secretory cells, and / or blood cells. (Aspect 19) 19. The method of any one of aspects 14 to 18, wherein the pluripotent stem cells are selected from induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs). (Aspect 20) The method of any one of embodiments 1 to 19, wherein the cell is human. (Aspect 21) 2. The method of embodiment 1, wherein the method is for insertion of a genetic sequence for gene therapy purposes, and optionally the genetic sequence encodes a protein comprising a wild-type protein, a variant protein, an antigen, an enzyme, a selectable marker, or a non-coding RNA molecule. (Aspect 22) A cell produced by the method of embodiment 1. (Aspect 23) A cell having a modified genome comprising an inducible cassette comprising a transcriptional regulatory protein-encoding gene inserted into a first gene safe harbor site; and a gene sequence operably linked to an inducible promoter inserted into a second gene safe harbor site, wherein the inducible promoter is regulated by the transcriptional regulatory protein, and the first and second sites are different. (Aspect 24) 24. Use of a cell according to embodiment 22 or embodiment 23 in therapy. (Aspect 25) 24. Use of a cell according to embodiment 22 or 23 for in vitro diagnosis. (Aspect 26) 24. Use of the cells of embodiment 22 or 23 for tissue engineering, optionally cultivated meat. (Aspect 27) 1. A method for generating muscle cells from pluripotent stem cells, comprising: a) targeted insertion of a transcriptional regulatory protein-encoding gene into a first gene safe harbor site; and b) targeted insertion of the MYOD1 gene operably linked to an inducible promoter into a second gene safe harbor site, wherein the inducible promoter is regulated by the transcriptional regulatory protein; wherein the first and second genetic safe harbor sites are different; The method comprising culturing the cells in the presence of retinoic acid. (Aspect 28) 1. A method for generating oligodendrocytes from pluripotent stem cells, comprising: a) targeted insertion of a transcriptional regulatory protein-encoding gene into a first gene safe harbor site; and b) targeted insertion of a SOX10 gene operably linked to an inducible promoter into a second gene safe harbor site, wherein the inducible promoter is regulated by a transcriptional regulatory protein. wherein the first and second genetic safe harbor sites are different; The method comprising culturing the cells in the presence of retinoic acid. (Aspect 29) 1. A method for reducing the transcription and / or translation of an endogenous gene in a cell, comprising: a) targeted insertion of a transcriptional regulatory protein-encoding gene into a first gene safe harbor site; and b) targeted insertion of an inducible cassette into a second genetic safe harbor site, said inducible cassette comprising DNA encoding a non-coding RNA sequence operably linked to an inducible promoter, said promoter being regulated by a transcriptional regulatory protein, and said non-coding RNA sequence repressing the transcription or translation of an endogenous gene; Including, The method, wherein the first and second genetic safe harbor sites are different. (Aspect 30) A method for knocking out an endogenous gene in a cell, a) targeted insertion of a transcriptional regulatory protein-encoding gene and a Cas9-encoding gene into a first gene safe harbor site; and b) targeted insertion of an inducible cassette into a second genetic safe harbor site, said inducible cassette comprising a guide RNA operably linked to an inducible promoter, said promoter being regulated by said transcriptional regulatory protein, and said gRNA sequence targeting an endogenous gene; Including, The method, wherein the first and second genetic safe harbor sites are different. (Aspect 31) 30. The method of any one of aspects 1 to 21 or 27 to 29, wherein a further induction cassette or transgene is inserted into a further GSH that is different from said first and second GSH. (Aspect 32) Optimized tetR sequences including sequences having 80%, 85%, 90%, 95% or 99% homology to SEQ ID NO:6. (Aspect 33) 1. A method for generating muscle cells from pluripotent stem cells that express MYOD1, the method comprising culturing the cells in the presence of retinoic acid.

Claims

1. 1. A method for regulating transcription of a gene sequence in a cell ex vivo, comprising: a) targeted insertion of a transcriptional regulatory protein-encoding gene into a first gene safe harbor site; and b) targeted insertion of an inducible cassette into a second genetic safe harbor site, said inducible cassette comprising said genetic sequence operably linked to an inducible promoter, said promoter being regulated by said transcriptional regulatory protein; Including, the first and second genetic safe harbor sites are different; the gene sequence is a transgene encoding a protein; the cells are pluripotent stem cells, and The method, wherein the method is for forward programming of the pluripotent stem cells.

2. The ex vivo method of claim 1 , wherein the activity of the transcriptional regulatory protein is controlled by an exogenously supplied substance.

3. 3. The ex vivo method of claim 1 or claim 2, wherein the transcriptional regulatory protein is constitutively expressed.

4. 4. The ex vivo method of claim 1, wherein the transcriptional regulatory protein is selected from any one of tetracycline-responsive transcriptional activator protein (rtTa), tetracycline repressor (TetR), VgEcR synthetic receptor, or a hybrid transcriptional regulatory protein comprising the DNA-binding domain of yeast GAL4 protein, a truncated ligand-binding domain of human progesterone receptor, and the activation domain of human NF-κB.

5. The ex vivo method according to any one of claims 1 to 4, wherein the transcriptional regulatory protein is rtTA or a modified rtTA protein thereof.

6. The activity of the rtTA is regulated by tetracycline or a derivative thereof; and 6. The ex vivo method of claim 4 or claim 5, wherein the inducible promoter comprises a Tet response element (TRE).

7. 7. The ex vivo method of claim 6, wherein the derivative of tetracycline is doxycycline.

8. 8. The ex vivo method of any one of claims 1 to 7, wherein the first and second genetic safe harbor sites are selected from any two of the hROSA26 locus, the AAVS1 locus, the CLYBL gene, or the CCR5 gene.

9. 9. The ex vivo method of any one of claims 1 to 8, wherein the insertion of the transcriptional regulatory protein-encoding gene into the first gene safe harbor site occurs on both chromosomes of the cell.

10. 10. The ex vivo method of any one of claims 1 to 9, wherein insertion of said induction cassette into the second genetic safe harbor site occurs on both chromosomes of said cell.

11. Additional genetic material is inserted into the first genetic safe harbor site, the second genetic safe harbor site, or both the first genetic safe harbor site and the second genetic safe harbor site, and is one of the following: a) suicide gene; b) selection marker; c) a reporter gene; and d) Non-coding RNA genes 11. The ex vivo method according to any one of claims 1 to 10, wherein the method is selected from one or more of:

12. 12. The ex vivo method of any one of claims 1 to 11, wherein the cells are human, marsupial, non-human primate, camelid, or domestic animal cells.

13. 10. The ex vivo method of claim 1, wherein said method is for programming pluripotent stem cells into defined mature cells.

14. 2. The ex vivo method of claim 1, wherein the cells are pluripotent stem cells and the genetic sequence comprises at least one transgene for one or more master regulatory factors.

15. 15. The ex vivo method of claim 14, wherein said one or more master regulatory factors are transcription factors.

16. 16. The ex vivo method of claim 14 or claim 15, wherein transcription of said gene sequence results in forward programming of said cell to a defined mature cell type.

17. 17. The ex vivo method of claim 16, wherein the mature cell type is selected from any one of the following cell types: nerve cells, muscle cells, bone cells, chondrocytes, epithelial cells, secretory cells, and blood cells.

18. 18. The ex vivo method of any one of claims 1 to 17, wherein said pluripotent stem cells are selected from induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs).

19. A cell having a modified genome comprising an inducible cassette comprising: a transcriptional regulatory protein-encoding gene inserted into a first genetic safe harbor site; and one or more introduced genes encoding one or more proteins suitable for forward programming of pluripotent stem cells operably linked to an inducible promoter inserted into a second genetic safe harbor site, wherein the inducible promoter is regulated by the transcriptional regulatory protein, and the first and second sites are different.

20. 20. A pharmaceutical composition comprising the cells of claim 19 for use in therapy.

21. 20. A pharmaceutical composition comprising the cells of claim 19 for use in in vitro diagnosis.

22. Use of the cells described in claim 19 for cultivated meat.

23. 1. A method for generating muscle cells from pluripotent stem cells ex vivo, comprising: a) targeted insertion of a transcriptional regulatory protein-encoding gene into a first gene safe harbor site; and b) targeted insertion of the MYOD1 gene operably linked to an inducible promoter into a second gene safe harbor site, wherein the inducible promoter is regulated by the transcriptional regulatory protein; Including, the first and second genetic safe harbor sites are different; The method comprising culturing the cells in the presence of retinoic acid.

24. 1. A method for generating oligodendrocytes from pluripotent stem cells ex vivo, comprising: a) targeted insertion of a transcriptional regulatory protein-encoding gene into a first gene safe harbor site; and b) targeted insertion of a SOX10 gene operably linked to an inducible promoter into a second gene safe harbor site, wherein the inducible promoter is regulated by the transcriptional regulatory protein. Including, the first and second genetic safe harbor sites are different; The method comprising culturing the cells in the presence of retinoic acid.

25. 25. The ex vivo method of any one of claims 1 to 18, 23 or 24, wherein a further inducible cassette or transgene is inserted into a further GSH that is different from said first and second gene safe harbor (GSH).

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