PRECURSOR CELLS

FIELD OF THE INVENTION

[001] The present invention relates to a method for expression of a transgene of interest from neural precursor cells (NPCs) comprising the steps of: a) Providing neural precursor cells; b1) i) targeted insertion of a nucleotide sequence encoding a transcriptional regulator protein being under the control of a suitable promoter into a first genomic location; and ii) targeted insertion of a nucleotide sequence encoding a transgene of interest into a second genomic location, wherein the transgene of interest is operably linked to a suitable promoter, which is regulated by the transcriptional regulator protein; or b2) targeted insertion of the nucleotide sequence encoding a transgene of interest into a genomic location, wherein the transgene of interest is operably linked to a constitutive promoter, and c) expression of the transgene of interest. Further is provided a system for expression of a transgene of interest from neural precursor cells. Additionally, the present invention is directed to a method for expression of a transgene of interest from stem cells.

BACKGROUND OF THE INVENTION

[002] Human pluripotent stem cells (hPSCs; including human embryonic stem cells (hESCs, [1]) and human induced pluripotent stem cells (hiPSCs, [2])) provide unlimited opportunities for biomedical research and therapeutic applications. One of the key promises of stem cell technologies is the in vitro production of bulk quantities of specific human cell types with bona fide properties for biomedical research or therapeutic interventions.

[003] Robust methods for constitutive and inducible transgene expression in hPSCs are required to exploit the full research and therapeutic potential of hPSCs. Therefore, the inventors of the present invention have developed a powerful dual genomic safe harbour (GSH)-based targeting system for transgene expression in hPSCs [3,4], Moreover, the inventors have applied their transgene targeting system for the highly efficient generation of a variety of cell types from hPSCs by forward programming using safely targeted inducible expression of reprogramming factors [3], This strategy outperforms previous strategies for the generation of specific cell types, that were based on classical developmental differentiation or forward programming using virus-mediated transgene-delivery [5,6], [004] However, an unresolved issue and important bottleneck of said forward programming approach towards high-throughput and large screen applications are some of the characteristics related to hPSC maintenance cultures: They are pricy, subject to variability, and resource intensive. Additionally, the necessity for dual GSH-targeting in hPSCs to circumvent transgenesilencing of the Tet-ON system hinders the generation of robust reporter expression in starting and target cell populations, due to blockade of the two most commonly used GSH sites.

[005] Previously, robust protocols for the generation of multipotent neural precursor cells from hPSCs using solely small molecule combinations have been established (smNPCs, [7]). The derivation and maintenance of smNPCs has already been published [7] and is summarized in Figure 1. The inventors of the present invention have investigated smNPCs as expandable, without limits, cell population for controllable transcription by inserting transgenes into defined GSHs. To this aim, the inventors have developed a GSH-targeted controllable transcription system to smNPCs and utilized this for efficient constitutive or inducible reporter expression and for rapid and deterministic programming of smNPCs into, e.g., cortical neurons.

[006] The present invention aims at and addresses these needs described above.

SUMMARY OF THE INVENTION

[007] The above mentioned problems are solved by the subject-matter as defined in the claims and as defined herein.

[008] The inventors of the present invention have developed a method for expression of a transgene of interest from neural precursor cells (NPCs).

[009] Thus, the present invention relates to a method for expression of a transgene of interest from neural precursor cells (NPCs), comprising the steps of: a) Providing neural precursor cells; b1) i) targeted insertion of a nucleotide sequence encoding a transcriptional regulator protein being under the control of a suitable promoter, preferably a constitutive promoter, into a first genomic location; and ii) targeted insertion of a nucleotide sequence encoding a transgene of interest into a second genomic location, wherein the transgene of interest is operably linked to a suitable promoter, preferably an inducible promoter, which is regulated by the transcriptional regulator protein; or b2) targeted insertion of the nucleotide sequence encoding a transgene of interest into a genomic location, preferably a genomic safe harbour site, wherein the transgene of interest is operably linked to a constitutive promoter, and c) expression of the transgene of interest.

[0010] In one embodiment of the method for expression of a transgene of interest from neural precursor cells (NPCs), the transcriptional regulator protein of b1) i) is the reverse tetracycline transactivator (rtTA) and the activity thereof is controlled by doxycycline, tetracycline or a derivative thereof.

[0011] In one further embodiment of the method for expression of a transgene of interest from neural precursor cells (NPCs), the suitable promoter of b1) ii) is an inducible promoter, preferably an inducible promoter including a Tet Responsive Element (TRE), more preferably a Tet Responsive Element (TRE) comprising or consisting of SEQ ID NO: 1.

[0012] In one embodiment of the method for expression of a transgene of interest from neural precursor cells (NPCs), the suitable promoter of b1) i) is a constitutive promoter, preferably a constitutive CAG promoter, more preferably a constitutive CAG promoter comprising or consisting of the SEQ ID NO: 2.

[0013] In one further embodiment of said method of the present invention, the constitutive promoter of b2) is a constitutive CAG promoter, more preferably a constitutive CAG promoter comprising or consisting of the SEQ ID NO: 2.

[0014] In one further embodiment of said method of the present invention, the first genomic location of b1) i) is a first genomic safe harbour site.

[0015] In one embodiment of said method of the present invention, the second genomic location of b1) ii) is a second genomic safe harbour site.

[0016] In one further embodiment of said method of the present invention, the first and the second genomic locations of b1), preferably the first and second genomic safe harbour sites of b1), are the same.

[0017] In one embodiment of said method of the present invention, the first and the second genomic locations of b1), preferably the first and second genomic safe harbour sites of b1), are different.

[0018] In one embodiment of said method of the present invention, step c) further comprises culturing the neural precursor cells under suitable conditions.

[0019] In one embodiment of said method of the present invention, the targeted insertion of the nucleotide sequence encoding the transcriptional regulator protein into the first genomic location of b1) i) and the targeted insertion of the nucleotide sequence encoding the transgene of interest into the second genomic location of b1) ii) is done in inverse directions.

[0020] In one further embodiment of said method of the present invention, the transgene of interest encodes the enhanced green fluorescence protein (EGFP).

[0021] In one embodiment of said method of the present invention, the transgene of interest encodes one or more transcription factor(s). It is preferred for said embodiment that the one or more transcription factor(s) is/ are selected from the group consisting of neurogenin-2 (NGN2, NEUROG2), ASCL1, NEUROD1, LMX1A, FOXA2, NURR1, PITX3, LHX3, HB9, ISL1 , DLX2, SOX10, OLIG2, NKX6.2, NKX6.1, NKX2.2, NFIA, NFIB, SOX9, SPI1, CEBPB, IRF8, RUNX1 , SALL1.

[0022] In one even more preferred embodiment thereof, the one or more transcription factor is NGN2, ASCL1 or NEUROD1. It is even more preferred for said embodiment that the one or more transcription factor is NGN2, ASCL1 or NEUROD1 , and the neural precursor cells are differentiated into neurons, preferably excitatory glutamatergic neurons, after step c).

[0023] In another even more preferred embodiment thereof, the one or more transcription factors are NGN2, ASCL1 or NEUROD1 and LMX1 A and optionally one or more of the group consisting of FOXA2, NLIRR1 and PITX3. It is even more preferred for said embodiment that the one or more transcription factors are NGN2, ASCL1 or NEUROD1 and LMX1A and optionally one or more of the group consisting of FOXA2, NLIRR1 and PITX3, and the neural precursor cells are differentiated into dopaminergic midbrain neurons after step c).

[0024] In another even more preferred embodiment thereof, the one or more transcription factors are NGN2, ASCL1 or NEUROD1 and LHX3 and optionally HB9 and/ or ISL1. It is even more preferred for said embodiment that the one or more transcription factors are NGN2, ASCL1 or NEUROD1 and LHX3 and optionally HB9 and/ or ISL1, and the neural precursor cells are differentiated into cholinergic motor neurons after step c).

[0025] In another even more preferred embodiment thereof, the one or more transcription factors are ASCL1 and DLX2. It is even more preferred for said embodiment that the one or more transcription factors are ASCL1 and DLX2, and the neural precursor cells are differentiated into GABAergic inhibitory neurons after step c).

[0026] In another even more preferred embodiment thereof, the one or more transcription factors are SOX10 and one or more of the group consisting of OLIG2, NKX6.2, NKX6.1 and NKX2.2. It is even more preferred for said embodiment that the one or more transcription factors are SOX10 and one or more of the group consisting of OLIG2, NKX6.2, NKX6.1 and NKX2.2, and the neural precursor cells are differentiated into oligodendrocytes after step c).

[0027] In another even more preferred embodiment thereof, the one or more transcription factors are NFIA and optionally NFIB and/ or SOX9. It is even more preferred for said embodiment that the one or more transcription factors are NFIA and optionally NFIB and/ or SOX9, and the neural precursor cells are differentiated into astrocytes after step c).

[0028] In another even more preferred embodiment thereof, the one or more transcription factor is SOX10. It is even more preferred for said embodiment that the one or more transcription factor is SOX10, and the neural precursor cells are differentiated into neural crest-derived cells after step c).

[0029] In another even more preferred embodiment thereof, the one or more transcription factor(s) is/ are SPI1 and optionally one or more of the group consisting of CEBPB, IRF8, RLINX1 and SALL1. It is even more preferred for said embodiment that the one or more transcription factor(s) is/ are SPI1 and optionally one or more of the group consisting of CEBPB, IRF8, RLINX1 and SALL1 , and the neural precursor cells are differentiated into microglia after step c).

[0030] In one preferred embodiment of said method of the present invention, wherein the method comprises step b1), the transgene of interest encodes the transcription factor NGN2 and the method further comprises the generation of neurons from the neural precursor cells.

[0031] In one further preferred embodiment of said method of the present invention, wherein the method comprises step b1), the transgene of interest encodes the transcription factor NGN2, ASCL1 or NEUROD1, and the method further comprises the generation of neurons, preferably excitatory glutamatergic neurons, from the neural precursor cells.

[0032] In one preferred embodiment of said method of the present invention, wherein the method comprises step b1), the transgene of interest encodes the one or more transcription factors NGN2, ASCL1 or NEUROD1 and LMX1A and optionally one or more of the group consisting of FOXA2, NLIRR1 and PITX3, and the method further comprises the generation of dopaminergic midbrain neurons from the neural precursor cells.

[0033] In one further preferred embodiment of said method of the present invention, wherein the method comprises step b1), the transgene of interest encodes the one or more transcription factors NGN2, ASCL1 or NEUROD1 and LHX3 and optionally HB9 and/ or ISL1, and the method further comprises the generation of cholinergic motor neurons from the neural precursor cells.

[0034] In one preferred embodiment of said method of the present invention, wherein the method comprises step b1), the transgene of interest encodes the transcription factors ASCL1 and DLX2, and the method further comprises the generation of GABAergic inhibitory neurons from the neural precursor cells.

[0035] In one further preferred embodiment of said method of the present invention, wherein the method comprises step b1), the transgene of interest encodes SOX10 and one or more of the group consisting of OLIG2, NKX6.2, NKX6.1 and NKX2.2, and the method further comprises the generation of oligodendrocytes from the neural precursor cells. [0036] In one preferred embodiment of said method of the present invention, wherein the method comprises step b1), the transgene of interest encodes NFIA and optionally NFIB and/or SOX9, and the method further comprises the generation of astrocytes from the neural precursor cells.

[0037] In one further preferred embodiment of said method of the present invention, wherein the method comprises step b1), the transgene of interest encodes SOX10, and the method further comprises the generation of neural crest-derived cells from the neural precursor cells.

[0038] In one preferred embodiment of said method of the present invention, wherein the method comprises step b1), the transgene of interest encodes SPI1 and optionally one or more of the group consisting of CEBPB, IRF8, RLINX1 and SALL1, and the method further comprises the generation of microglia from the neural precursor cells.

[0039] In one preferred embodiment of the method for expression of a transgene of interest from neural precursor cells (NPCs), step a) comprises culturing stem cells including exposure of the stem cells to at least one growth factor(s), cytokine(s) and/ or small molecule(s) for differentiating the stem cells into neural precursor cells. It is more preferred for said embodiment that the at least one growth factor(s), cytokine(s) and/ or small molecule(s) is/ are selected from the group consisting of SB431542, dorsomorphin, CHIR99021, doxycycline, purmorphamine, cAMP, BDNF and NT3. Thus, in one even more preferred embodiment thereof, the at least one small molecule(s) is/ are selected from the group consisting of SB431542, CHIR99021, doxycycline, dorsomorphin, purmorphamine and cAMP.

[0040] In another preferred embodiment of the method for expression of a transgene of interest from neural precursor cells (NPCs), step a) is without the use of one or more growth factor(s).

[0041] In one preferred embodiment of the method for expression of a transgene of interest from neural precursor cells (NPCs), said first and said second genomic location of b1) or the genomic location of b2) is/ are selected from the group consisting of the hROSA26 locus (SEQ ID NO: 61), the AAVS1 locus (SEQ ID NO: 62), the CLYBL gene (SEQ ID NO: 63), the CCR5 gene (SEQ ID NO: 64), the HPRT gene (SEQ ID NO: 65) or genes with the site ID 325 on chromosome 8 (SEQ ID NO: 66), site ID 227 on chromosome 1 (SEQ ID NO: 67), site ID 229 on chromosome 2 (SEQ ID NO: 68), site ID 255 on chromosome 5 (SEQ ID NO: 69), site ID 259 on chromosome 14 (SEQ ID NO: 70), site ID 263 on chromosome X (SEQ ID NO: 71), site ID 303 on chromosome 2 (SEQ ID NO: 72), site ID 231 on chromosome 4 (SEQ ID NO: 73), site ID 315 on chromosome 5 (SEQ ID NO: 74), site ID 307 on chromosome 16 (SEQ ID NO: 75), site ID 285 on chromosome 6 (SEQ ID NO: 76), site ID 233 on chromosome 6 (SEQ ID NO: 77), site ID 311 on chromosome 134 (SEQ ID NO: 78), site ID 301 on chromosome 7 (SEQ ID NO: 79), site ID 293 on chromosome 8 (SEQ ID NO: 80), site ID 319 on chromosome 11 (SEQ ID NO: 81), site ID 329 on chromosome 12 (SEQ ID NO: 82) and site ID 313 on chromosome X (SEQ ID NO: 83). [0042] In a further aspect, the present invention also provides a system for expression of a transgene of interest from neural precursor cells, comprising: i) Neural precursor cells; ii) a nucleotide sequence encoding a transcriptional regulator protein, a nucleotide sequence of a constitutive promoter, wherein the transcriptional regulator protein is under the control of the constitutive promoter; a nucleotide sequence encoding a transgene of interest, and a nucleotide sequence encoding an inducible promoter, which is regulated by the transcriptional regulator protein and wherein the transgene of interest is operably linked to the inducible promoter; or iii) a nucleotide sequence encoding a transgene of interest, a nucleotide sequence of a constitutive promoter, wherein the transgene of interest is operably linked to the constitutive promoter.

[0043] In another aspect, the present invention also provides a vector comprising or consisting of one or more of the sequence(s) set forth in SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 , SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, or SEQ ID NO: 87.

[0044] The present invention also provides in a further aspect a method for expression of a transgene of interest from stem cells, comprising the steps of: a) Providing stem cells, preferably pluripotent stem cells, induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs); b) i) targeted insertion of a nucleotide sequence encoding a transcriptional regulator protein being under the control of a first constitutive promoter, into a first genomic location, preferably the hROSA26 locus (SEQ ID NO: 61); and ii) targeted insertion of a nucleotide sequence encoding a first transgene of interest into a second genomic location, preferably the AAVS1 locus (SEQ ID NO: 62), wherein the first transgene of interest is operably linked to an inducible promoter, which is regulated by the transcriptional regulator protein; and iii) targeted insertion of the nucleotide sequence of a second transgene of interest into a third genomic location, preferably the CLYBL locus (SEQ ID NO: 63), wherein the second transgene of interest is operably linked to a second constitutive promoter, and c) expression of the first and /or second transgene of interest. It is further preferred for said method that it further comprises the differentiation of the stem cells into microglia.

BRIEF DESCRIPTION OF THE FIGURES

[0045] Figure 1 shows a schematic overview of a protocol of the prior art for NPC generation from human pluripotent stem cells (hPSCs). hPSCs are detached and resuspended in human neural progenitor cell (hNPC) medium containing SB43152 (SB) and dorsomorphin (DM) for neural induction and CHIR99021 (CHIR) and purmorphamine (PMA) for epithelium formation. After 4 days SB and DM are withdrawn. After 6 days, embryoid bodies are selected depending on their neuroepithelial outgrowth, disrupted and plated on Matrigel-coated culture dishes. After a maximum of 5 passages, the small molecule neural progenitor cell (smNPC) line is established and can be maintained under standard culture conditions.

[0046] Figure 2 shows a scheme of the hROSA26 genomic safe harbour targeting for constitutive EGFP expression according to the present invention. Figure 2A shows in the top scheme the hROSA26 donor plasmid, in the middle scheme the hROSA26 wild-type locus and in the bottom scheme the targeted genomic hROSA26 with an integrated CAG-EGFP expression cassette. Figure 2B shows phase contrast and EGFP images of neural progenitor cells targeted with CAG- EGFP.

[0047] Figure 3 shows an AAVS1 targeting strategy containing both elements of the doxycycline- inducible Tet-On system. Figure 3A, top scheme, shows the AAVS1 donor plasmid, the middle scheme shows the AAVS1 wild-type locus and the bottom scheme shows the targeted AAVS1 genomic locus containing both, the TRE-EGFP cassette and the CAG-rtTA cassette in an inverse direction. Figure 3B shows the map of the AAVS1 donor plasmid. Figure 3C presents a scheme showing the two options of reporter protein expression in hNPCs: NPCs with doxycycline inducible EGFP expression achieved via integration of both elements of the Tet-On system into the AAVS1 GSH, as depicted in this figure or hNPCs with constitutive EGFP expression achieved by integration of the CAG-EGFP cassette into the ROSA26 GSH as depicted in Figure 2. Figure 3D shows three independent hNPC lines that were transfected with the AAVS1 donor plasmid, thus, generating doxycycline-inducible EGFP expression with corresponding values of mean fluorescence intensity (MFI) measured by flow cytometry, showing no leakage of EGFP expression in all three cell lines, when cultivated without doxycycline.

[0048] Figure 4 shows a scheme of the AAVS1 targeting strategy containing both elements of the doxycycline-inducible Tet-On system for the generation of glutamatergic neurons by overexpression of Ngn2. Figure 4A, top scheme, shows the AAVS1 donor plasmid, the middle scheme shows the AAVS1 wild-type locus, and the bottom scheme shows the targeted AAVS1 genomic locus containing both, the TRE-Ngn2 cassette and the CAG-rtTA cassette in inverse directions. Figure 4B shows a map of the AAVS1 donor plasmid. Figure 4C presents a scheme showing applications for this single step targeting system: Direct cell programming of hNPCs into i- neurons by overexpression of Ngn2 or generation of other target cells by overexpression of different transcription factors. Figure 4D shows phase contrast microscope images of i-neurons 7 and 18 days after initial doxycycline supplementation. Figure 4E shows immunocytochemistry of 4 i-neuron cell lines 14 days after induction with doxycycline. All 4 cell lines express the panneuronal marker pill-tubulin (TUBB3) and the microtubule-associated protein tau mainly found in axons.

[0049] Figure 5 shows the generation of a reporter i-neuron cell line. Figure 5A, top scheme, shows the donor plasmid, the middle scheme shows the AAVS1 wild-type locus, and the bottom scheme shows the targeted AAVS1 genomic locus containing both, the TRE-Ngn2 cassette and the CAG-rtTA cassette in inverse directions. Figure 5B, top scheme, shows the hROSA26 donor plasmid, the middle scheme shows the hROSA26 wild-type locus, the bottom scheme shows the targeted hROSA26 locus with integrated CAG-EGFP expression cassette. Figure 5C shows the phase contrast and EGFP images of i-neurons constitutively expressing EGFP. Figure 5D shows a flow cytometry analysis of i-neurons without ROSA26 targeting serving as negative control, NPCs containing dox-inducible Ngn2 (AAVS1) and constitutive EGFP (ROSA26) and i-neurons (14 days old) with constitutive EGFP expression.

[0050] Figure 6 shows the generation of an inducible i-microglia reporter cell line. Figure 6A, top scheme, shows the CLYBL donor plasmid, the middle scheme shows the CLYBL wild-type locus and the bottom scheme shows the CLYBL locus targeted with a CAG-mCherry cassette. Figure 6B, top scheme, shows the hROSA26 donor plasmid, the middle scheme shows the ROSA26 wild-type locus and the bottom scheme shows the hROSA26 locus with integrated CAG-rtTA cassette. Figure 6C, top scheme, shows the AAVS1 donor plasmid, the middle scheme shows the AAVS1 wild-type locus and the bottom scheme shows the targeted AAVS2 locus with integrated TRE-SPI1-CEBPP cassette. Figure 6D shows the phase contrast and mCherry image of i-MGL-hPSCs and mature i-MGL, that constitutively express mCherry. Figure 6E shows the coculture of i-MGL (mCherry) and i-neuron (EGFP) reporter cell lines.

[0051] Figure 7: Figure 7A shows a scheme of the AAVS1 targeting in hNPCs using a single vector containing a puromycin resistance cassette for antibiotic selection, both elements of the Tet- ON system in inverse directions and the transcription factor NGN2. Figure 7B shows a scheme of the protocol for neuron generation out of hNPCs by dox induction. Phase contrast images show morphological changes during induction from hNPC colonies towards neuronal networks.

[0052] Figure 8 shows the vector map of ROSA26-guideA_Cas9n (SEQ ID NO: 14).

[0053] Figure 9 shows the vector map of ROSA26-guideB_Cas9n (SEQ ID NO: 17).

[0054] Figure 10 shows the vector map of pZFN-AAVS1-L-ELD (SEQ ID NO: 19).

[0055] Figure 11 shows the vector map of pZFN-AAVS1-R_KKR (SEQ ID NO: 21).

[0056] Figure 12 shows the vector map of pUC_ROSA_n-CAG-rtTA (SEQ ID NO: 23).

[0057] Figure 13 shows the vector map of pAAVS1_single[CAG]-(EGFP)_INV (SEQ ID NO: 10).

[0058] Figure 14 shows the vector map of pUC_AAVS1_p-single[CAG]-(Ngn2) (SEQ ID NO: 6).

[0059] Figure 15 shows the vector map of pCLYBL_CAG-mCherry_hygro (SEQ ID NO: 24).

[0060] Figure 16 shows the vector map of pZT-C13-L1 (SEQ ID NO: 30).

[0061] Figure 17 shows the vector map of pZT-C13-R1 (SEQ ID NO: 28).

DETAILED DESCRIPTION OF THE INVENTION

[0062] In a first aspect, the present invention relates to a method for expression of a transgene of interest from neural precursor cells (NPCs), comprising the steps of: a) Providing neural precursor cells; b1) i) targeted insertion of a nucleotide sequence encoding a transcriptional regulator protein being under the control of a suitable promoter, preferably a constitutive promoter, into a first genomic location; and ii) targeted insertion of a nucleotide sequence encoding a transgene of interest into a second genomic location, wherein the transgene of interest is operably linked to a suitable promoter, preferably an inducible promoter, which is regulated by the transcriptional regulator protein; or b2) targeted insertion of the nucleotide sequence encoding a transgene of interest into a genomic location, preferably a genomic safe harbour site, wherein the transgene of interest is operably linked to a constitutive promoter, and c) expression of the transgene of interest.

[0063] In one embodiment, the method for expression of a transgene of interest from neural precursor cells (NPCs) comprises the following steps: a) Providing neural precursor cells; b1) i) targeted insertion of a nucleotide sequence encoding a transcriptional regulator protein being under the control of a constitutive promoter into a first genomic location; and ii) targeted insertion of a nucleotide sequence encoding a transgene of interest into a second genomic location, wherein the transgene of interest is operably linked to an inducible promoter, which is regulated by the transcriptional regulator protein; or b2) targeted insertion of the nucleotide sequence encoding a transgene of interest into a genomic location, preferably a genomic safe harbour site, wherein the transgene of interest is operably linked to a constitutive promoter, and c) expression of the transgene of interest.

[0064] In one further embodiment, the method for expression of a transgene of interest from neural precursor cells (NPCs) comprises the following steps: a) Providing neural precursor cells; b1) i) targeted insertion of a nucleotide sequence encoding a transcriptional regulator protein being under the control of a constitutive promoter into a first genomic location; and ii) targeted insertion of a nucleotide sequence encoding a transgene of interest into a second genomic location, wherein the transgene of interest is operably linked to an inducible promoter, which is regulated by the transcriptional regulator protein; or b2) targeted insertion of the nucleotide sequence encoding a transgene of interest into a genomic safe harbour site, wherein the transgene of interest is operably linked to a constitutive promoter, and c) expression of the transgene of interest.

[0065] In one embodiment, the method for expression of a transgene of interest from neural precursor cells (NPCs), comprises the following steps: a) Providing neural precursor cells; b1) i) targeted insertion of a nucleotide sequence encoding a transcriptional regulator protein being under the control of a suitable promoter, preferably a constitutive promoter, into a first genomic location; and ii) targeted insertion of a nucleotide sequence encoding a transgene of interest into a second genomic location, wherein the transgene of interest is operably linked to a suitable promoter, preferably an inducible promoter, which is regulated by the transcriptional regulator protein; and c) expression of the transgene of interest.

[0066] In one further embodiment, the method for expression of a transgene of interest from neural precursor cells (NPCs) comprises the following steps: a) Providing neural precursor cells; b1) i) targeted insertion of a nucleotide sequence encoding a transcriptional regulator protein being under the control of a constitutive promoter into a first genomic location; and ii) targeted insertion of a nucleotide sequence encoding a transgene of interest into a second genomic location, wherein the transgene of interest is operably linked to an inducible promoter, which is regulated by the transcriptional regulator protein; and c) expression of the transgene of interest.

[0067] In one further embodiment, the method for expression of a transgene of interest from neural precursor cells (NPCs), comprises the following steps: a) Providing neural precursor cells; b2) targeted insertion of the nucleotide sequence encoding a transgene of interest into a genomic location, preferably a genomic safe harbour site, wherein the transgene of interest is operably linked to a constitutive promoter, and c) expression of the transgene of interest.

[0068] In one further embodiment, the method for expression of a transgene of interest from neural precursor cells (NPCs), comprises the following steps: a) Providing neural precursor cells; b2) targeted insertion of the nucleotide sequence encoding a transgene of interest into a genomic safe harbour site, wherein the transgene of interest is operably linked to a constitutive promoter, and c) expression of the transgene of interest.

[0069] The principal cell types of the central nervous system are neurons (and hundreds of neuronal subtypes), oligodendrocytes, and astrocytes. The term “neural precursor cells” as used herein and in the context of the present invention, represent the in vitro correlate of the developmental precursor of all these cell types. Thus, forward programming of NPCs into any of these cell types should be feasible and highly efficient. Additionally, due to their proliferative and renewal capacity, multipotency, and ease of culture, NPCs may represent an ideal starting cell population for reprogramming even into cells with a non-neuroectodermal origin, i.e. all other cell types of the entire organism.

[0070] “Transgene of interest”, as used herein, means any nucleic acid molecule encoding a biological product, namely either a transcript of interest or useful transcript such as an mRNA, an rRNA, a tRNA, a ribozyme or an aptazyme, or a protein, a polypeptide or a peptide of therapeutic or experimental interest. According to the invention, the transgene of interest includes a gDNA, a cDNA or DNAs.

[0071] The term “providing” as used herein and in the context of the present invention, means the supply of something, specifically, here, the supply of NPCs.

[0072] As used within the present invention, the term “targeted insertion” means the insertion into a genomic location or genomic safe harbour (GSH) site as defined herein. Any suitable technique for insertion of a polynucleotide into a specific sequence may be used, and several are described in the art. Suitable techniques include any method known to a person skilled in the art, which introduces a break at the desired location and permits recombination of the vector into the gap. Thus, a crucial first step for targeted site-specific genomic modification is the creation of a doublestrand DNA break (DSB) at the genomic locus to be modified. Distinct cellular repair mechanisms can be exploited to repair the DSB and to introduce the desired sequence, and these are non- homologous end joining repair (NHEJ), which is more prone to error; and homologous recombination repair (HR) mediated by a donor DNA template, that can be used to insert inducible cassettes. Several techniques exist to allow customized site-specific generation of DSB in the genome. Many of these involve the use of customized endonucleases, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) or the clustered regularly interspaced short palindromic repeats/ CRISPR associated protein (CRISPR/Cas9) system (Gaj et al., 2013). Zinc finger nucleases are artificial enzymes, which are generated by fusion of a zinc-finger DNA-binding domain to the nuclease domain of the restriction enzyme Fokl. The latter has a non-specific cleavage domain, which must dimerize in order to cleave DNA. This means that two ZFN monomers are required to allow dimerization of the Fokl domains and to cleave the DNA. The DNA binding domain may be designed to target any genomic sequence of interest, may be a tandem array of Cys2His2 zinc fingers, each of which recognises three contiguous nucleotides in the target sequence. The two binding sites are separated by 5-7 bp to allow optimal dimerization of the Fokl domains. The enzyme thus is able to cleave DNA at a specific site, and target specificity is increased by ensuring that two proximal DNA-binding events must occur to achieve a double-strand break. Transcription activator-like effector nucleases, or TALENs, are dimeric transcription factor/ nucleases. They are made by fusing a TAL effector DNA- binding domain to a DNA cleavage domain (a nuclease). Transcription activator-like effectors (TALENs) can be engineered to bind practically any desired DNA sequence, so when combined with a nuclease, DNA can be cut at specific locations. TAL effectors are proteins that are secreted by Xanthomonas bacteria, the DNA binding domain of which contains a repeated highly conserved 33-34 amino acid sequence with divergent 12 ^th and 13 ^th amino acids. These two positions are highly variable and show a strong correlation with specific nucleotide recognition. This straightforward relationship between amino acid sequence and DNA recognition has allowed for the engineering of specific DNA-binding domains by selecting a combination of repeat segments containing appropriate residues at the two variable positions. TALENs are thus built from arrays of 33 to 35 amino acid modules, each of which targets a single nucleotide. By selecting the array of the modules, almost any sequence may be targeted. Three types of CRISPR mechanisms have been identified, of which type II is best studied. The CRISPR/Cas9 system (type II system) utilises the Cas9 nuclease to make a double-stranded break in DNA at a site determined by a short guide RNA. The CRISPR/Cas system is a prokaryotic immune system that confers resistance to foreign genetic elements. CRISPR are segments of prokaryotic DNA containing short repetitions of base sequences. Each repetition is followed by short segments of "protospacer DNA" from previous exposures to foreign genetic elements. CRISPR spacers recognize and cut the exogenous genetic elements using RNA interference. The CRISPR immune response occurs through two steps: CRISPR-RNA (crRNA) biogenesis and crRNA-guided interference. CrRNA molecules are composed of a variable sequence transcribed from the protospacer DNA and a CRISP repeat. Each crRNA molecule then hybridizes with a second RNA, known as the trans-activating CRISPR RNA (tracrRNA) and together these two eventually form a complex with the nuclease Cas9. The protospacer DNA encoded section of the crRNA directs Cas9 to cleave complementary target DNA sequences, if they are adjacent to short sequences known as protospacer adjacent motifs (PAMs). This natural system has been engineered and exploited to introduce DSB breaks in specific sites in genomic DNA, amongst many other applications. In particular, the CRIPSR type II system from Streptococcus pyogenes may be used. At its simplest, the CRISPR/Cas9 system comprises two components that are delivered to the cell to provide genome editing: The Cas9 nuclease itself and a small guide RNA (gRNA). The gRNA is a fusion of a customised, site-specific crRNA (directed to the target sequence) and a standardized tracrRNA. Once a DSB has been made, a donor template with homology to the targeted locus is supplied. The DSB may be repaired by the homology- directed repair (HDR) pathway allowing for precise insertions to be made. Derivatives of this system are also possible. Mutant forms of Cas9 are available, such as Cas9D10A, with only nickase activity. This means, it cleaves only one DNA strand, and does not activate NHEJ. Instead, when provided with a homologous repair template, DNA repairs are conducted via the high-fidelity HDR pathway only. Cas9D10A may be used in paired Cas9 complexes designed to generate adjacent DNA nicks in conjunction with two sgRNAs, complementary to the adjacent area on opposite strands of the target site, which may be particularly advantageous. The elements for making the double-strand DNA break may be introduced in one or more vectors such as plasmids for expression in the cell. Thus, any method of making specific, targeted double strand breaks in the genome in order to allow the insertion of a nucleotide sequence/ gene/ inducible cassette may be used in the method of the present invention. It may be preferred that the method of the present invention utilises for inserting the gene/ inducible cassette one or more of ZFNs, TALENs and/or CRISPR/Cas9 systems or any derivative thereof. Once the DSB has been made by any appropriate means, the gene/ inducible cassette for insertion may be supplied in any suitable fashion as described below. The gene/ inducible cassette and associated genetic material form the donor DNA for repair of the DNA at the DSB and are inserted using standard cellular repair machinery/ pathways. How the break is initiated will alter which pathway is used to repair the damage, as noted above. However, this is also within the knowledge of a person skilled in the art.

[0073] As used within the present invention, the term “gene” means the basic physical unit heredity, a linear sequence of nucleotides along a segment of DNA that provides the coded instructions for synthesis of RNA, which, when translated into protein, leads to the expression of hereditary character.

[0074] As used within the present invention, the term “nucleotide sequence” refers to a succession of bases in a DNA segment forming a gene as defined above. [0075] As used within the present invention, the term “transcriptional regulator protein” means a protein that binds to DNA, preferably sequence-specifically to a DNA site located in or near a promoter, and either facilitating the binding of the transcription machinery to the promoter, and thus transcription of the DNA sequence (a transcriptional activator) or blocks this process (a transcriptional repressor). Such entities are also known as transcription factors. The DNA sequence that a transcriptional regulator protein binds to is called a transcription factor-binding site or response element, and these are found in or near the promoter of the regulated DNA sequence. Transcriptional activator proteins bind to a response element and promote gene expression. Transcriptional repressor proteins bind to a response element and prevent gene expression. Transcriptional regulator proteins may be activated or deactivated by a number of mechanisms including binding of a substance, interaction with other transcription factors (e.g., homo- or heterodimerization) or coregulatory proteins, phosphorylation, and/or methylation. The transcriptional regulator may be controlled by activation or deactivation. If the transcriptional regulator protein is a transcriptional activator protein, it is preferred that the transcriptional activator protein requires activation. This activation may be through any suitable means, but it is preferred that the transcriptional regulator protein is activated through the addition of an exogenous substance to the respective cells. The supply of an exogenous substance to the cells can be controlled, and thus the activation of the transcriptional regulator protein can be controlled. Such transcriptional regulator proteins are also called inducible transcriptional regulator proteins.

[0076] As used within the present invention, the term “inducible promoter” means a nucleotide sequence, which initiates and regulates transcription of a polynucleotide. An "inducible promoter" is a nucleotide sequence, wherein expression of a genetic sequence operably linked to the promoter is controlled by an analyte, co-factor, regulatory protein, etc. In one embodiment of the method of the present invention, the control is affected by the transcriptional regulator protein. It is intended that the term "promoter" or "control element" includes full-length promoter regions and functional (e.g., controls transcription or translation) segments of these regions.

[0077] It is preferred that the nucleotide sequence encoding the transcriptional regulator protein is operably linked to a constitutive promoter. Alternatively, the first genomic location or GSH can be selected such that it already has a constitutive promoter that can also drive expression of the transcriptional regulator protein gene and any associated genetic material. Constitutive promoters ensure sustained and high-level gene expression. Commonly used constitutive promoters include the human p-actin promoter (ACTB), cytomegalovirus (CMV), elongation factor-la (EFla), phosphoglycerate kinase (PGK) and ubiquitin C (UbC). The CAG promoter is a strong synthetic promoter frequently used to drive high levels of gene expression, which is also preferred as constitutive promoter. [0078] The terms “being under the control of a promoter” or “being operably linked to a promoter’ means that an expression of the respective nucleotide sequence is enabled.

[0079] As used within the present invention, the term “genomic location” and then more specifically “genomic safe harbour site” means a genetic site, which allows the insertion of genetic material without deleterious effects for the cell and permits transcription of the inserted genetic material. Those skilled in the art may use these simplified criteria to identify a suitable genomic location or GSH, and/or the more formal criteria. Insertions specifically within genomic locations or genomic safe harbour sites (GSH) are preferred over random genome integration, since this is expected to be a safer modification of the genome, and is less likely to lead to unwanted side effects, such as silencing natural gene expression or causing mutations that lead to cancerous cell types. Thus, a genomic location or genomic safe harbour site is a locus within the genome, wherein a gene or other genetic material may be inserted without any deleterious effects on the cell or on the inserted genetic material. Most beneficial is a genomic location or GSH site in which expression of the inserted gene sequence is not perturbed by any read-through expression from neighbouring genes and expression of the inducible cassette, minimizes interference with the endogenous transcription programme. More formal criteria have been proposed that assist in the determination of whether a particular locus is not only a genomic location, but specifically refers to a GSH site [9], These criteria include a site that is (i) > 300 kb from any cancer-related gene on all Oncogenes list, (ii)

> 300 kb from any miRNA/ other functional small RNAs, (iii) > 50 kb from any 5' gene end, (iv)

> 50 kb away from any replication origin, (v) > 50 kb away from any ultra-conserved element, (vi) low transcriptional activity (no mRNA ± 25 kb), (vii) not in copy number variable region (viii) in open chromatin (DHS signal ± 1 kb) and (ix) unique (1 copy in human genome). It may not be necessary to satisfy all of these proposed criteria, since GSH already identified do not fulfil all of these criteria. It is preferred, that a suitable GSH may satisfy at least 3, 4, 5, 6, 7 or 8 and most preferably all nine of these criteria. In the methods of the present invention, insertions occur at different genomic locations or GSHs.

[0080] The first genomic location or GSH can be any suitable genomic location or GSH. It is modified by insertion of nucleotide sequence encoding a transcriptional regulator protein. It may be a GSH with an endogenous promoter that is constitutively expressed, which will result in the inserted transcriptional regulator protein being constitutively expressed. A suitable first genomic location or GSH is the hROSA26 site (SEQ ID NO: 61) for human cells. In a further embodiment of the present invention, the inserted transcriptional regulator protein, operably linked to a promoter, is a constitutive promoter. A constitutive promoter can be, for example, used in conjunction with an insertion in the hROSA26 site (SEQ ID NO: 61).

[0081] The second genomic location or GSH is modified by the insertion of a nucleotide sequence encoding a transgene of interest, which comprises a coding sequence operably linked to a suitable promoter, preferably an inducible promoter. Other genetic material may also be inserted. The genetic sequence, operably linked to an inducible promoter, is preferably a DNA sequence. The transcription is controlled using the inducible promoter.

[0082] The method for expression of a transgene of interest from neural precursor cells (NPCs) according to the present invention enables modified GSH-targeting for controllable transcription in hPSC-derived neural precursor cells, e.g. smNPCs. The inventors of the present invention have used smNPCs as expandable (without limits) cell population for controllable transcription by inserting transgenes into defined GSHs. To this aim, the inventors have utilized this for efficient constitutive or inducible reporter expression and for rapid and deterministic programming of smNPCs into, e.g., cortical neurons. The use of smNPCs instead of hPSCs as starting cell type for controllable transcription and cell programming has several advantages, namely, the maintenance culture is cheaper (medium components), less resource intensive (e.g. easier handling in laboratory practice, fewer medium changes), less variable (less tissue culture expertise is required by the researcher). Specifically, for neural cell types, faster maturation may be achieved [8],

[0083] The method for expression of a transgene of interest from neural precursor cells (NPCs) according to the present invention thus also enables constitutive and inducible reporter expression in smNPCs, reporter target cell production or cell programming in smNPCs. The latter is, for example, exemplified by the rapid and deterministic production of neurons. Reporter target cell production is exemplified herein by rapid and deterministic forward programming of EGFP smNPCs into neurons, with all starting and target cells expressing the fluorescent reporter, thus providing a powerful tool for many downstream applications.

[0084] In one embodiment of the method for expression of a transgene of interest from neural precursor cells (NPCs), the transcriptional regulator protein of b1) i) is the reverse tetracycline transactivator (rtTA) and the activity thereof is controlled by doxycycline, tetracycline or a derivative thereof.

[0085] As used within the present invention, the term “reverse tetracycline transactivator (rtTA)” means a transcriptional activator protein induced by tetracycline or a derivate thereof. Tetracycline- controlled transcriptional activation is a method of inducible gene expression where transcription is reversibly turned on or off in the presence of the antibiotic tetracycline or one of its derivatives (e.g. doxycycline, which is more stable). In this system, the transcriptional activator protein may be tetracycline-responsive transcriptional activator protein (rtTA) or a derivative thereof. The transcriptional regulator protein of the present invention may be an rtTA. The rtTA protein is able to 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), which together form a tetracycline response element (TRE). There are two forms of this system, depending on whether the addition of tetracycline or a derivative activates (Tet-On) or deactivates (Tet-Off) the rtTA protein. The Tet-ON system, in which doxycycline activates the rtTA protein, may also be used in one embodiment of the method of the present invention.

[0086] The Tet-On system is composed of two components; (1) the constitutively expressed tetracycline - responsive transcriptional activator protein (rtTA) and the rtTA sensitive inducible promoter (Tet Responsive Element, TRE). This may be bound by tetracycline or its more stable derivatives, including doxycycline (dox), resulting in activation of rtTA, allowing it to bind to TRE sequences and inducing expression of TRE-controlled genes. The use of this may be preferred in the method of the present invention. Thus, the transcriptional regulator protein of the method of the present invention may be the tetracycline-responsive transcriptional activator protein (rtTA), which can be activated or deactivated by the antibiotic tetracycline or one of its derivatives, which are supplied exogenously. If the transcriptional regulator protein is rtTA, then the inducible promoter inserted into the second genomic location includes the tetracycline response element (TRE). The exogenously supplied substance may be the antibiotic tetracycline or one of its derivatives, like doxycycline, preferably tetracycline or doxycycline. Variants and modified rtTA proteins may be used in the method of the present invention. These may include Tet-On Advanced transactivator (also known as rtTA2S-M2) and Tet-On 3G (also known as rtTA-V16, derived from rtTA2S-S2).

[0087] In one embodiment of the method for expression of a transgene of interest from neural precursor cells (NPCs), the suitable promoter of b1) ii) is an inducible promoter, preferably an inducible promoter including a Tet Responsive Element (TRE), more preferably a Tet Responsive Element (TRE) comprising or consisting of SEQ ID NO: 1. In one embodiment of the method for expression of a transgene of interest from neural precursor cells (NPCs), the suitable promoter of b1) ii) is an inducible promoter. In one preferred embodiment of the method for expression of a transgene of interest from neural precursor cells (NPCs), the suitable promoter of b1) ii) is an inducible promoter including a Tet Responsive Element (TRE). More preferably, the Tet Responsive Element (TRE) comprises or consists of SEQ ID NO: 1. It is even more preferred in one embodiment of the method for expression of a transgene of interest from neural precursor cells (NPCs) that the suitable promoter of b1) ii) is an inducible promoter including a Tet Responsive Element (TRE) consisting of SEQ ID NO: 1.

[0088] As used within the present invention, the term “Tet Responsive Element (TRE)” means a bacterial TetO sequence of 7 repeats of 19 bp separated by spacer sequences, together with a minimal promoter. Variants and modifications of the TRE sequence are possible, since the minimal promoter can be any suitable promoter. Preferably, the minimal promoter shows no or minimal expression levels in the absence of rtTA binding. The inducible promoter inserted into the second genomic location may thus comprise a TRE.

[0089] In one embodiment of the method for expression of a transgene of interest from neural precursor cells (NPCs), the suitable promoter of b1) i) is a constitutive promoter, preferably a constitutive CAG promoter, more preferably a constitutive CAG promoter comprising or consisting of the SEQ ID NO: 2. In one embodiment of the method for expression of a transgene of interest from neural precursor cells (NPCs), the suitable promoter of b1) i) is a constitutive promoter. In one preferred embodiment of the method for expression of a transgene of interest from neural precursor cells (NPCs), the suitable promoter of b1) i) is a constitutive CAG promoter. In one more preferred embodiment of the method for expression of a transgene of interest from neural precursor cells (NPCs), the suitable promoter of b1) i) is a constitutive CAG promoter comprising SEQ ID NO: 2. In one even more preferred embodiment of the method for expression of a transgene of interest from neural precursor cells (NPCs), the suitable promoter of b1) i) is a constitutive CAG promoter consisting of SEQ ID NO: 2.

[0090] In one further embodiment of said method of the present invention, the constitutive promoter of b2) is a constitutive CAG promoter, preferably a constitutive CAG promoter comprising or consisting of the SEQ ID NO: 2. In one embodiment of said method of the present invention, the constitutive promoter of b2) is a constitutive CAG promoter. In one preferred embodiment of said method of the present invention, the constitutive promoter of b2) is a constitutive CAG promoter comprising or consisting of the SEQ ID NO: 2. In one more preferred embodiment of said method of the present invention, the constitutive promoter of b2) is a constitutive CAG promoter comprising SEQ ID NO: 2. In one even more preferred embodiment of said method of the present invention, the constitutive promoter of b2) is a constitutive CAG promoter consisting of SEQ ID NO: 2.

[0091] In one further embodiment of said method of the present invention, the first genomic location of b1) i) is a first genomic safe harbour site.

[0092] In one embodiment of said method of the present invention, the second genomic location of b1) ii) is a second genomic safe harbour site.

[0093] In one further embodiment of said method of the present invention, the first genomic location of b1) i) is a first genomic safe harbour site and first genomic location of b1) i) is a first genomic safe harbour site. [0094] In one further embodiment of said method of the present invention, the first and the second genomic locations of b1), preferably the first and second genomic safe harbour sites of b1), are the same.

[0095] In one embodiment of said method of the present invention, the first and the second genomic locations of b1) are the same.

[0096] In one preferred embodiment of said method of the present invention, the first and the second genomic locations of b1) are a first and a second genomic safe harbour sites of b1) and are the same.

[0097] In this embodiment, wherein the first and the second genomic locations or the first and second genomic safe harbour sites are the same may comprise the use of an “all-in-one” inducible overexpression cassette that contains both components of the Tet-ON system (1. the transactivator rtTA being under the control of the strong constitutive CAG promoter, and 2. the transgene of interest being under the control of the strong inducible TRE promoter, which can be inserted into a single genomic location or single genomic safe harbor site (GSH), e.g. the AAVS1 , without being subject to transgene-silencing as observed in hPSCs, when using all-in-one genomic location/ GSH-targeting strategies. Further, the proliferative capacity and thus limitlessness of the starting/ source cell type is not compromised. The restricted developmental potency of NPCs towards ectodermal lineages does not represent a limitation for cell programming into other cell types as germ layer boundaries can be crossed by cellular reprogramming technologies. This “all- in-one”-strategy as described herein means that all transgene elements are integrated and expressed from one allele (in case of heterozygous targeting) or both alleles (in case of homozygous targeting) of one specific genomic safe harbour site. In theory, the two elements of the Tet-ON system could also be expressed separately from the two alleles of a single genomic safe harbour site, i.e. the transactivator rtTA is expressed from allele 1, and the inducible transgene of interest is expressed from allele 2. This approach is not meant by our all-in-one strategy.

[0098] In one embodiment of said method of the present invention, the first and the second genomic locations of b1), preferably the first and second genomic safe harbour sites of b1), are different. In one embodiment of said method of the present invention, the first and the second genomic locations of b1) are different. In one preferred embodiment of said method of the present invention, the first and the second genomic locations of b1) are a first genomic safe harbour site and a second genomic safe harbour site of b1), which are different.

[0099] In one further embodiment of said method of the present invention, step c) further comprises culturing the neural precursor cells under suitable conditions. In this embodiment, the term “suitable conditions” may mean any conditions that allow the provided neural precursor cells to be able to express the transgene of interest and to be able to differentiate into a desired cell type later on.

[00100] In one embodiment of said method of the present invention, the targeted insertion of the nucleotide sequence encoding the transcriptional regulator protein into the first genomic location of b1) i) and the targeted insertion of the nucleotide sequence encoding the transgene of interest into the second genomic location of b1) ii) is done in inverse directions.

[00101] In one further embodiment of said method of the present invention, the transgene of interest encodes the enhanced green fluorescence protein (EGFP). The EGFP is a basic (constitutively fluorescent) green fluorescent protein published in 1996, derived from Aequorea victoria. It is a rapidly-maturing weak dimer with moderate acid sensitivity. It allowed the practical use of GFPs in mammalian cells. EGFP has an extinction coefficient (denoted E) of 55,000 M" ¹ cm" ¹ . The fluorescence quantum yield (QY) of EGFP is 0.60. The relative brightness, expressed as E*QY, is 33,000 M" ¹ cm" ¹ .

[00102] In one embodiment of said method of the present invention, the transgene of interest encodes one or more transcription factor(s). The term “transcription factor (TF)” may also be called sequence-specific DNA-binding factor. As used within the context of the present invention, the term “transcription factor” means a protein that binds to DNA, preferably sequence- specifically to a DNA site located in or near a promoter, and either facilitating the binding of the transcription machinery to the promoter, and thus transcription of the DNA sequence (a transcriptional activator) or blocks this process (a transcriptional repressor). In the context of the present invention, a transcription factor is a desired genetic sequence, preferably a DNA sequence that is to be transferred into a cell together with an inducible cassette. The introduction of an inducible cassette into the genome has the potential to change the phenotype of that cell by addition of a genetic sequence that permits gene expression.

[00103] It is preferred for said embodiment that the one or more transcription factor(s) is/ are selected from the group consisting of neurogenin-2 (NGN2, NEUROG2), ASCL1, NEUROD1, LMX1A, FOXA2, NURR1, PITX3, LHX3, HB9, ISL1, DLX2, SOX10, OLIG2, NKX6.2, NKX6.1, NKX2.2, NFIA, NFIB, SOX9, SPI1 , CEBPB, IRF8, RUNX1, SALL1.

[00104] The coding sequence of NEUROG2 (= NGN2) is given herein as SEQ ID NO: 37 (see also NCBI Reference Sequence NM_024019.4). The coding sequence of ASCL1 is given herein as SEQ ID NO: 38 (see also NCBI Reference Sequence NM_004316.4). The coding sequence of NEUROD1 is given herein as SEQ ID NO: 39 (see also NCBI Reference Sequence: NM_002500.5). The coding sequence of LMX1A is given herein as SEQ ID NO: 40 (see also NCBI Reference Sequence: NM_177398.4). The coding sequence of FOXA2 is given herein as SEQ ID NO: 41 (see also NCBI Reference Sequence: NM_021784.5). The coding sequence of NR4A2 (= NLIRR1) is given herein as SEQ ID NO: 42 (see also NCBI Reference Sequence: NM_006186.4). The coding sequence of PITX3 is given herein as SEQ ID NO: 43 (see also NCBI Reference Sequence: NM_005029.4). The coding sequence of LHX3 is given herein as SEQ ID NO: 44 (see also NCBI Reference Sequence: NM_014564.5). The coding sequence of MNX1 (= HB9) is given herein as SEQ ID NO: 45 (see also NCBI Reference Sequence: NM_005515.4). The coding sequence of ISL1 is given herein as SEQ ID NO: 46 (NCBI Reference Sequence: NM_002202.3). The coding sequence of DLX2 is given herein as SEQ ID NO: 47 (see also NCBI Reference Sequence: NM_004405.4). The coding sequence of SOX10 is given herein as SEQ ID NO: 48 (see also NCBI Reference Sequence: NM_006941.4). The coding sequence of OLIG2 is given herein as SEQ ID NO: 49 (see also NCBI Reference Sequence: NM_005806.4). The coding sequence of NKX6.2 is given herein as SEQ ID NO: 50 (NCBI Reference Sequence: NM_177400.3). The coding sequence of NKX6.1 is given herein as SEQ ID NO: 51 (see also NCBI Reference Sequence: NM_006168.3). The coding sequence of NKX2.2 is given herein as SEQ ID NO: 52 (see also NCBI Reference Sequence: NM_002509.4). The coding sequence of NFIA is given herein as SEQ ID NO: 53 (see also NCBI Reference Sequence: NM_001134673.4). The coding sequence of NFIB is given herein as SEQ ID NO: 54 (see also NCBI Reference Sequence: NM_001190737.2). The coding sequence of SOX9 is given herein as SEQ ID NO: 55 (see also NCBI Reference Sequence: NM_000346.4). The coding sequence of SPI1 is given herein as SEQ ID NO: 56 (see also NCBI Reference Sequence: NM_003120.3). The coding sequence of CEBPB is given herein as SEQ ID NO: 57 (see also NCBI Reference Sequence: NM_005194.4). The coding sequence of IRF8 is given herein as SEQ ID NO: 58 (NCBI Reference Sequence: NM_001363907.1). The coding sequence of RLINX1 is given herein as SEQ ID NO: 59 (see also NCBI Reference Sequence: NM_001754.5). The coding sequence of SALL1 is given herein as SEQ ID NO: 60 (see also NCBI Reference Sequence: NM_002968.3).

[00105] Neurogenin-2 is a protein that in humans is encoded by the NEUROG2 gene. Neurogenin-2 is a member of the neurogenin subfamily of basic helix-loop-helix (bHLH) transcription factor genes that play an important role in neurogenesis.

[00106] ASCL1 is also called Achaete-scute homolog 1. It is a protein that in humans is encoded by the ASCL1 gene. Because it was discovered subsequent to studies on its homolog in Drosophila, the Achaete-scute complex, it was originally named MASH-1 for mammalian achaete scute homolog-1. This gene encodes a member of the basic helix-loop-helix (BHLH) family of transcription factors. The protein activates transcription by binding to the E-box (5'-CANNTG-3'). Dimerization with other BHLH proteins is required for efficient DNA binding. This protein plays a role in the neuronal commitment and differentiation and in the generation of olfactory and autonomic neurons.

[00107] NEUROD1 is also called neurogenic differentiation 1 (NeuroDI) or p2. It is a transcription factor of the NeuroD-type. It is encoded by the human gene NEUROD1. It is a member of the NeuroD family of basic helix-loop-helix (bHLH) transcription factors. The protein forms heterodimers with other bHLH proteins and activates transcription of genes that contain a specific DNA sequence known as the E-box. It regulates expression of the insulin gene, and mutations in this gene result in type II diabetes mellitus. NeuroDI is found to convert reactive glial cells into functional neurons in the mouse brain in vivo.

[00108] LMX1A is also known as LIM homeobox transcription factor 1. It is a protein, which in humans is encoded by the LMX1A gene and is a LIM homeobox transcription factor that binds an A/T-rich sequence in the insulin promoter and stimulates transcription of insulin.

[00109] FOXA2 is also known as forkhead box protein A2 (FOXA2) or hepatocyte nuclear factor 3-beta (HNF-3B). It is a transcription factor that plays an important role during development, in mature tissues and, when dysregulated or mutated, also in cancer. FOXA2 belongs to a subfamily of the forkhead box (FOX) transcription factors, the other members being FOXA1 and FOXA3. This subfamily of mammalian FOX proteins was first identified because of their ability to bind DNA in rat liver nuclear extracts. The proteins were therefore originally named hepatocyte nuclear factor 3 alpha, beta and gamma. These transcription factors contain a forkhead domain (also known as the winged-helix domain) flanked by sequences necessary for nuclear localization. Their N- and C-termini are also conserved and serve as transactivation domains.

[00110] NURR1 is also known as the nuclear receptor related 1 protein (NURR1) or as NR4A2 (nuclear receptor subfamily 4, group A, member 2). It is a protein that in humans is encoded by the NR4A2 gene. NURR1 is a member of the nuclear receptor family of intracellular transcription factors. NURR1 plays a key role in the maintenance of the dopaminergic system of the brain. Four transcript variants encoding four distinct isoforms have been identified for this gene. [00111] PITX3 is also known as pituitary homeobox 3. It is a protein that in humans is encoded by the PITX3 gene. This gene encodes a member of the RIEG/PITX homeobox family, which is in the bicoid class of homeodomain proteins. Members of this family act as transcription factors. This protein is involved in lens formation during eye development, and the specification and terminal differentiation of mesencephalic dopamine neurons in the substantia nigra compacta.

[00112] LHX3 is also known as LIM/homeobox protein Lhx3. It is a protein that in humans is encoded by the LHX3 gene. LHX3 is a member of the LIM-type homeodomain protein family of transcription factors that feature two LIM domains in their amino terminus (N-terminal) and a centrally located homeodomain used to interact with specific DNA elements on target genes. [00113] The homeobox gene HLXB9 encodes for the transcription factor HB9, which is essential for pancreatic as well as motor neuronal development. Beside its physiological expression pattern, aberrant HB9 expression has been observed in several neoplasias.

[00114] ISL1 is also known as insulin gene enhancer protein ISL-1. It is a protein that in humans is encoded by the ISL1 gene. This gene encodes a transcription factor containing two N- terminal LIM domains and one C-terminal homeodomain. The encoded protein plays an important role in the embryogenesis of pancreatic islets of Langerhans. In mouse embryos, a deficiency of this gene results in failure to undergo neural tube motor neuron differentiation. It is a protein involved in the retinal ganglion cell differentiation. This protein interacts with another POLI4F2 (transcription factor) that further enhances the development of the retina. This protein also cooperates with other proteins such as LHX3 and LDB1 proteins.

[00115] DLX2 is also known as homeobox protein DLX-2, which is a protein that in humans is encoded by the DLX2 gene. Many vertebrate homeo box-containing genes have been identified on the basis of their sequence similarity with Drosophila developmental genes. Members of the Dlx gene family contain a homeobox that is related to that of Distal-less (Dll), a gene expressed in the head and limbs of the developing fruit fly.

[00116] The transcription factor SOX10 is also called sex-determining region Y-box 10. It is part of a gene family of approximately 20 members. Structurally they are characterized by a DNA- binding HMG (High Motility Group) box domain. The different SOX gene members exert various biological functions such as sex determination and neuronal development.

[00117] OLIG2 is also called oligodendrocyte transcription factor (OLIG2), which is a basic helix-loop-helix (bHLH) transcription factor, encoded by the Olig2 gene. The protein is of 329 amino acids in length, 32kDa in size and contains one basic helix-loop-helix DNA-binding domain.

[00118] NKX6.2 is also called homeobox protein Nkx-6.2, a protein that in humans is encoded by the NKX6-2 gene.

[00119] NKX6.1 is a factor for IL-6-regulated growth and tumor formation in basal-like breast cancer. A significant relationship was observed between NKX6.1 and EMT marker expression levels, and NKX6.1 knockdown inhibited cell invasion, and overexpression of NKX6.1 promotes cell proliferation in vitro.

[00120] NKX2.2 is also called homeobox protein Nkx-2.2, which is a protein that in humans is encoded by the NKX2-2 gene. Homeobox protein Nkx-2.2 contains a homeobox domain and may be involved in the morphogenesis of the central nervous system. This gene is found on chromosome 20 near NKX2-4, and these two genes appear to be duplicated on chromosome 14 in the form of TITF1 and NKX2-8.

[00121] NFIA is also called nuclear factor 1 A-type, which is a protein that is encoded by the NFIA gene. Nuclear factor I (NFI) proteins constitute a family of dimeric DNA-binding proteins with similar, and possibly identical, DNA-binding specificity. They function as cellular transcription factors and as replication factors for adenovirus DNA replication. Diversity in this protein family is generated by multiple genes, differential splicing, and heterodimerization.

[00122] NFIB is a transcription factor that regulates the expression of lung differentiation programmes and is crucial for pulmonary maturation.

[00123] SOX9 is involved in organogenesis of the liver and pancreas. During hepatogenesis, SOX9 expression is confined to the bile duct, while hepatocytes do not express SOX9, and this expression pattern persists in adulthood.

[00124] SPI1 is also called transcription factor Pll.1, which is a protein that in humans is encoded by the SPI1 gene. This gene encodes an ETS-domain transcription factor that activates gene expression during myeloid and B-lymphoid cell development. The nuclear protein binds to a purine-rich sequence known as the Pll-box found on enhancers of target genes, and regulates their expression in coordination with other transcription factors and cofactors. The protein can also regulate alternative splicing of target genes. Multiple transcript variants encoding different isoforms have been found for this gene.

[00125] The transcription factor CEBPB is a critical mediator of steroid-stimulated proliferation and differentiation of uterine epithelial and endometrial stromal cells. Impaired decidualization in CEBPB-deficient mice was attributed to the inability of endometrial cells to complete their cell cycle.

[00126] IRF8 is a transcription factor that plays critical roles in the regulation of lineage commitment and in myeloid cell maturation including the decision for a common myeloid progenitor (CMP) to differentiate into a monocyte precursor cell.

[00127] RLINX1 is also called runt-related transcription factor 1, acute myeloid leukemia 1 protein (AML1) or core-binding factor subunit alpha-2 (CBFA2), a protein encoded by the RLINX1 gene. RLINX1 is a transcription factor that regulates the differentiation of hematopoietic stem cells into mature blood cells. In addition, it plays a major role in the development of the neurons that transmit pain. It belongs to the runt-related transcription factor (RLINX) family of genes, which are also called core binding factor-a (CBFa). RLINX proteins form a heterodimeric complex with CBFp which confers increased DNA binding and stability to the complex.

[00128] SALL1 is also called Sal-like 1 (Drosophila), which is a protein encoded by the SALL1 gene. As the full name suggests, it is one of the human versions of the spalt (sal) gene known in Drosophila. The protein encoded by this gene is a zinc finger transcriptional repressor and may be part of the NuRD histone deacetylase (HDAC) complex.

[00129] In one even more preferred embodiment thereof, the one or more transcription factor is NGN2, ASCL1 or NEUROD1. It is even more preferred for said embodiment that the one or more transcription factor is NGN2, ASCL1 or NEUROD1 , and the neural precursor cells are differentiated into neurons, preferably excitatory glutamatergic neurons, after step c).

[00130] In another even more preferred embodiment thereof, the one or more transcription factors are NGN2, ASCL1 or NEUROD1 and LMX1A and optionally one or more of the group consisting of FOXA2, NLIRR1 and PITX3. It is even more preferred for said embodiment that the one or more transcription factors are NGN2, ASCL1 or NEUROD1 and LMX1A and optionally one or more of the group consisting of FOXA2, NLIRR1 and PITX3, and the neural precursor cells are differentiated into dopaminergic midbrain neurons after step c).

[00131] In another even more preferred embodiment thereof, the one or more transcription factors are NGN2, ASCL1 or NEUROD1 and LHX3 and optionally HB9 and/ or ISL1. It is even more preferred for said embodiment that the one or more transcription factors are NGN2, ASCL1 or NEUROD1 and LHX3 and optionally HB9 and/ or ISL1, and the neural precursor cells are differentiated into cholinergic motor neurons after step c).

[00132] In another even more preferred embodiment thereof, the one or more transcription factors are ASCL1 and DLX2. It is even more preferred for said embodiment that the one or more transcription factors are ASCL1 and DLX2, and the neural precursor cells are differentiated into GABAergic inhibitory neurons after step c).

[00133] In another even more preferred embodiment thereof, the one or more transcription factors are SOX10 and one or more of the group consisting of OLIG2, NKX6.2, NKX6.1 and NKX2.2. It is even more preferred for said embodiment that the one or more transcription factors are SOX10 and one or more of the group consisting of OLIG2, NKX6.2, NKX6.1 and NKX2.2, and the neural precursor cells are differentiated into oligodendrocytes after step c).

[00134] In another even more preferred embodiment thereof, the one or more transcription factors are NFIA and optionally NFIB and/or SOX9. It is even more preferred for said embodiment that the one or more transcription factors are NFIA and optionally NFIB and/or SOX9, and the neural precursor cells are differentiated into astrocytes after step c).

[00135] In another even more preferred embodiment thereof, the one or more transcription factor is SOX10. It is even more preferred for said embodiment that the one or more transcription factor is SOX10, and the neural precursor cells are differentiated into neural crest-derived cells after step c).

[00136] In another even more preferred embodiment thereof, the one or more transcription factor(s) is/ are SPI1 and optionally one or more of the group consisting of CEBPB, IRF8, RLINX1 and SALL1. It is even more preferred for said embodiment that the one or more transcription factor(s) is/ are SPI1 and optionally one or more of the group consisting of CEBPB, IRF8, RLINX1 and SALL1, and the neural precursor cells are differentiated into microglia after step c). As used within the present invention, the term “microglia” means a mature cell type being a distinct cell population of the central nervous system. As defined in Comparative Anatomy and Histology, “microglia” is the resident histiocytic-type cell and the key innate immune effector of the CNS. They are often described as either resting (i.e. , ramified) or activated, but these terms fail to convey the dynamic remodeling of their fine processes and constitutive immunosurveillance activity. Microglia are generated during early embryonic stages and reside in the brain throughout adult live.

[00137] In one preferred embodiment of said method of the present invention, wherein the method comprises step b1), the transgene of interest encodes the transcription factor NGN2 and the method further comprises the generation of neurons from the neural precursor cells.

[00138] In one further preferred embodiment of said method of the present invention, wherein the method comprises step b1), the transgene of interest encodes the transcription factor NGN2, ASCL1 or NEUROD1, and the method further comprises the generation of neurons, preferably excitatory glutamatergic neurons, from the neural precursor cells. In one further preferred embodiment of said method of the present invention, wherein the method comprises step b1), the transgene of interest encodes the transcription factor NGN2, ASCL1 or NEUROD1, and the method further comprises the generation of excitatory glutamatergic neurons from the neural precursor cells.

[00139] In one preferred embodiment of said method of the present invention, wherein the method comprises step b1), the transgene of interest encodes the one or more transcription factors NGN2, ASCL1 or NEUROD1 and LMX1A and optionally one or more of the group consisting of FOXA2, NLIRR1 and PITX3, and wherein the method further comprises the generation of dopaminergic midbrain neurons from the neural precursor cells.

[00140] In one further preferred embodiment of said method of the present invention, wherein the method comprises step b1), the transgene of interest encodes the one or more transcription factors NGN2, ASCL1 or NEUROD1 and LHX3 and optionally HB9 and/ or ISL1, and the method further comprises the generation of cholinergic motor neurons from the neural precursor cells.

[00141] In one preferred embodiment of said method of the present invention, wherein the method comprises step b1), the transgene of interest encodes the transcription factors ASCL1 and DLX2, and the method further comprises the generation of GABAergic inhibitory neurons from the neural precursor cells.

[00142] In one further preferred embodiment of said method of the present invention, wherein the method comprises step b1), the transgene of interest encodes SOX10 and one or more of the group consisting of OLIG2, NKX6.2, NKX6.1 and NKX2.2, and the method further comprises the generation of oligodendrocytes from the neural precursor cells. [00143] In one preferred embodiment of said method of the present invention, wherein the method comprises step b1), the transgene of interest encodes NFIA and optionally NFIB and/or SOX9, and the method further comprises the generation of astrocytes from the neural precursor cells.

[00144] In one further preferred embodiment of said method of the present invention, wherein the method comprises step b1), the transgene of interest encodes SOX10, and the method further comprises the generation of neural crest-derived cells from the neural precursor cells.

[00145] In one preferred embodiment of said method of the present invention, wherein the method comprises step b1), the transgene of interest encodes SPI1 and optionally one or more of the group consisting of CEBPB, IRF8, RLINX1 and SALL1, and the method further comprises the generation of microglia from the neural precursor cells.

[00146] Thus, in summary, neurons may be created by the use of any of the three proneuronal bHLH TFs: NEUROG2 (= NGN2) or ASCL1 or NEUROD1. These three transcription factors induce a common neuronal default phenotype, i.e. excitatory glutamatergic neurons. Other neuronal subtypes are created by the combined expression of any of the TFs NEUROG2 (= NGN2) or ASCL1 or NEUROD1 (also called herein “proneuronal TFs “) with other subtype specific TFs: For example, dopaminergic midbrain neurons may be created by the proneuronal TFs + LMX1A +/- FOXA2 +/- NLIRR1 +/- PITX3, cholinergic motor neurons by the proneuronal TFs + LHX3 +/- HB9 +/- ISL1. GABAergic inhibitory neurons may be created by the two TFs ASCL1 + DLX2, oligodendrocytes by using SOX10 +/- OLIG2 +/- NKX6.2 +/- NKX6.1 +/- NKX2.2, astrocytes by using NFIA +/- NFIB +/- SOX9. Non-neuroectodermal lineages, like neural crest-derived cells, are created by SOX10. Microglia may be created by the following TFs: SPI1 +/- CEBPB +/- IRF8 +/- RUNX1 +/- SALL1.

[00147] In one preferred embodiment of the method for expression of a transgene of interest from neural precursor cells (NPCs), step a) comprises culturing stem cells including exposure of the stem cells to at least one growth factor(s), cytokine(s) and/ or small molecule(s) for differentiating the stem cells into neural precursor cells. It is more preferred for said embodiment that the at least one growth factor(s), cytokine(s) and/ or small molecule(s) is/ are selected from the group consisting of SB431542, dorsomorphin, CHIR99021, doxycycline, purmorphamine, cAMP, BDNF and NT3. Thus, in one even more preferred embodiment thereof, the at least one small molecule(s) is/ are selected from the group consisting of SB431542, CHIR99021, doxycycline, dorsomorphin, purmorphamine and cAMP.

[00148] As used within the present invention, the term “culturing” means the growth of microorganisms such as bacteria and yeast, or human, plant, or animal cells under suitable conditions ensuring the growth, which are knowledge of the person skilled in the art.

[00149] As used within the present invention, the term “stem cell” means a type of cell that is able to divide for producing more cells or to develop into a cell that has a particular purpose. In the present invention, the used stem cell might be a pluripotent stem cell. Pluripotent stem cells have the potential to differentiate into almost any cell 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 a blastocyst, an early-stage preimplantation embryo. Induced pluripotent stem cells (iPSCs) are adult cells that have been genetically reprogrammed to an embryonic stem celllike state by being forced to express genes and factors important for maintaining the defining properties of embryonic stem cells.

[00150] As used within the present invention, the term “growth factor” means a signaling molecule that controls cell activities in an autocrine, paracrine or endocrine manner. As used herein, in the context of the present invention, the term “growth factor” may be used interchangeably with “cytokine”. Growth factors or cytokines are produced by different cell types of the organism and exert their biological functions by binding to specific receptors and activating associated downstream signaling pathways which in turn, regulate gene transcription in the nucleus and ultimately stimulate a biological response, including regulatory cellular processes like cell division, cell survival, cell differentiation, adhesion and migration.

[00151] As used within the present invention, the term “small molecule” means a bioactive molecule that is naturally or artificially produced and is capable of diffusion through the cell membrane and is able to regulate signaling pathways.

[00152] SB431542 is characterized by the molecular formula C ₂ 2H ₁₆ N ₄ O3 and the following structural formula:

[00153] Dorsomorphin is characterized by the molecular formula C24H25N5O2HCI and the following structural formula:

[00154] CHIR99021 , as used in the present invention, means an amino pyrimidine derivative that is an extremely potent inhibitor of glycogen synthase kinase 3, inhibiting GSK3P (IC ₅₀ = 6.7 nM) and GSK3a (IC ₅₀ = 10 nM). It is characterized by the molecular formula C22H ₁₈ CI ₂ N ₈ and the following structural formula:

[00155] Doxycycline is characterized by the molecular formula C22H24N2O8 and the following structural formula:

[00156] Purmorphamine is characterized by the molecular formula C3iH ₃₂ N ₆ O2 and the following structural formula: [00157] cAMP is characterized by the molecular formula Cist^NsNaOsP and the following structural formula:

[00158] BDNF is also called Peprotech 450-02 and the sequence of this growth factor is given herein as SEQ ID NO: 3.

[00159] NT3 is also called Peprotech 450-03 and the sequence of this growth factor is given herein as SEQ ID NO: 4.

[00160] In another preferred embodiment of the method for expression of a transgene of interest from neural precursor cells (NPCs), step a) is without the use of one or more growth factor(s).

[00161] In one preferred embodiment of the method for expression of a transgene of interest from neural precursor cells (NPCs), said first and said second genomic location of b1) or the genomic location of b2) is/ are selected from the group consisting of the hROSA26 locus (SEQ ID NO: 61), the AAVS1 locus (SEQ ID NO: 62), the CLYBL gene (SEQ ID NO: 63), the CCR5 gene (SEQ ID NO: 64), the HPRT gene (SEQ ID NO: 65) or genes with the site ID 325 on chromosome 8 (SEQ ID NO: 66), site ID 227 on chromosome 1 (SEQ ID NO: 67), site ID 229 on chromosome 2 (SEQ ID NO: 68), site ID 255 on chromosome 5 (SEQ ID NO: 69), site ID 259 on chromosome 14 (SEQ ID NO: 70), site ID 263 on chromosome X (SEQ ID NO: 71), site ID 303 on chromosome 2 (SEQ ID NO: 72), site ID 231 on chromosome 4 (SEQ ID NO: 73), site ID 315 on chromosome 5 (SEQ ID NO: 74), site ID 307 on chromosome 16 (SEQ ID NO: 75), site ID 285 on chromosome 6 (SEQ ID NO: 76), site ID 233 on chromosome 6 (SEQ ID NO: 77), site ID 311 on chromosome 134 (SEQ ID NO: 78), site ID 301 on chromosome 7 (SEQ ID NO: 79), site ID 293 on chromosome 8 (SEQ ID NO: 80), site ID 319 on chromosome 11 (SEQ ID NO: 81), site ID 329 on chromosome 12 (SEQ ID NO: 82) and site ID 313 on chromosome X (SEQ ID NO: 83). In one more preferred embodiment of the method for expression of a transgene of interest from neural precursor cells (NPCs), said first and said second genomic location of b1) or the genomic location of b2) is/ are the hROSA26 locus (SEQ ID NO: 61). In one more preferred embodiment of the method for expression of a transgene of interest from neural precursor cells (NPCs), said first and said second genomic location of b1) or the genomic location of b2) is/ are the AAVS1 locus (SEQ ID NO: 62). In one more preferred embodiment of the method for expression of a transgene of interest from neural precursor cells (NPCs), said first and said second genomic location of b1) or the genomic location of b2) is/ are the CLYBL gene (SEQ ID NO: 63). In one more preferred embodiment of the method for expression of a transgene of interest from neural precursor cells (NPCs), said first and said second genomic location of b1) or the genomic location of b2) is/ are the CCR5 gene (SEQ ID NO: 64).

[00162] 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, which is expressed uniformly and ubiquitously in human tissues. This site serves as a specific integration locus for AAV serotype 2. AAVS1 has been shown to be a favourable environment for transcription, since it comprises an open chromatin structure and native chromosomal insulators that enable resistance of the inducible cassettes against silencing. There are no known adverse effects on a cell resulting from disruption of the PPP1R12C gene. Moreover, an inducible cassette inserted into this site remains transcriptionally active in many diverse cell types.

[00163] The hROSA26 site may also be a genetic location for inserting genetic material into this locus. The hROSA26 locus is on chromosome 3 (3p25.3), and can be found within the Ensembl database (GenBank: CR624523). The exact genomic co-ordinates of the integration site are 3:9396280-9396303: Ensembl. The integration site lies within the open reading frame (ORF) of the THUMPD3 long non-coding RNA (reverse strand). Since the hROSA26 site has an endogenous promoter, the inserted genetic material may take advantage of that endogenous promoter, or alternatively, may be inserted operably linked to a promoter.

[00164] Intron 2 of the Citrate Lyase Beta-like (CLYBL) gene, on the long arm of Chromosome 13, is one of the identified integration hot-spots of the phage derived phiC31 integrase. Studies have demonstrated that randomly inserted inducible cassettes into this locus are stable and expressed. It has been shown that insertion of inducible cassettes at this genetic location does not perturb local gene expression. CLYBL thus provides a GSH which may be used in the method of the present invention.

[00165] CCR5, which is located on chromosome 3 (position 3p21.31) is a gene, which codes for HIV-1 major co-receptor. Interest in the use of this site as a genomic location arises from the null mutation in this gene that appears to have no adverse effects, but predisposes to HIV-1 infection resistance. Zinc-finger nucleases that target the third exon have been developed, thus allowing for insertion of genetic material at this locus.

[00166] The following SHS sites may be used in any of the methods of the present invention. They were published by Pellenz et al., 2019 [9], and fulfil five out of nine criteria listed above: Site ID 325 on chromosome 8:68,720,172-68,720,191; site ID 227 on chromosome 1 :231 ,999,396- 231 ,999,415; site ID 229 on chromosome 2:45,708,354-45,708,373; site ID 255 on chromosome 5:19,069,307-19,069,326; site ID 259 on chromosome 14:92,099,558-92,099,577; site ID 263 on chromosome X:12, 590, 812-12, 590, 831 ; site ID 303 on chromosome 2:77,263,930-77,263,949; site ID 317 on chromosome 2:77,263,930-77,263,949; site ID 231 on chromosome 4:58,976,613- 58,976,632; site ID 315 on chromosome 5:7,577,728-7,577,747; site ID 307 on chromosome 16:19,323,777-19,323,796; site ID 285 on chromosome 6:89,574,320-89,574,339; site ID 233 on chromosome 6:114,713,905-114,713,924; site ID 311 on chromosome 6:134,385,946- 134,385,965; site ID 301 on chromosome 7:113,327,685-113,327,704; site ID 293 on chromosome 8:40,727,927-40,727,946; site ID 319 on chromosome 11:32,680,546-32,680,565; site ID 329 on chromosome 12:126,152,581-126,152,600; and site ID 313 on chromosome X:16,059,732- 16,059,751.

[00167] In a further aspect, the present invention also provides a system for expression of a transgene of interest from neural precursor cells, comprising: i) Neural precursor cells; ii) a nucleotide sequence encoding a transcriptional regulator protein, a nucleotide sequence of a constitutive promoter, wherein the transcriptional regulator protein is under the control of the constitutive promoter; a nucleotide sequence encoding a transgene of interest, and a nucleotide sequence encoding an inducible promoter, which is regulated by the transcriptional regulator protein and wherein the transgene of interest is operably linked to the inducible promoter; or iii) a nucleotide sequence encoding a transgene of interest, a nucleotide sequence of a constitutive promoter, wherein the transgene of interest is operably linked to the constitutive promoter.

[00168] In another aspect, the present invention also provides a vector comprising or consisting of one or more of the sequence(s) set forth in SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 , SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 , SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, or SEQ ID NO: 87. The definitions provided herein also apply for this aspect of the present invention. [00169] The present invention also provides in a further aspect a method for expression of a transgene of interest from stem cells, comprising the steps of: a) Providing stem cells, preferably pluripotent stem cells, induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs); b) i) targeted insertion of a nucleotide sequence encoding a transcriptional regulator protein being under the control of a first constitutive promoter, into a first genomic location, preferably the hROSA26 locus (SEQ ID NO. 61); and ii) targeted insertion of a nucleotide sequence encoding a first transgene of interest into a second genomic location, preferably the AAVS1 locus (SEQ ID NO: 62), wherein the first transgene of interest is operably linked to an inducible promoter, which is regulated by the transcriptional regulator protein; and iii) targeted insertion of the nucleotide sequence of a second transgene of interest into a third genomic location, preferably the CLYBL locus (SEQ ID NO: 63), wherein the second transgene of interest is operably linked to a second constitutive promoter, and c) expression of the first and /or second transgene of interest.

[00170] In one embodiment, the method for expression of a transgene of interest from stem cells, comprises the following steps: a) Providing pluripotent stem cells; b) i) targeted insertion of a nucleotide sequence encoding a transcriptional regulator protein being under the control of a first constitutive promoter, into a first genomic location, preferably the hROSA26 locus (SEQ ID NO: 61); and ii) targeted insertion of a nucleotide sequence encoding a first transgene of interest into a second genomic location, preferably the AAVS1 locus (SEQ ID NO: 62), wherein the first transgene of interest is operably linked to an inducible promoter, which is regulated by the transcriptional regulator protein; and iii) targeted insertion of the nucleotide sequence of a second transgene of interest into a third genomic location, preferably the CLYBL locus (SEQ ID NO: 63), wherein the second transgene of interest is operably linked to a second constitutive promoter, and c) expression of the first and /or second transgene of interest.

[00171] In one embodiment, the method for expression of a transgene of interest from stem cells, comprises the following steps: a) Providing induced pluripotent stem cells (iPSCs); b) i) targeted insertion of a nucleotide sequence encoding a transcriptional regulator protein being under the control of a first constitutive promoter, into a first genomic location, preferably the hROSA26 locus (SEQ ID NO: 61); and ii) targeted insertion of a nucleotide sequence encoding a first transgene of interest into a second genomic location, preferably the AAVS1 locus (SEQ ID NO: 62), wherein the first transgene of interest is operably linked to an inducible promoter, which is regulated by the transcriptional regulator protein; and iii) targeted insertion of the nucleotide sequence of a second transgene of interest into a third genomic location, preferably the CLYBL locus (SEQ ID NO: 63), wherein the second transgene of interest is operably linked to a second constitutive promoter, and c) expression of the first and /or second transgene of interest.

[00172] In one embodiment, the method for expression of a transgene of interest from stem cells, comprises the following steps: a) Providing embryonic stem cells (ESCs); b) i) targeted insertion of a nucleotide sequence encoding a transcriptional regulator protein being under the control of a first constitutive promoter, into a first genomic location, preferably the hROSA26 locus (SEQ ID NO: 61); and ii) targeted insertion of a nucleotide sequence encoding a first transgene of interest into a second genomic location, preferably the AAVS1 locus (SEQ ID NO: 62), wherein the first transgene of interest is operably linked to an inducible promoter, which is regulated by the transcriptional regulator protein; and iii) targeted insertion of the nucleotide sequence of a second transgene of interest into a third genomic location, preferably the CLYBL locus (SEQ ID NO: 63), wherein the second transgene of interest is operably linked to a second constitutive promoter, and c) expression of the first and /or second transgene of interest.

[00173] It is further preferred for said method that the method further comprises the differentiation of the stem cells into microglia.

[00174] Said method for expression of a transgene of interest from stem cells according to the present invention presents an extension of GSH-targeting strategy in hPSCs. The inventors extended their dual GSH-targeting method by using a triple GSH-targeting method for forward programming of reporter hPSCs into reporter target cells. This is exemplified by rapid and deterministic forward programming of mCherry hPSCs into microglia-like cells, with all starting and target cells expressing the fluorescent reporter, thus, providing a powerful tool for many downstream applications.

[00175] In one embodiment of the method for expression of a transgene of interest from stem cells according to the present invention, said stem cell is preferably a pluripotent stem cell, more preferably an induced pluripotent stem cell (iPSC) or an embryonic stem cell (ESC).

[00176] Within the present invention, the term “pluripotent stem cell” means cells having the potential to differentiate into almost any cell 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 a blastocyst, an early-stage preimplantation embryo. Induced pluripotent stem cells (iPSCs) are adult cells that have been genetically reprogrammed to an embryonic stem cell-like state by being forced to express genes and factors important for maintaining the defining properties of embryonic stem cells.

[00177] “Induced pluripotent stem cell” are a type of pluripotent stem cell, also known as iPS cells or iPSCs, which can be generated directly from a somatic cell. iPSCs can be derived directly from adult tissues.

[00178] In some embodiments of the method for expression of a transgene of interest from stem cells according to the present invention, it may be preferred that the used stem cell is an embryonic stem cell or stem cell line. Numerous embryonic stem cell lines are now available, for example, WA01 (HI), WA09 (H9), KhES-1 , KhES-2 and KhES-3. Stem cell lines, which have been derived without destroying an embryo, are available. The present invention does not extend to any methods which involve the destruction of human embryos.

[00179] The definitions provided herein also apply for this aspect of the present invention which is directed to a method for expression of a transgene of interest from stem cells.

[00180] In the context of the present invention and as used herein, the following abbreviations apply: hPSC = human pluripotent stem cell; TGF i = transforming growth factor pi ; FGF2 = fibroblast growth factor; hNPC = human neural progenitor cell; smNPC = small molecule neural progenitor cell; SB = SB43152; DM = Dorsomorphin; CHIR = CHIR99021 ; PM = Purmorphamine; Chr. = Chromosome; hROSA26 = human ROSA26 locus on chromosome 6 (human orthologue of the mouse reverse oriented splice acceptor (Rosa26)); 5‘-HAR = homology arm; 3’-HAR = homology arm; SA = splice acceptor; Neo = neomycin resistance cassette; pA = polyadenylation; CAG = constitutive CAG promoter; EGFP = enhanced green fluorescence protein; R26-prom = ROSA26 endogenous promoter; AAVS1 = human AAVS1 locus on chromosome 19 (adeno-associated virus integration site 1); T2A = 2A peptide of the thosea asigna virus; rtTA = reverse tetracycline transactivator; TRE = Tet-responsive element promoter; Puro = puromycin resistance cassette; MFI = mean fluorescence intensity; iEGFP = inducible EGFP expression; cEGFP = constitutive EGFP expression; TLIBB3 = pi I l-tubulin; TAU = microtubule associated protein tau; Hygro = hygromycin resistance cassette.

* * *

[00181] It is noted that as used herein, the singular forms “a”, “an”, and “the”, include plural references unless the context clearly indicates otherwise. Thus, for example, reference to “a reagent” includes one or more of such different reagents and reference to “the method” includes reference to equivalent steps and methods known to those of ordinary skill in the art that could be modified or substituted for the methods described herein.

[00182] Unless otherwise indicated, the term “at least” preceding a series of elements is to be understood to refer to every element in the series. The term “at least one” refers, if not particularly defined differently, to one or more such as two, three, four, five, six, seven, eight, nine, ten or more. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

[00183] The term “and/or” wherever used herein includes the meaning of “and”, “or” and “all or any other combination of the elements connected by said term”.

[00184] The term “less than” or in turn “more than” does not include the concrete number. [00185] For example, less than 20 mean less than the number indicated. Similarly, “more than” or “greater than” means more than or greater than the indicated number, e.g. more than 80 % means more than or greater than the indicated number of 80 %.

[00186] Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps, but not the exclusion of any other integer or step or group of integer or step. When used herein the term “comprising” can be substituted with the term “containing” or “including” or sometimes when used herein with the term “having”. When used herein “consisting of” excludes any element, step, or ingredient not specified.

[00187] The term “including” means “including but not limited to”. “Including” and “including but not limited to” are used interchangeably.

[00188] The term “about” means plus or minus 10%, preferably plus or minus 5%, more preferably plus or minus 2%, most preferably plus or minus 1%.

[00189] Throughout the description and the claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

[00190] It should be understood that this invention is not limited to the particular methodology, protocols, material, reagents, and substances, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims.

[00191] All publications cited throughout the text of this specification (including all patents, patent application, scientific publications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. To the extent the material incorporated by reference contradicts or is inconsistent with this specification, the specification will supersede any such material.

[00192] The content of all documents and patent documents cited herein is incorporated by reference in their entirety.

[00193] A better understanding of the present invention and of its advantages will be gained from the following examples, offered for illustrative purposes only. The examples are not intended to limit the scope of the present invention in any way. The invention is further characterized by the following items:

1. A method for expression of a transgene of interest from neural precursor cells (NPCs), comprising the steps of: a) Providing neural precursor cells; b1) i) targeted insertion of a nucleotide sequence encoding a transcriptional regulator protein being under the control of a suitable promoter, preferably a constitutive promoter, into a first genomic location; and ii) targeted insertion of a nucleotide sequence encoding a transgene of interest into a second genomic location, wherein the transgene of interest is operably linked to a suitable promoter, preferably an inducible promoter, which is regulated by the transcriptional regulator protein; or b2) targeted insertion of the nucleotide sequence encoding a transgene of interest into a genomic location, preferably a genomic safe harbour site, wherein the transgene of interest is operably linked to a constitutive promoter, and c) expression of the transgene of interest.

2. Method according to item 1 , wherein the transcriptional regulator protein of b1) i) is the reverse tetracycline transactivator (rtTA) and the activity thereof is controlled by doxycycline, tetracycline or a derivative thereof, preferably a reverse tetracycline transactivator (rtTA) comprising or consisting of SEQ ID NO: 5.

3. Method according to item 1 or item 2, wherein the suitable promoter of b1) ii) is an inducible promoter, preferably an inducible promoter including a Tet Responsive Element (TRE), more preferably a Tet Responsive Element (TRE) comprising or consisting of SEQ ID NO: 1.

4. Method according to any one of the previous items, wherein the suitable promoter of b1) i) is a constitutive promoter, preferably a constitutive CAG promoter, more preferably a constitutive CAG promoter comprising or consisting of the SEQ ID NO: 2.

5. Method according to any one of the previous items, wherein the constitutive promoter of b2) is a constitutive CAG promoter, preferably a constitutive CAG promoter comprising or consisting of the SEQ ID NO: 2. 6. Method according to any one of the previous items, wherein the first genomic location of b1) i) is a first genomic safe harbour site.

7. Method according to any one of the previous items, wherein the second genomic location of b1) ii) is a second genomic safe harbour site.

8. Method according to any one of the previous items, wherein the first and the second genomic locations of b1), preferably the first and second genomic safe harbour sites of b1), are the same.

9. Method according to any one of the previous items, wherein the first and the second genomic locations of b1), preferably the first and second genomic safe harbour sites of b1), are different.

10. Method according to any one of the previous items, wherein step c) further comprises culturing the neural precursor cells under suitable conditions.

11. Method according to any one of the previous items, wherein the targeted insertion of the nucleotide sequence encoding the transcriptional regulator protein into the first genomic location of b1) i) and the targeted insertion of the nucleotide sequence encoding the transgene of interest into the second genomic location of b1) ii) is done in inverse directions.

12. Method according to any one of the previous items, wherein the transgene of interest encodes the enhanced green fluorescence protein (EGFP).

13. Method according to any one of the previous items, wherein the transgene of interest encodes one or more transcription factor(s).

14. Method according to item 13, wherein the one or more transcription factor(s) is/ are selected from the group consisting of neurogenin-2 (NGN2, NEUROG2), ASCL1, NEUROD1, LMX1A, FOXA2, NURR1, PITX3, LHX3, HB9, ISL1 , DLX2, SOX10, OLIG2, NKX6.2, NKX6.1, NKX2.2, NFIA, NFIB, SOX9, SPI1, CEBPB, IRF8, RUNX1 and SALL1.

15. Method according to item 13 or item 14, wherein the one or more transcription factor is NGN2, ASCL1 or NEURODI . 16. Method according to item 13 or item 14, wherein the one or more transcription factor is NGN2, ASCL1 or NEUROD1, and wherein the neural precursor cells are differentiated into neurons, preferably excitatory glutamatergic neurons, after step c).

17. Method according to item 13 or item 14, wherein the one or more transcription factors are NGN2, ASCL1 or NEUROD1 and LMX1A and optionally one or more of the group consisting of FOXA2, NURR1 and PITX3.

18. Method according to item 13 or item 14, wherein the one or more transcription factors are NGN2, ASCL1 or NEUROD1 and LMX1A and optionally one or more of the group consisting of FOXA2, NLIRR1 and PITX3, and wherein the neural precursor cells are differentiated into dopaminergic midbrain neurons after step c).

19. Method according to item 13 or item 14, wherein the one or more transcription factors are NGN2, ASCL1 or NEUROD1 and LHX3 and optionally HB9 and/ or ISL1.

20. Method according to item 13 or item 14, wherein the one or more transcription factors are NGN2, ASCL1 or NEUROD1 and LHX3 and optionally HB9 and/ or ISL1 , and wherein the neural precursor cells are differentiated into cholinergic motor neurons after step c).

21. Method according to item 13 or item 14, wherein the one or more transcription factors are ASCL1 and DLX2.

22. Method according to item 13 or item 14, wherein the one or more transcription factors are ASCL1 and DLX2, and wherein the neural precursor cells are differentiated into GABAergic inhibitory neurons after step c).

23. Method according to item 13 or item 14, wherein the one or more transcription factors are SOXIO and one or more of the group consisting of OLIG2, NKX6.2, NKX6.1 and NKX2.2.

24. Method according to item 13 or item 14, wherein the one or more transcription factors are SOX10 and one or more of the group consisting of OLIG2, NKX6.2, NKX6.1 and NKX2.2, and wherein the neural precursor cells are differentiated into oligodendrocytes after step c).

25. Method according to item 13 or item 14, wherein the one or more transcription factors are NFIA and optionally NFIB and/ or SOX9. 26. Method according to item 13 or item 14, wherein the one or more transcription factors are NFIA and optionally NFIB and/ or SOX9, and wherein the neural precursor cells are differentiated into astrocytes after step c).

27. Method according to item 13 or item 14, wherein the one or more transcription factor is SOX10.

28. Method according to item 13 or item 14, wherein the one or more transcription factor is SOX10, and wherein the neural precursor cells are differentiated into neural crest-derived cells after step c).

29. Method according to item 13 or item 14, wherein the one or more transcription factors is/ are SPI1 and optionally one or more of the group consisting of CEBPB, IRF8, RLINX1 and SALLI.

30. Method according to item 13 or item 14, wherein the one or more transcription factors is/ are SPI1 and optionally one or more of the group consisting of CEBPB, IRF8, RLINX1 and SALL1, and wherein the neural precursor cells are differentiated into microglia after step c).

31. Method according to any one of the previous items, comprising step b1), wherein the transgene of interest encodes the transcription factor NGN2 and wherein the method further comprises the generation of neurons from the neural precursor cells.

32. Method according to any one of the previous items, comprising step b1), wherein the transgene of interest encodes the transcription factor NGN2, ASCL1 or NEUROD1, and wherein the method further comprises the generation of neurons, preferably excitatory glutamatergic neurons, from the neural precursor cells.

33. Method according to any one of the previous items, comprising step b1), wherein the transgene of interest encodes the one or more transcription factors NGN2, ASCL1 or NEUROD1 and LMX1A and optionally one or more of the group consisting of FOXA2, NLIRR1 and PITX3, and wherein the method further comprises the generation of dopaminergic midbrain neurons from the neural precursor cells.

34. Method according to any one of the previous items, comprising step b1), wherein the transgene of interest encodes the one or more transcription factors NGN2, ASCL1 or NEUROD1 and LHX3 and optionally HB9 and/ or ISL1 , and wherein the method further comprises the generation of cholinergic motor neurons from the neural precursor cells.

35. Method according to any one of the previous items, comprising step b1), wherein the transgene of interest encodes the transcription factors ASCL1 and DLX2, and wherein the method further comprises the generation of GABAergic inhibitory neurons from the neural precursor cells.

36. Method according to any one of the previous items, comprising step b1), wherein the transgene of interest encodes SOX10 and one or more of the group consisting of OLIG2, NKX6.2, NKX6.1 and NKX2.2, and wherein the method further comprises the generation of oligodendrocytes from the neural precursor cells.

37. Method according to any one of the previous items, comprising step b1), wherein the transgene of interest encodes NFIA and optionally NFIB and/ or SOX9, and wherein the method further comprises the generation of astrocytes from the neural precursor cells.

38. Method according to any one of the previous items, comprising step b1), wherein the transgene of interest encodes SOX10, and wherein the method further comprises the generation of neural crest-derived cells from the neural precursor cells.

39. Method according to any one of the previous items, comprising step b1), wherein the transgene of interest encodes SPI1 and optionally one or more of the group consisting of CEBPB, IRF8, RLINX1 and SALL1 , and wherein the method further comprises the generation of microglia from the neural precursor cells.

40. Method according to any one of the previous items, wherein step a) comprises culturing stem cells including exposure of the stem cells to at least one growth factor(s), cytokine(s) and/ or small molecule(s) for differentiating the stem cells into neural precursor cells.

41. Method according to item 40, wherein the at least one growth factor(s), cytokine(s) and/ or small molecule(s) is selected from the group consisting of SB431542, dorsomorphin, CHIR99021, doxycycline, purmorphamine, cAMP, BDNF and NT3. 42. Method according to item 40 or item 41, wherein the at least one small molecule(s) is/ are selected from the group consisting of SB431542, CHIR99021, doxycycline, dorsomorphin, purmorphamine and cAMP.

43. Method according to any one of the previous items, wherein the step a) is without the use of one or more growth factor(s).

44. Method of any one of the previous items, wherein said first and said second genomic location of b1) or the genomic location of b2) is/ are selected from the group consisting of the hROSA26 locus (SEQ ID NO: 61), the AAVS1 locus (SEQ ID NO: 62), the CLYBL gene (SEQ ID NO: 63), the CCR5 gene (SEQ ID NO: 64), the HPRT gene (SEQ ID NO: 65) or genes with the site ID 325 on chromosome 8 (SEQ ID NO: 66), site ID 227 on chromosome 1 (SEQ ID NO: 67), site ID 229 on chromosome 2 (SEQ ID NO: 68), site ID 255 on chromosome 5 (SEQ ID NO: 69), site ID 259 on chromosome 14 (SEQ ID NO: 70), site ID 263 on chromosome X (SEQ ID NO: 71), site ID 303 on chromosome 2 (SEQ ID NO: 72), site ID 231 on chromosome 4 (SEQ ID NO: 73), site ID 315 on chromosome 5 (SEQ ID NO: 74), site ID 307 on chromosome 16 (SEQ ID NO: 75), site ID 285 on chromosome 6 (SEQ ID NO: 76), site ID 233 on chromosome 6 (SEQ ID NO: 77), site ID 311 on chromosome 134 (SEQ ID NO: 78), site ID 301 on chromosome 7 (SEQ ID NO: 79), site ID 293 on chromosome 8 (SEQ ID NO: 80), site ID 319 on chromosome 11 (SEQ ID NO: 81), site ID 329 on chromosome 12 (SEQ ID NO: 82) and site ID 313 on chromosome X (SEQ ID NO: 83).

45. System for expression of a transgene of interest from neural precursor cells, comprising: i) Neural precursor cells; ii) a nucleotide sequence encoding a transcriptional regulator protein, a nucleotide sequence of a constitutive promoter, wherein the transcriptional regulator protein is under the control of the constitutive promoter; a nucleotide sequence encoding a transgene of interest, and a nucleotide sequence encoding an inducible promoter, which is regulated by the transcriptional regulator protein and wherein the transgene of interest is operably linked to the inducible promoter; or iii) a nucleotide sequence encoding a transgene of interest, a nucleotide sequence of a constitutive promoter, wherein the transgene of interest is operably linked to the constitutive promoter.

46. Vector comprising or consisting of one or more of the sequence(s) set forth in SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, or SEQ ID NO: 87.

47. A method for expression of a transgene of interest from stem cells, comprising the steps of: a) Providing stem cells, preferably pluripotent stem cells, induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs); b) i) targeted insertion of a nucleotide sequence encoding a transcriptional regulator protein being under the control of a first constitutive promoter, into a first genomic location, preferably the hROSA26 locus (SEQ ID NO: 61); and ii) targeted insertion of a nucleotide sequence encoding a first transgene of interest into a second genomic location, preferably the AAVS1 locus (SEQ ID NO: 62), wherein the first transgene of interest is operably linked to an inducible promoter, which is regulated by the transcriptional regulator protein; and iii) targeted insertion of the nucleotide sequence of a second transgene of interest into a third genomic location, preferably the CLYBL locus (SEQ ID NO: 63), wherein the second transgene of interest is operably linked to a second constitutive promoter, and c) expression of the first and/ or second transgene of interest.

48. Method according to item 47, wherein the method further comprises the differentiation of the stem cells into microglia.

EXAMPLES OF THE INVENTION

[00194] The following Examples illustrate the invention, but are not to be construed as limiting the scope of the invention.

[00195] Materials and Methods:

[00196] Nucleofection:

[00197] Nucleofection was performed using an Amaxa Nucleofector II (Lonza). First, nucleofection solution was prepared by adding the provided supplement. For detachment of the cells, culture medium was aspirated and cells were incubated in Accutase for 5 min at 37°C. Cells were washed off using split medium in a ratio of 1 :10. Cells were counted using a Neubauer chamber and trypan blue for detection of dead cells. 2 x 10 ⁶ cells were resuspended in 100 pl nucleofection mix containing the plasmids to be introduced into the cell (4 pg DNA per plasmid). The cell suspension was transferred into a nucleofection cuvette and pulsed with the program B-016. After 5 min incubation at RT, 500 pl pre-warmed pluripotency medium containing ROCK inhibitor (5 ng/ml) was added. After another incubation period of 5 min at RT cells were plated onto two Matrigel-coated 10 cm culture dishes. ROCK inhibitor was withdrawn after 24 h and medium was changed every 24 h. As soon as colonies reached an appropriate size (after 4-5 days), antibiotic selection with the respective substance was carried out for approximately 3-5 days. As soon as the remaining colonies reached a certain size, colonies were picked and transferred to separate 24-wells. Each clone was expanded and checked for the correct genotype.

[00198] ROSA26:

[00199] Targeting of the hROSA26 locus (SEQ ID NO: 61) was performed by transfecting the following plasmids: ROSA_guideA (SEQ ID NO: 14), ROSA_guideB (SEQ ID NO: 17) and pUC_ROSA_n_CAG-rtTA (SEQ ID NO: 25) (ROSA donor plasmid). Antibiotic selection was carried out using neomycin (G418, Sigma #4727878001) in a concentration of 25-100 pg/ml depending on the transfected cell line.

[00200] AAVS1 :

[00201] Targeting of the AAVS1 locus (SEQ ID NO: 62) was carried out using the already targeted cell line with rtTA (SEQ ID NO: 5) integrated into the hROSA26 locus (SEQ ID NO: 61). The donor plasmid pUC_AAVS1_pResp(PU.1-CEBPB) (SEQ ID NO: 84) was introduced into the cell in conjunction with two zinc-finger nucleases, pZFN-AAVS1-R-KKR (SEQ ID NO: 22) and pZFN- AAVS1-L-ELD (SEQ ID NO: 19). Antibiotic selection was performed using puromycin (ThermoFisher #A1113803) in a concentration of 0.3-0.75 pg/ml depending on the cell line.

[00202] CLYBL:

[00203] CLYBL targeting was performed with a double targeted cell line using two TALEN plasmids pZT-C13-R1 (SEQ ID NO: 28) and pZT-C13-L1 (SEQ ID NO: 30) and the donor plasmid pC13N-iCAG_mCherry_hygro (SEQ ID NO: 86) or pC13N-iCAG_copGFP_hygro (SEQ ID NO: 87). Antibiotic selection was performed using hygromycin in a concentration of 50-100 pg/ml.

[00204] Example 1

[00205] Firstly, we targeted a CAG-EGFP expression cassette (described previously in Bertero et al., Fig. 2A) into the hROSA26 GSH of smNPCs. Subsequent immunofluorescent imaging demonstrated EGFP-expression in all cells (see Fig. 2B).

[00206] Secondly, we generated an “all-in-one” AAVS1 targeting vector for inducible EGFP expression by molecular cloning. The vector contains EGFP under control of the inducible promoter (TRE = Tet-responsive-element, SEQ ID NO: 1) of the Tet-ON system in inverse orientation followed by two polyadenylation sites (SEQ ID NO: 88 and SEQ ID NO: 89) placed in head-to-head orientation and a CAG-promoter (SEQ ID NO: 2) driven rtTA transactivater transgene (see Fig. 3A). The inventors targeted this vector into the AAVS1 GSH of smNPCs. The inventors observed no leaky expression in the absence of doxycycline. Immunofluorescent imaging taken 24 hours after addition of doxycycline to the culture medium demonstrated homogeneous EGFP expression (coding sequence given herein as SEQ ID NO: 11) in smNPCs (see Fig. 3B).

[00207] Thirdly, the inventors generated an “all-in-one” AAVS1 targeting vector for inducible expression of the transcription factor NGN2 (also known as NEUROG2, SEQ ID NO: 37) by molecular cloning (see Fig. 4A). The inventors targeted the inducible NGN2 expression system into four independent smNPC lines. In all cases, addition of doxycycline resulted in rapid and deterministic programming into neurons as demonstrated by phase contrast images and immunocytochemistry for typical neuronal marker proteins, such as [33-tubulin and tau (see Fig. 4B).

[00208] Fourthly, the inventors combined the steps one (see Fig. 2) and three (see Fig. 4) to generate a dual GSH-targeted smNPCs with constitutive EGFP-expression (coding sequence given herein as SEQ ID NO: 11) and inducible NGN2-expression (SEQ ID NO: 37). This strategy allowed reporter cell production with reporter expression being resistant to transgene silencing ensuring reporter expression in 100 % of both starting and target cells (see Fig. 5).

[00209] Fifthly, in extension to our previous dual GSH targeting system in hPSCs, the inventors generated a triple GSH targeting system for robust reporter gene expression in starting and target cells. To this aim, the inventors generated hiPSC lines with three different transgene cassettes, each in a different GSHs: GSH 1 (CYLBL, SEQ ID NO: 63): reporter (exemplified by copEGFP and mCherry; SEQ ID NO: 25); GSH 2 hROSA26, SEQ ID NO: 61): rtTA (transactivator of the Tet-ON system, SEQ ID NO: 5); GSH 3 (AAVS1, SEQ ID NO: 62): inducible transgene (exemplified by SPI1-T2A-CEBPB, corresponding to PU.1-CEBPB, SEQ ID NO: 85);

[00210] The inventors therefore have demonstrated that this strategy allows forward programming of 100 % reporter positive starting cells into 100 % reporter positive target cells. This is exemplified by the generation of EGFP- (coding sequence given herein as SEQ ID NO: 11) or mCherry (coding sequence given herein as SEQ ID NO: 25) expressing macrophages and microglia.

[00211] Example 2: Induction of genetically modified hNPCs for hiNeuron generation

[00212] Targeted human hNPCs were cultured on Matrigel-coated 12-well culture dishes. Seeding densities varied between different cell lines from 5 x 10 ⁴ to 3 x 10 ⁵ cells per well. One day after seeding, hNPCs were induced with neuron medium containing dox (1 pg/ml). After 14 to 28 days of neuronal network formation, cells were used for further experiments or MGLs were added for coculture. After addition of MGLs, cells were further cultivated in MGL medium without dox.

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